PROMOTERS FROM CORYNEBACTERIUM GLUTAMICUM AND USES THEREOF IN REGULATING ANCILLARY GENE EXPRESSION

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 5, 2018, is named ZMG-004_PCT_SL.txt and is 645,695 bytes in size.

BACKGROUND
Field

The disclosure relates to native promoters comprising polynucleotides isolated from Corynebacterium glutamicum, and mutant promoters derived therefrom, host cells and recombinant vectors comprising the promoters, and methods of modifying the expression of ancillary target genes and producing biomolecules comprising culturing the host cells.

Description of the Related Art

Strains of industrial important bacteria play a significant role in the production of biomolecules. For example, coryneform bacteria, in particular Corynebacterium glutamicum, can be cultured to produce biomolecules such as amino acids, organic acids, vitamins, nucleosides and nucleotides. Continuous efforts are being made to improve production processes. Said processes may be improved with respect to fermentation related measures such as, for example, stirring and oxygen supply, or the composition of nutrient media, such as, for example, sugar concentration during fermentation, nutrient feeding schedules, pH balance, metabolite removal, or the work-up into the product form, for example by means of ion exchange chromatography, or the intrinsic performance characteristics of the microorganism itself.

Performance characteristics can include, for example, yield, titer, productivity, by-product elimination, tolerance to process excursions, optimal growth temperature and growth rate. One way to improve performance of a microbial strain is to increase the expression of genes that control the production of a metabolite. Increasing expression of a gene can increase the activity of an enzyme that is encoded by that gene. Increasing enzyme activity can increase the rate of synthesis of the metabolic products made by the pathway to which that enzyme belongs. In some instances, increasing the rate of production of a metabolite can unbalance other cellular processes and inhibit growth of a microbial culture. Sometimes, down regulating activity is important to improve performance of a strain. For example, re-directing flux away from by-products can improve yield. Accordingly, fine-tuning of expression levels of the various components simultaneously within a metabolic pathway is often necessary.

Promoters regulate the rate at which genes are transcribed and can influence transcription in a variety of ways. Constitutive promoters, for example, direct the transcription of their associated genes at a constant rate regardless of the internal or external cellular conditions, while regulatable promoters increase or decrease the rate at which a gene is transcribed depending on the internal and/or the external cellular conditions, e.g. growth rate, temperature, responses to specific environmental chemicals, and the like. Promoters can be isolated from their normal cellular contexts and engineered to regulate the expression of virtually any gene, enabling the effective modification of cellular growth, product yield and/or other phenotypes of interest.

For the production of a target biomolecule, a promoter is typically functionally linked to a heterologous target gene that is a component of the biosynthetic pathway that makes the target biomolecule in the host cell. For example for production of lysine, a component of the lysine biosynthetic pathway (e.g., as defined in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway M00030) can be functionally linked to a heterologous promoter. The universe of such on-pathway components is finite and well-explored, and the potential for further optimization by modulating expression or activity of on-pathway target genes to optimize target biomolecule production is limited. However, the potential impact on the productivity and yield of such biomolecules afforded by operably linking heterologous promoters to one or more ancillary target genes, and thereby modulate expression of such target genes, in industrially important host strains has been largely unexplored. Thus, there remains a need in the art for methods and compositions to screen for, identify, and use ancillary target genes that can be modulated to increase or decrease expression or activity and thereby improve target biomolecule production.

BRIEF SUMMARY

The present disclosure addresses these and other needs in the art. In brief, the present disclosure is directed to a host cell containing a promoter polynucleotide sequence functionally linked to at least one heterologous ancillary target gene, wherein the ancillary target gene is not a component of the biosynthetic pathway for producing the target biomolecule. The present disclosure provides methods for screening for, identifying, and using a promoter polynucleotide operably linked to a heterologous ancillary target gene to improve production of a target biomolecule.

In preferred embodiments, the promoter polynucleotide comprises a sequence selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the promoter polynucleotide consists of a sequence selected from:

SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:7.

In some embodiments, the ancillary target gene is a gene that is classified under GOslim term GO:0003674; GO:0003677; GO:0008150; GO:0034641; or GO:0009058. Preferably, the ancillary target gene is a gene that is classified under, or under at least, 2, 3, 4, or 5 of the following GOslim terms GO:0003674; GO:0003677; GO:0008150; GO:0034641; or GO:0009058. In some embodiments, the ancillary target gene is selected from the genes of one or more, or all, of the following KEGG entries: M00010, M00002, M00007, M00580, or M00005.

In some embodiments, the ancillary target gene is not a component of a biosynthesis pathway comprising genes of one or more, or all, of the following KEGG entries: M00016; M00525; M00526; M00527; M00030; M00433 M00031; M00020; M00018; M00021; M00338; M00609; M00017; M00019; M00535; M00570; M00432; M00015; M00028; M00763; M00026; M00022; M00023; M00024; M00025; and M00040.

In one embodiment, the disclosure provides a host cell containing at least a first and a second promoter polynucleotide sequence, wherein the first promoter is functionally linked to a first heterologous target gene, wherein the first heterologous target gene is a component of a biosynthetic pathway for producing a target biomolecule, and the second promoter is functionally linked to a second heterologous ancillary target gene that is not a component of the biosynthetic pathway for producing the target biomolecule. In some embodiments, the first promoter can be a native promoter comprising polynucleotides isolated from Corynebacterium glutamicum, and/or a mutant promoter derived therefrom, which can each be encoded by short DNA sequences, ideally less than 100 base pairs, while the second promoter comprises a sequence selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, both the first and the second promoter comprise a sequence selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the promoter polynucleotide consists of a sequence selected from: SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:7.

One embodiment of the present disclosure relates to host cells comprising the first and/or second promoter polynucleotides described herein. One embodiment of the present disclosure relates to recombinant vectors comprising the first promoter polynucleotide and/or second promoter polynucleotide described herein. In some embodiments, the first promoter polynucleotide is functionally linked to a first on-pathway target gene. In some embodiments, the second promoter polynucleotide is functionally linked to a first or second ancillary target gene. One embodiment of the present disclosure relates to host cells comprising the combinations of promoter polynucleotides described herein. One embodiment of the present disclosure relates to recombinant vectors comprising the combinations of promoter polynucleotides described herein. In some embodiments, each promoter polynucleotide is functionally linked to a different target gene. Preferably, as described and demonstrated in more detail herein, the target genes are not part of the same metabolic pathway. In some embodiments, a first set of target genes are part of the same metabolic pathway and a second set of target genes are part of a different pathway. One embodiment of the present disclosure relates to host cells transformed with the recombinant vectors described herein.

One embodiment of the present disclosure relates to host cells comprising at least one promoter polynucleotide functionally linked to an ancillary target gene; wherein the promoter polynucleotide comprises a sequence selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein when the promoter polynucleotide comprises a sequence selected from: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, the target gene is other than the promoter polynucleotide's endogenous gene. In some embodiments, the host cell comprises at least two promoter polynucleotides, wherein each promoter polynucleotide is functionally linked to a different target gene. One embodiment of the present disclosure relates to recombinant vectors comprising at least one promoter polynucleotide functionally linked to an ancillary target gene; wherein the promoter polynucleotide comprises a sequence selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein when the promoter polynucleotide comprises a sequence selected from: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, the target gene is other than the promoter polynucleotide's endogenous gene.

In some embodiments, the recombinant vector comprises at least two promoter polynucleotides, wherein each promoter polynucleotide is functionally linked to a different target gene. Preferably, as described and demonstrated more fully herein, the target genes are not part of the same metabolic pathway. For example, one target gene can be an on-pathway target gene for production of a target biomolecule, and the second target gene can be an ancillary target gene.

One embodiment of the present disclosure relates to host cells transformed with the recombinant vectors described herein. In some cases, the transformed host cells comprise a combination of promoter polynucleotides functionally linked to a heterologous ancillary target gene or at least one heterologous ancillary target gene, wherein said combination of promoter polynucleotides comprises a promoter ladder. The individual promoter polynucleotides can be in different transformed host cells and operably linked to the same heterologous ancillary target gene sequence. In some embodiments, said combination of promoter polynucleotides comprises at least one first promoter polynucleotide, and at least one second promoter polynucleotide. In some embodiments, the first promoter polynucleotide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:7 and the second promoter polynucleotide is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8 In some embodiments, said first and second promoter polynucleotide are in different host cells of a plurality of host cells and operably linked to the same heterologous ancillary target gene sequence. In some cases, the transformed host cells comprise a combination of promoter polynucleotides comprising a promoter ladder of two, three, four, five, six, seven, and/or eight different promoter polynucleotides. In some cases, said first, second, third, fourth, fifth, sixth, and/or seventh promoter polynucleotide are in different host cells of the plurality of transformed host cells and operably linked to the same heterologous ancillary target gene sequence.

In some cases, the transformed host cells comprising the combination of promoter polynucleotides functionally linked to a heterologous ancillary target gene or at least one heterologous ancillary target gene, wherein said combination of promoter polynucleotides comprises a promoter ladder, further comprises a promoter polynucleotide operably linked to an on-pathway, a shell 1, and/or a shell 2 heterologous target gene. In some cases, each of the transformed host cells, substantially all of the transformed host cells, or a majority of the transformed host cells comprises a promoter polynucleotide operably linked to an on-pathway, a shell 1, and/or a shell 2 heterologous target gene.

One embodiment of the present disclosure relates to methods of modifying the expression of one or more ancillary target genes, comprising culturing a host cell described herein, wherein the modification of each ancillary target gene is independently selected from: up-regulating and down-regulating. Preferably, the ancillary target gene does not code for one or more polypeptides or proteins of a biosynthetic pathway of biomolecules such as an amino acid, organic acid, nucleic acid, protein, or polymer. For example, in some embodiments, the ancillary target gene may code for one or more polypeptides or proteins of the biosynthetic pathway of a transcription factor, a signaling molecule, a component of the citric acid cycle, or a component of glycolysis.

Another embodiment of the present disclosure relates to methods of producing a biomolecule comprising culturing a host cell described herein, under conditions suitable for producing the biomolecule. In some embodiments the ancillary target gene directly or indirectly enhances the biosynthesis of a biomolecule selected from: amino acids, organic acids, flavors and fragrances, biofuels, proteins and enzymes, polymers/monomers and other biomaterials, lipids, nucleic acids, small molecule therapeutics, protein or peptide therapeutics, fine chemicals, and nutraceuticals. In preferred embodiments, the biomolecule is an L-amino acid. In specific embodiments, the L-amino acid is lysine.

In some embodiments, the host cell belongs to the genus Corynebacterium. In some embodiments, the host cell is Corynebacterium glutamicum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a diagram of the genetic and biochemical pathway for the biosynthesis of the amino acid L-lysine. Genes that divert intermediates in the biosynthetic pathway (e.g., pck, odx, icd, and hom) are underlined.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the disclosure. However, one skilled in the art will understand that the disclosure may be practiced without these details.

Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is as “including, but not limited to”.

As used herein, the term “recombinant nucleic acid molecule” refers to a recombinant DNA molecule or a recombinant RNA molecule. A recombinant nucleic acid molecule is any nucleic acid molecule containing joined nucleic acid molecules from different original sources and not naturally attached together. Recombinant RNA molecules include RNA molecules transcribed from recombinant DNA molecules. In particular, a recombinant nucleic acid molecule includes a nucleic acid molecule comprising a promoter of SEQ ID NOs:1 to 8 functionally linked to a heterologous target gene.

As used herein, the term “heterologous target gene” refers to any gene or coding sequence that is not controlled in its natural state (e.g., within a non-genetically modified cell) by the promoter to which it is operably linked in a particular genome. As provided herein, all target genes functionally linked to non-naturally occurring promoters are considered “heterologous target genes”. More specifically, as promoter polynucleotide sequences of SEQ ID NOs:1, 5, and 7 do not occur in nature, all functionally linked target gene sequences are “heterologous target gene” sequences. Similarly, all, e.g., naturally occurring, target genes in a host cell that are functionally linked with a promoter that is naturally occurring in the host cell but is not normally functionally linked to said target gene in a wild-type organism are “heterologous target genes.” As used herein, a heterologous target gene can include one or more target genes that are part of an operon. That is, the endogenous promoter of an operon is replaced with a promoter polynucleotide sequence having a nucleic sequence of SEQ ID NOs:1 to 8. As used herein, the term “promoter polynucleotide sequence” refers to nucleic acids having a sequence as recited in the associated SEQ ID NO.

A “metabolic pathway” or “biosynthetic pathway” is a series of substrate to product conversion reactions, each of which is catalysed by a gene product (e.g., an enzyme), wherein the product of one conversion reaction acts as the substrate for the next conversion reaction and which includes the conversion reactions from a feedstock to a target biomolecule. In some embodiments, the metabolic pathway is a pathway module as defined in the Kyoto Encyclopedia of Genes and Genomes KEGG database. As used herein, reference to the KEGG database, including maps and pathway modules therein, refers to the database as it is publicly available on the priority date of the present application.

An “on-pathway” heterologous target gene is a heterologous target gene that encodes a gene product (e.g., an enzyme or a component of a multi-enzyme complex) that is in the metabolic pathway by which the target biomolecule is produced in the organism in which it is present. Conventionally, the genes targeted for modification are those genes that are judged to be “on-pathway,” i.e., the genes for the metabolic enzymes known to be part of, or branching into or off of, the biosynthetic pathway for the molecule of interest (Keasling, JD. “Manufacturing molecules through metabolic engineering.” Science, 2010). Methods such as flux balance analysis (“FBA”) (Segre et al, “Analysis of optimality in natural and perturbed metabolic networks.” PNAS, 2002) are known that can automate the discovery of such genes.

An “ancillary” or “off-pathway” heterologous target gene, or heterologous target gene that is “not a component of a biosynthetic pathway for production of a target molecule” and the like is a heterologous target gene that does not encode a gene product (e.g., an enzyme or a component of a multi-enzyme complex) that is in the metabolic pathway by which the target biomolecule is produced in the organism in which it is present.

For example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule L-lysine is a gene that is not disclosed in KEGG pathway module M00016, M00030, M00031, M00433, M00525, M00526, or M00527, or preferably all thereof. As another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule serine is a gene that is not disclosed in KEGG pathway module M00020. As another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule threonine is a gene that is not disclosed in KEGG pathway module M00018. As another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule cysteine is a gene that is not disclosed in KEGG pathway module M00021, M00338, or M00609, or preferably all thereof.

As yet another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule valine and/or isoleucine is a gene that is not disclosed in KEGG pathway module M00019. As yet another example, an ancillary off-pathway heterologous target gene for production of the target biomolecule isoleucine is a gene that is not disclosed in KEGG pathway module M00535, or M00570, or preferably all thereof. As yet another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule leucine is a gene that is not disclosed in KEGG pathway module M00432.

As yet another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule proline is a gene that is not disclosed in KEGG pathway module M00015. As yet another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule ornithine is a gene that is not disclosed in KEGG pathway module M00028, M00763, or preferably all thereof. As yet another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule histidine is a gene that is not disclosed in KEGG pathway module M00026.

As yet another example, aromatic amino acids such as tryptophan, tyrosine, and phenylalanine are produced via the shikimate pathway. Thus, an ancillary or off-pathway heterologous target gene for production of the target biomolecule shikimate or an amino acid that is a biosynthetic product of the shikimate pathway (e.g., one or more of the target biomolecules tryptophan, tyrosine, or phenylalanine) is a gene that is not disclosed in KEGG pathway module M00022. As yet another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule tryptophan is a gene that is not disclosed in KEGG pathway module M00022. As yet another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule phenylalanine is a gene that is not disclosed in KEGG pathway module M00024. As yet another example, an ancillary or off-pathway heterologous target gene for production of the target biomolecule tyrosine is a gene that is not disclosed in KEGG pathway module M00025, M00040, or the combination thereof.

As yet another example, in some embodiments, in the context of producing an L-lysine target biomolecule, a heterologous target gene that is a component of the biosynthetic pathway that produces L-lysine is one of the following genes, or an endogenous functional ortholog thereof in the organism in which it is present, asd, ask, aspB, cg0931, dapA, dapB, dapD, dapE, dapF, ddh, fbp, hom, icd, lysA, lysE, odx, pck, pgi, ppc, ptsG, pyc, tkt, or zwf. Accordingly, in the context of producing an L-lysine target biomolecule, an ancillary or off-pathway heterologous target gene is a gene that is not one of the following genes, or an endogenous functional ortholog thereof in the organism in which it is present, asd, ask, aspB, cg0931, dapA, dapB, dapD, dapE, dapF, ddh, fbp, hom, icd, lysA, lysE, odx, pck, pgi, ppc, ptsG, pyc, tkt, or zwf.

In some embodiments, target genes are divided into priority levels, called “shells” and promoter polynucleotides are operably linked to one or more heterologous target genes of a shell, wherein the shell is comprised genes that are indirectly involved in target molecule production. As used herein, “shell 1” genes are genes that encode biosynthetic enzymes directly involved in a selected metabolic pathway. “Shell 2” genes include genes encoding for non-shell 1 enzymes or other proteins within the biosynthetic pathway responsible for product diversion or feedback signaling. “Shell 3” genes include regulatory genes responsible for modulating expression of the biosynthetic pathway or for regulating carbon flux within the host cell. “Shell 4” genes are the genes of a target organism that are not assigned to any one of shells 1-3. Example 5 describes allocation of genes in C. glutamicum into shells for systematic genome-wide perturbation of lysine production.

In some cases, an ancillary heterologous target gene is a “shell 2,” “shell 3,” and/or “shell 4” heterologous target gene for production of a target molecule. In some cases, an ancillary heterologous target gene is a “shell 3” and/or “shell 4” heterologous target gene for production of a target molecule. In some cases, the ancillary heterologous target gene is a “shell 3” heterologous target gene for production of a target molecule. In some cases, the ancillary heterologous target gene is a “shell 4” heterologous target gene for production of a target molecule. In some cases, the ancillary heterologous target gene is a “shell 2” heterologous target gene for production of a target molecule.

Exemplary target genes and their shell designation in the context of lysine production in C. glutamicum are provided in Table 10 below.

Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Polynucleotides Having Promoter Activity

Native C. glutamicum promoters were identified that satisfy both of the following criteria: 1) represent a ladder of constitutive promoters, i.e., a plurality of promoters with incrementally increasing levels of promoter activity; and 2) encoded by short DNA sequences, ideally less than 100 base pairs. A published data set describing global gene expression levels in C. glutamicum ATCC 13032 (Lee et al., Biotechnol Lett (2013) 35:709-717) was examined to identify genes that were constitutively expressed across different growth conditions. Genes whose expression level remained constant (defined as a ratio of expression between 0.33 and 3) across two growth conditions, namely chemostat growth in minimal media with and without the addition of hydrogen peroxide satisfied the first criterion. A published data set describing the C. glutamicum ATCC 13032 transcriptome (Pfeifer-Sancar et al., BMC Genomics 2013, 14:888) was examined to find genes with compact promoters, i.e. those consisting of the 60 base pair core promoter region and a 5′ untranslated region between 26 and 40 base pairs in length. The two data sets were cross-referenced to identify promoters that satisfied both criteria. The following five wild-type promoters were identified (Table 1).

TABLE 1

Promoters of C. glutamicum Having Increasing Levels of Expression

and Constituent Expression Under Different Growth Conditions

Strain
SEQ ID NO
Mean Activity

Pcg1860-eyfp
2
89243

Pcg0007-eyfp
3
44527

Pcg0755-eyfp
4
43592

Pcg3381-eyfp
6
4723

Pcg3121-eyfp
8
98

The wild-type promoters Pcg1860, and Pcg3121 are not described in the literature. The wild-type promoter Pcg0007-gyrB is also not described in the literature, however, Neumann and Quiñones, (J Basic Microbiol. 1997; 37(1):53-69) describes regulation of gyrB gene expression in E. coli. The wild type promoter Pcg0755 is a known part of the methionine biosynthesis pathway (Suda et al., Appl Microbiol Biotechnol (2008) 81:505-513; and Rey et al., Journal of Biotechnology 103 (2003) 51-65). The wild-type promoter Pcg3381 is a tatA homolog. The tatA pathway in Corynebacterium is described by Kikuchi et al., Applied and Environmental Microbiology, November 2006, p. 7183-7192. The strong constitutive promoter Pcg0007 was chosen for mutagenesis. Four out of six positions in the predicted ˜10 element (TAAGAT) of Pcg0007 were randomized to generate both stronger and attenuated promoter variants (SEQ ID NOs 1, 5, and 7).

Following the identification of promoters comprising SEQ ID NOs: 1-8, the present inventors determined that one or more such promoters can be functionally linked to one or more heterologous target genes of a biosynthetic pathway to increase the production of a target biomolecule produced by that biosynthetic pathway in a host cell. The identification and characterization of promoters of SEQ ID NOs: 1-8, and their use in upregaulting and/or downregulating expression of one or more on-pathway heterologous target genes to produce a target biomolecule are further described in PCT Appl. No. PCT/US16/65464, filed Dec. 7, 2016, the contents of which are hereby incorporated by reference in the entirety and for all purposes, including but not limited to the promoters of SEQ ID NO:1-8; vectors, expression cassettes, and host cells comprising said promoters, whether or not operably linked to a heterologous target gene, and methods and compositions for production of target biomolecules (e.g., using a promoter of SEQ ID NO:1-8).

Additionally, the present inventors surprisingly discovered that functionally linking one or more such promoters to one or more ancillary or off-pathway heterologous target genes can be used to increase production of the target biomolecule or further increase production of the target biomolecule.

For example, in some embodiments, functionally linking one or more such promoters to one or more ancillary heterologous target genes can be used to increase production of the target biomolecule in a strain background that does not have a promoter functionally linked to a heterologous target gene that is a component of the biosynthetic pathway that produces the target biomolecule. Additionally, in some embodiments, functionally linking one or more such promoters to one or more ancillary heterologous target genes can be used to increase production of the target biomolecule in a strain background that also comprises one or more promoters functionally linked to one or more heterologous target genes that are components of the biosynthetic pathway that produces the target biomolecule.

In some cases, the one or more promoters functionally linked to one or more heterologous target genes that are components of the biosynthetic pathway for production of a target biomolecule can be selected from SEQ ID NOs:1-8, SEQ ID NOs: 1, 5, and 7, and other promoters known in the art. Similarly, in some cases, the one or more promoters functionally linked to one or more ancillary heterologous target genes that are not components of the biosynthetic pathway for production of a target biomolecule can be selected from SEQ ID NOs:1-8, SEQ ID NOs: 1, 5, and 7, and other promoters known in the art.

Accordingly, one embodiment of the present disclosure relates to native promoters comprising polynucleotides isolated from C. glutamicum, and mutant promoters derived therefrom that together represent a ladder of constitutive promoters with incrementally increasing levels of promoter activity, wherein one or more of the ladder of promoters is functionally linked to a heterologous ancillary target gene for production of a target biomolecule. In some embodiments, a C. glutamicum promoter can be encoded by a short DNA sequence. In some embodiments a C. glutamicum promoter can be encoded by a DNA sequence of less than 100 base pairs. The promoters can be used in any strain background, including strains that also include a promoter functionally linked to a heterologous target gene that is in a biosynthetic pathway for production of a target biomolecule.

One embodiment of the present disclosure relates to a promoter polynucleotide comprising a sequence selected from: SEQ ID NO:1 (Pcg0007_lib_39), SEQ ID NO:2 (Pcg1860), SEQ ID NO:3 (Pcg0007), SEQ ID NO:4 (Pcg0755), SEQ ID NO:5 (Pcg0007_lib_265), SEQ ID NO:6 (Pcg3381), SEQ ID NO:7 (Pcg0007_lib_119), or SEQ ID NO:8 (Pcg3121). In another embodiment, the present specification provides for, and includes, a promoter polynucleotide comprising of SEQ ID NO:1 functionally linked to at least one heterologous ancillary target gene. In an embodiment, the present specification provides for, and includes, a promoter polynucleotide of SEQ ID NO:2 functionally linked to at least one heterologous ancillary target gene. In another embodiment, the present specification provides for, and includes, a promoter polynucleotide of SEQ ID NO:3 functionally linked to at least one heterologous ancillary target gene. In another embodiment, the present specification provides for, and includes, a promoter polynucleotide of SEQ ID NO:4 functionally linked to at least one heterologous ancillary target gene. In another embodiment, the present specification provides for, and includes, a promoter polynucleotide of SEQ ID NO:5 functionally linked to at least one heterologous ancillary target gene. In another embodiment, the present specification provides for, and includes, a promoter polynucleotide comprising of SEQ ID NO:5 functionally linked to at least one heterologous ancillary target gene. In another embodiment, the present specification provides for, and includes, a promoter polynucleotide of SEQ ID NO:7 functionally linked to at least one heterologous ancillary target gene. In another embodiment, the present specification provides for, and includes, a promoter polynucleotide of SEQ ID NO:8 functionally linked to at least one heterologous ancillary target gene.

As used herein, a “promoter cassette” refers to the polynucleotide sequences comprising a promoter polynucleotide of SEQ ID NOs:1 to 8 functionally linked to at least one heterologous ancillary target gene. In certain embodiments of the present disclosure, a “promoter cassette” may further include one or more of a linker polynucleotide, a transcription terminator following the ancillary target gene, a ribosome binding site upstream of the start codon of the ancillary target gene, and combinations of each.

One embodiment of the present disclosure relates to a promoter polynucleotide consisting of a sequence selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In an embodiment, the present specification provides for, and includes a promoter polynucleotide sequence of SEQ ID NO:1. In an embodiment, the present specification provides for, and includes a promoter polynucleotide sequence of SEQ ID NO:5. In an embodiment, the present specification provides for, and includes a promoter polynucleotide sequence of SEQ ID NO:7. As used herein, a promoter cassette may be described by reference to the promoter name followed by the name of the heterologous target gene that is functionally linked to it. For example, the promoter of SEQ ID NO: 2, entitled Pcg1860, functionally linked to the gene zwf encoding the off-pathway glucose-6-phosphate 1-dehydrogenase gene is referenced as Pcg1860-zwf. Similarly, Pcg0007_39-lysA is the 0007_39 promoter of SEQ ID NO:1 functionally linked to target gene lysA encoding the polypeptide diaminopimelate decarboxylase.

One embodiment of the present disclosure relates to combinations of the promoter polynucleotides described herein. In this context the term “combinations of promoter polynucleotides” refers to two or more polynucleotides that may be present as separate isolated sequences, as components of separate polynucleotide molecules, or as components of the same polynucleotide molecule, and combinations thereof. Examples of polynucleotide molecules include chromosomes and plasmids.

The disclosure also relates to an isolated promoter polynucleotide, which essentially consists of a polynucleotide having the nucleotide sequence depicted in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In an embodiment, the present specification provides for, and includes an isolated promoter polynucleotide of SEQ ID NO:1. In an embodiment, the present specification provides for, and includes an isolated promoter polynucleotide of SEQ ID NO:5. In an embodiment, the present specification provides for, and includes an isolated promoter polynucleotide of SEQ ID NO:7.

The term “essentially” in this context means that a polynucleotide of no more than 1,000, no more than 800, no more than 700, no more than 600, no more than 500 or no more than 400 nucleotides in length; and a polynucleotide of no more than 15,000, no more than 10,000, no more than 7,500, no more than 5,000, no more than 2,500, no more than 1,000, no more than 800, no more than 700, no more than 600, no more than 500, or no more than 400 nucleotides in length have been added to the 5′ end and 3′ end, respectively, of the polynucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

Any useful combination of the features from the preceding two lists of polynucleotides added to the 5′ end and 3′ end, respectively, of the polynucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, is in accordance with the invention here. “Useful combination” means, for example, a combination of features which results in an efficient recombination being carried out. The use of additions of the same length flanking a DNA region to be replaced facilitates the transfer of the region by homologous recombination in the experimental procedure. Relatively long flanking homologous regions are advantageous for efficient recombination between circular DNA molecules but cloning of the replacement vector is made more difficult with increasing length of the flanks (Wang et al., Molecular Biotechnology, 432:43-53 (2006)). The specification provides for, and includes, homologous regions flanking a promoter polynucleotide sequence of SEQ ID NOs:1 to 8 functionally linked to at least one heterologous ancillary target gene (e.g., the “promoter cassette”) to direct homologous recombination and replacement of a target gene sequence. In an embodiment, the homologous regions are direct repeat regions. In an embodiment, the homologous regions comprises between 500 base pairs (bp) and 5000 bp each of the target gene sequence flanking the promoter cassette. In an embodiment, the homologous regions comprises at least 500 bp each of the target gene sequence flanking the promoter cassette. In an embodiment, the homologous regions comprises at least 1000 bp (1 Kb) each of the target gene sequence flanking the promoter cassette. In an embodiment, the homologous regions comprises at least 2 Kb each of the target gene sequence flanking the promoter cassette. In an embodiment, the homologous regions comprises at least 5 Kb each of the target gene sequence flanking the promoter cassette.

The disclosure furthermore relates to an isolated promoter polynucleotide, which consists of the nucleotide sequence depicted in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In an embodiment, the isolated promoter polynucleotide consists of the polynucleotide sequence of SEQ ID NO:1. In an embodiment, the isolate promoter polynucleotide consists of the polynucleotide sequence of SEQ ID NO:5. In an embodiment, the isolate promoter polynucleotide consists of the polynucleotide sequence of SEQ ID NO:7.

Details regarding the biochemistry and chemical structure of polynucleotides as present in living things such as microorganisms, for example, can be found inter alia in the text book “Biochemie” [Biochemistry] by Berg et al. (Spektrum Akademischer Verlag Heidelberg Berlin, Germany, 2003; ISBN 3-8274-1303-6).

Polynucleotides consisting of deoxyribonucleotide monomers containing the nucleobases or bases adenine (A), guanine (G), cytosine (C) and thymine (T) are referred to as deoxyribo-polynucleotides or deoxyribonucleic acid (DNA). Polynucleotides consisting of ribonucleotide monomers containing the nucleobases or bases adenine (A), guanine (G), cytosine (C) and uracil (U) are referred to as ribopolynucleotides or ribonucleic acid (RNA). The monomers in said polynucleotides are covalently linked to one another by a 3′,5′-phosphodiester bond.

A “promoter polynucleotide” or a “promoter” or a “polynucleotide having promoter activity” means a polynucleotide, preferably deoxyribopolynucleotide, or a nucleic acid, preferably deoxyribonucleic acid (DNA), which when functionally linked to a polynucleotide to be transcribed determines the point and frequency of initiation of transcription of the coding polynucleotide, thereby enabling the strength of expression of the controlled polynucleotide to be influenced. The term “promoter ladder” as used herein refers to a plurality of promoters with incrementally increasing levels of promoter activity. The term “promoter activity” as used herein refers to the ability of the promoter to initiate transcription of an polynucleotide sequence into mRNA. Methods of assessing promoter activity are well known to those of skill in the art and include, for example the methods described in Example 2 of PCT/US16/65464. The term “constitutive promoter” as used herein refers to a promoter that directs the transcription of its associated gene at a constant rate regardless of the internal or external cellular conditions. In some cases, the promoters of the promoter ladder exhibit a range of promoter strengths in response to a stimuli (e.g., in response to induction with a chemical agent, heat, cold, stress, phosphate starvation, etc.). In some cases, the promoters of the promoter ladder exhibit a range of constitutive promoter strengths.

Owing to the double-stranded structure of DNA, the strand complementary to the strand in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 of the sequence listing is likewise a subject of the invention.

Kits

One embodiment of the present disclosure relates to kits comprising a first promoter polynucleotide comprising a sequence selected from: SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:7, and a suitable storage means for the polynucleotide. In some embodiments, the first promoter polynucleotide consists of a sequence selected from: SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:7. In some embodiments, the kits comprise combinations of promoter polynucleotides comprising at least two first promoter polynucleotides described herein. In some embodiments, the kits comprise combinations of promoter polynucleotides comprising at least one first promoter polynucleotide described herein, and at least one second promoter polynucleotide comprising a sequence selected from: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. In some embodiments, the kits comprise combinations of promoter polynucleotides comprising at least one first promoter polynucleotide described herein, and at least one second promoter polynucleotide consisting of a sequence selected from: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

Target Genes

One embodiment of the present disclosure relates to methods of modulating the expression of a heterologous target gene, comprising culturing a host cell transformed with a recombinant vector comprising a promoter polynucleotide as described herein. Heterologous target genes are polynucleotides the expression of which are controlled by the promoters described herein. The heterologous target genes may be coding polynucleotides which code for one or more polypeptide(s) or non-coding polynucleotides such as non-coding RNAs. A polynucleotide coding for a protein/polypeptide essentially consists of a start codon selected from the group consisting of ATG, GTG and TTG, preferably ATG or GTG, particularly preferably ATG, a protein-encoding sequence and one or more stop codon(s) selected from the group consisting of TAA, TAG and TGA. The heterologous target genes can be “on-pathway,” or “off-pathway,” or a combination thereof.

“Transcription” means the process by which a complementary RNA molecule is produced starting from a DNA template. This process involves proteins such as RNA polymerase, “sigma factors” and transcriptional regulatory proteins. Where the target gene is a coding polynucleotide, the synthesized RNA (messenger RNA, mRNA) then serves as a template in the process of translation which subsequently yields the polypeptide or protein.

“Functionally linked” means in this context the sequential arrangement of the promoter polynucleotide according to the disclosure with a further oligo- or polynucleotide, resulting in transcription of said further polynucleotide to produce a sense RNA transcript.

If the further polynucleotide is a target gene which codes for a polypeptide/protein and consists of the coding region for a polypeptide, starting with a start codon, including the stop codon and, where appropriate, including a transcription termination sequence, “functionally linked” then means the sequential arrangement of the promoter polynucleotide according to the invention with the target gene, resulting in transcription of said target gene and translation of the synthesized RNA.

If the target gene codes for a plurality of proteins/polypeptides, each gene may be preceded by a ribosome-binding site. Where appropriate, a termination sequence is located downstream of the last gene.

The target gene preferably codes for one or more polypeptides or proteins of the biosynthetic pathway of biomolecules, preferably selected from the group of proteinogenic amino acids, non-proteinogenic amino acids, vitamins, nucleosides, nucleotides and organic acids. The target gene preferably consists of one or more of the one-pathway and/or off-pathway target genes listed in Table 1 of EP 1 108 790 A2 which is hereby incorporated by reference.

The present specification provides for, and includes, recombinant nucleic acid molecules comprising a promoter polynucleotide sequence selected from the group consisting of SEQ ID NOs:1 to 8 functionally linked to any one of the heterologous target genes identifiable in the Kyoto Encyclopedia of Genes and Genomes (KEGG) as genes involved in metabolic and biosynthetic pathways. The KEGG database is available on the internet at genome.jp/kegg.

In preferred embodiments, the target biomolecule is an amino acid, a protein, or a carbohydrate polymer, and one or more of the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more ancillary target genes of the citric acid cycle. In some cases, the ancillary target genes are selected from the genes in KEGG pathway M00010. In one embodiment, the target biomolecule is an amino acid, a protein, or a carbohydrate polymer and one or more of the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more ancillary target genes of the glycolysis pathway. In some cases, the ancillary target genes are selected from the genes in KEGG pathway M00002. In one embodiment, the target biomolecule is an amino acid, a protein, or a carbohydrate polymer and one or more of the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more ancillary target genes of the pentose phosphate pathway. In some cases, the ancillary target genes are selected from the genes in KEGG pathway M00007, or M00580, or the combination thereof.

In one embodiment, the target biomolecule is an amino acid, a protein, or a carbohydrate polymer and one or more of the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more ancillary target genes of the PRPP biosynthesis pathway. In some cases, the ancillary target genes are selected from the genes in KEGG pathway M00005. In some cases, the target biomolecule is a specific amino acid or a set of amino acids, and one or more of the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more ancillary target genes selected from a metabolic pathway for production of a different amino acid or set of amino acids.

In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the lysine biosynthesis pathway as represented in KEGG map number 00300. In an embodiment, the one or more on-pathway target genes are selected from the Lysine succinyl-DAP biosynthesis pathway, M00016. In an embodiment, the one or more on-pathway target genes are selected from the lysine acetyl-DAP biosynthesis pathway, M00525. In an embodiment, the one or more on-pathway target genes are selected from the lysine DAP dehydrogenase biosynthesis pathway, M00526. In an embodiment, the one or more on-pathway target genes are selected from the lysine DAP aminotransferase biosynthesis pathway, M00527. In an embodiment, the one or more on-pathway target genes are selected from the AAA pathway biosynthesis pathway, M00030. In an embodiment, the one or more on-pathway target genes are selected from the lysine biosynthesis pathway from 2-oxoglutarate, M00433 or the lysine biosynthesis pathway mediated by LysW, M00031.

The present disclosure provides for, and includes, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the serine biosynthesis pathway comprising genes of entry M00020. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the threonine biosynthesis pathway comprising genes of KEGG entry M00018. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the cysteine biosynthesis pathway comprising genes of KEGG entry M00021. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the cysteine biosynthesis pathway comprising genes of KEGG entry M00338. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the cysteine biosynthesis pathway comprising genes of KEGG entry M00609. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the methionine biosynthesis pathway comprising genes of KEGG entry M00017. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the valine/isoleucine biosynthesis pathway comprising genes of KEGG entry M00019. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the isoleucine biosynthesis pathway comprising genes of KEGG entry M00535. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the isoleucine biosynthesis pathway comprising genes of KEGG entry M00570. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the leucine biosynthesis pathway comprising genes of KEGG entry M00432. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the proline biosynthesis pathway comprising genes of KEGG entry M00015. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the ornithine biosynthesis pathway comprising genes of KEGG entry M00028. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the ornithine biosynthesis pathway comprising genes of KEGG entry M00763. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the histidine biosynthesis pathway comprising genes of KEGG entry M00026. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the shikimate biosynthesis pathway comprising genes of KEGG entry M00022. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the tryptophan biosynthesis pathway comprising genes of entry M00023. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the phenylalanine biosynthesis pathway comprising genes of KEGG entry M00024. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the tyrosine biosynthesis pathway comprising genes of KEGG entry M00025. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes of the tyrosine biosynthesis pathway comprising genes of KEGG entry M00040.

In a preferred embodiment, one or more of the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes described herein and one or more promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more ancillary target genes described herein, e.g., in a host cell, a genome of a host cell, an expression cassette, and/or a polynucleotide vector. In another embodiment, one or more of the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more on-pathway target genes described herein and one or more other promoter polynucleotide sequences are functionally linked to one or more ancillary target genes described herein, e.g., in a host cell, a genome of a host cell, an expression cassette, and/or a polynucleotide vector. In yet another embodiment, one or more of the promoter polynucleotide sequences of SEQ ID NOs:1 to 8 are functionally linked to one or more ancillary target genes described herein and one or more other promoter polynucleotide sequences are functionally linked to one or more on-pathway target genes described herein, e.g., in a host cell, a genome of a host cell, an expression cassette, and/or a polynucleotide vector.

The present disclosure provides for, and includes, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the serine biosynthesis pathway comprising genes of entry M00020. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the threonine biosynthesis pathway comprising genes of KEGG entry M00018. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the cysteine biosynthesis pathway comprising genes of KEGG entry M00021. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the cysteine biosynthesis pathway comprising genes of KEGG entry M00338. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the cysteine biosynthesis pathway comprising genes of KEGG entry M00609. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the methionine biosynthesis pathway comprising genes of KEGG entry M00017. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the valine/isoleucine biosynthesis pathway comprising genes of KEGG entry M00019. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the isoleucine biosynthesis pathway comprising genes of KEGG entry M00535. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the isoleucine biosynthesis pathway comprising genes of KEGG entry M00570. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the leucine biosynthesis pathway comprising genes of KEGG entry M00432. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the proline biosynthesis pathway comprising genes of KEGG entry M00015. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the ornithine biosynthesis pathway comprising genes of KEGG entry M00028. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the ornithine biosynthesis pathway comprising genes of KEGG entry M00763. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the histidine biosynthesis pathway comprising genes of KEGG entry M00026. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the shikimate biosynthesis pathway comprising genes of KEGG entry M00022. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the tryptophan biosynthesis pathway comprising genes of entry M00023. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the phenylalanine biosynthesis pathway comprising genes of KEGG entry M00024. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the tyrosine biosynthesis pathway comprising genes of KEGG entry M00025. In an embodiment, the promoter polynucleotide sequences of SEQ ID NOs:1, 5 or 7 are functionally linked to one or more target genes of the tyrosine biosynthesis pathway comprising genes of KEGG entry M00040.

The present specification provides for, and includes, recombinant nucleic acid molecules comprising a promoter polynucleotide sequence selected from the group consisting of SEQ ID NOs:1 to 8 functionally linked to any one of the heterologous on- or off-pathway target genes from Corynebacterium glutamicum ATCC 13032 provided in Table 2 or any Corynebacterium glutamicum equivalent thereof. Sequence start and end positions correspond to genomic nucleotide accession NC 003450.3. It will be understood by those of ordinary skill in the art that corresponding genes exist in other strains of C. glutamicum and may be readily identified from Table 2. In an embodiment, the present specification provides for, and includes a recombinant nucleic acid molecule comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant nucleic acid molecule comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant nucleic acid molecule comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant nucleic acid molecule comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant nucleic acid molecule comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant nucleic acid molecule comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant nucleic acid molecule comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant nucleic acid molecule comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 2.

TABLE 2

Target genes from Corynebacterium glutamicum according to the present specification

Gene ID
Symbol
Aliases
Description
start
end
orientation

1021315
NCgl0248
NCgl0248,
aspartate-semialdehyde
270660
271694
plus

Cgl0252
dehydrogenase

1021300
NCgl0223
NCgl0223,
prephenate dehydrogenase
241880
242902
minus

Cgl0226

1021294
NCgl0247
NCgl0247,
aspartate kinase
269371
270636
plus

Cgl0251

1021282
NCgl0215
NCgl0215,
Aminotransferase
232257
233282
minus

Cgl0218

1021250
NCgl0181
NCgl0181,
glutamine 2-oxoglutarate
195240
199772
plus

Cgl0184
aminotransferase large

subunit

1021247
gltD
NCgl0182,
glutamate synthase
199772
201292
plus

Cgl0185

1021203
aroE
NCgl0409,
quinate/shikimate
446538
447389
plus

Cgl0424
dehydrogenase

1021149
NCgl0245
NCgl0245,
2-isopropylmalate synthase
266151
267896
minus

Cgl0248

1021136
gpmA
NCgl0390,
2,3-bisphosphoglycerate-
425177
425923
plus

Cgl0402
dependent phosphoglycerate

mutase

1021131
NCgl0408
NCgl0408,
3-dehydroquinate
446087
446524
plus

Cgl0423
dehydratase

1021078
NCgl0398
NCgl0398,
pyrroline-5-carboxylate
434877
435698
plus

Cgl0410
reductase

1020978
trpA
NCgl2932,
tryptophan synthase subunit
3239333
3240175
plus

Cgl3035
alpha

1020976
NCgl2931
NCgl2931,
tryptophan synthase subunit
3238083
3239336
plus

Cgl3034
beta

1020975
NCgl2930
NCgl2930,
bifunctional indole-3-
3236642
3238066
plus

trpC, trpF
glycerol phosphate

synthase/phospho-

ribosylanthranilate

isomerase

1020974
trpD
NCgl2929,
anthranilate
3235603
3236649
plus

Cgl3032
phosphoribosyltransferase

1020973
NCgl2928
NCgl2928,
anthranilate synthase II
3234957
3235583
plus

Cgl3031

1020972
NCgl2927
NCgl2927,
anthranilate synthase I
3233404
3234960
plus

Cgl3029

1020852
NCgl2809
NCgl2809,
pyruvate kinase
3110462
3112321
minus

Cgl2910

1020842
NCgl2799
NCgl2799,
prephenate dehydratase
3098576
3099523
minus

Cgl2899

1020841
NCgl2798
NCgl2798,
phosphoglycerate mutase
3097902
3098573
minus

Cgl2898

1020788
NCgl2747
NCgl2747,
Aminotransferase
3030670
3031983
plus

Cgl2844

1020745
NCgl2704
NCgl2704,
Nucleosidase
2988212
2988772
minus

Cgl2802

1020729
NCgl2688
NCgl2688,
cystathionine gamma-
2972058
2973206
minus

Cgl2786
synthase

1020714
NCgl2673
NCgl2673,
fructose-bisphosphate
2954239
2955273
minus

Cgl2770
aldolase

1020594
NCgl2557
NCgl2557,
dihydrodipicolinate
2815459
2816397
plus

Cgl2646
synthase

1020564
NCgl2528
NCgl2528,
D-2-hydroxyisocaproate
2786754
2787716
minus

Cgl2617
dehydrogenase

1020509
NCgl2474
NCgl2474,
serine acetyltransferase
2723065
2723613
plus

Cgl2563

1020508
NCgl2473
NCgl2473,
cysteine synthase
2721905
2722861
plus

Cgl2562

1020471
NCgl2436
NCgl2436,
phosphoserine phosphatase
2669555
2670856
minus

Cgl2522

1020393
NCgl2360
NCgl2360,
cystathionine gamma-
2590310
2591470
minus

Cgl2446
synthase

1020370
NCgl2337
NCgl2337,
ribose-5-phosphate
2563930
2564403
minus

Cgl2423
isomerase B

1020307
NCgl2274
NCgl2274,
gamma-glutamyl kinase
2496668
2497777
minus

Cgl2356

1020305
proA
NCgl2272,
gamma-glutamyl phosphate
2494337
2495635
minus

Cgl2354
reductase

1020301
NCgl2268
NCgl2268,
fructose-2,6-bisphosphatase
2491149
2491859
minus

Cgl2350

1020260
NCgl2227
NCgl2227,
PLP-dependent
2444607
2445713
plus

Cgl2309
aminotransferase

1020188
NCgl2155
NCgl2155,
bifunctional RNase H/acid
2371410
2372558
minus

Cgl2236
phosphatase

1020181
NCgl2148
NCgl2148,
glutamine synthase
2362816
2364156
minus

Cgl2229

1020172
NCgl2139
NCgl2139,
threonine synthase
2353598
2355043
minus

Cgl2220

1020166
NCgl2133
NCgl2133,
glutamine synthase
2348830
2350263
plus

Cgl2214

1020155
NCgl2123
NCgl2123,
branched-chain amino acid
2335913
2337016
minus

Cgl2204
aminotransferase

1020130
NCgl2098
NCgl2098,
3-deoxy-7-
2307695
2309095
minus

Cgl2178
phosphoheptulonate

synthase

1020087
NCgl2055
NCgl2055,
cysteine synthase
2258360
2259313
minus

Cgl2136

1020086
NCgl2054
NCgl2054,
diaminopimelate
2255736
2257025
minus

Cgl2135
decarboxylase

1020080
NCgl2048
NCgl2048,
methionine synthase II
2247004
2248209
minus

Cgl2129

1020078
NCgl2046
NCgl2046,
threonine dehydratase
2244862
2246172
minus

Cgl2127

1020053
hisD
NCgl2021,
histidinol dehydrogenase
2217597
2218925
minus

Cgl2102

1020052
NCgl2020
NCgl2020,
histidinol-phosphate
2216491
2217591
minus

Cgl2101
aminotransferase

1020051
hisB
NCgl2019,
imidazoleglycerol-
2215866
2216474
minus

Cgl2100
phosphate dehydratase

1020048
hisH
NCgl2016,
imidazole glycerol
2212638
2213273
minus

Cgl2097
phosphate synthase subunit

HisH

1020047
NCgl2015
NCgl2015,
phosphoribosyl isomerase A
2211879
2212619
minus

Cgl2096

1020045
hisF
NCgl2013,
imidazole glycerol
2210270
2211046
minus

Cgl2094
phosphate synthase subunit

HisF

1020044
hisI
NCgl2012,
phosphoribosyl-AMP
2209917
2210273
minus

Cgl2093
cyclohydrolase

1020042
NCgl2010
NCgl2010,
indole-3-glycerol phosphate
2208364
2209149
minus

Cgl2091
synthase

1020040
NCgl2008
NCgl2008,
pyruvate kinase
2205665
2207092
minus

Cgl2089

1019930
NCgl1898
NCgl1898,
4-hydroxy-
2081188
2081934
minus

Cgl1973
tetrahydrodipicolinate

reductase

1019928
dapA
NCgl1896,
4-hydroxy-
2079278
2080183
minus

Cgl1971
tetrahydrodipicolinate

synthase

1019900
dapF
NCgl1868,
diaminopimelate epimerase
2051842
2052675
minus

Cgl1943

1019614
NCgl1583
NCgl1583,
L-serine deaminase
1744884
1746233
plus

Cgl1645

1019598
aroE
NCgl1567,
shikimate 5-dehydrogenase
1724609
1725439
minus

Cgl1629

1019592
NCgl1561
NCgl1561,
chorismate synthase
1719666
1720898
minus

Cgl1623

1019591
aroK
NCgl1560,
shikimate kinase
1719104
1719676
minus

Cgl1622

1019590
aroB
NCgl1559,
3-dehydroquinate synthase
1717935
1719032
minus

Cgl1621

1019571
NCgl1541
NCgl1541,
methionine
1699174
1700397
minus

Cgl1603
adenosyltransferase

1019566
NCgl1536
NCgl1536,
ribulose-phosphate 3-
1693259
1693918
minus

Cgl1598
epimerase

1019556
NCgl1526
NCgl1526,
glyceraldehyde-3-phosphate
1682621
1683625
minus

Cgl1588
dehydrogenase

1019555
pgk
NCgl1525,
phosphoglycerate kinase
1681187
1682404
minus

Cgl1587

1019554
tpiA
NCgl1524,
triosephosphate isomerase
1680329
1681108
minus

Cgl1586

1019550
NCgl1520
NCgl1520,
ornithine cyclodeaminase
1674120
1675268
minus

Cgl1582

1019543
NCgl1513
NCgl1513,
Transaldolase
1666673
1667755
plus

Cgl1575

1019542
NCgl1512
NCgl1512,
Transketolase
1664403
1666505
plus

Cgl1574

1019512
NCgl1482
NCgl1482,
aconitate hydratase
1626279
1629110
plus

Cgl1540

1019480
NCgl1450
NCgl1450,
methionine synthase I
1587570
1591235
minus

Cgl1507
cobalamin-binding subunit

1019478
hisE
NCgl1448,
phosphoribosyl-ATP
1586462
1586725
minus

Cgl1505
pyrophosphatase

1019477
hisG
NCgl1447,
ATP
1585600
1586445
minus

Cgl1504
phosphoribosyltransferase

1019377
NCgl1347
NCgl1347,
argininosuccinate lyase
1471477
1472910
plus

Cgl1401

1019376
NCgl1346
NCgl1346,
argininosuccinate synthase
1470211
1471416
plus

Cgl1400

1019374
NCgl1344
NCgl1344,
ornithine
1468565
1469524
plus

Cgl1398
carbamoyltransferase

1019373
argD
NCgl1343,
acetylornithine
1467376
1468551
plus

Cgl1397
aminotransferase

1019372
NCgl1342
NCgl1342,
acetylglutamate kinase
1466422
1467375
plus

Cgl1396

1019371
argJ
NCgl1341,
bifunctional ornithine
1465210
1466376
plus

Cgl1395
acetyltransferase/N-

acetylglutamate synthase

1019370
argC
NCgl1340,
N-acetyl-gamma-glutamyl-
1464053
1465126
plus

Cgl1394
phosphate reductase

1019293
leuD
NCgl1263,
3-isopropylmalate
1381902
1382495
plus

Cgl1316
dehydratase small subunit

1019292
NCgl1262
NCgl1262,
3-isopropylmalate
1380440
1381885
plus

Cgl1315
dehydratase large subunit

1019267
NCgl1237
NCgl1237,
3-isopropylmalate
1353489
1354511
plus

Cgl1286
dehydrogenase

1019265
NCgl1235
NCgl1235,
D-3-phosphoglycerate
1350855
1352447
plus

Cgl1284
dehydrogenase

1019254
NCgl1224
NCgl1224,
ketol-acid reductoisomerase
1340724
1341740
plus

Cgl1273

1019253
ilvH
NCgl1223,
acetolactate synthase small
1340025
1340543
plus

Cgl1272
subunit

1019252
NCgl1222
NCgl1222,
acetolactate synthase large
1338131
1340011
plus

Cgl1271
subunit

1019249
NCgl1219
NCgl1219,
dihydroxy-acid dehydratase
1333439
1335280
minus

Cgl1268

1019232
NCgl1202
NCgl1202,
6-phosphofructokinase
1315046
1316086
plus

Cgl1250

1019167
NCgl1137
NCgl1137,
homoserine kinase
1243855
1244784
plus

Cgl1184

1019166
NCgl1136
NCgl1136,
homoserine dehydrogenase
1242507
1243844
plus

Cgl1183

1019163
NCgl1133
NCgl1133,
diaminopimelate
1239929
1241266
plus

Cgl1180
decarboxylase

1019124
NCgl1094
NCgl1094,
5-
1188385
1190622
minus

Cgl1139
methyltetrahydropteroyl-

triglutamate--homocysteine S-

methyltransferase

1019117
aroE
NCgl1087,
shikimate 5-dehydrogenase
1180869
1181675
minus

Cgl1132

1019094
NCgl1064
NCgl1064,
succinyl-diaminopimelate
1155731
1156840
plus

Cgl1109
desuccinylase

1019093
NCgl1063
NCgl1063,
tetrahydrodipicolinate N-
1154726
1155676
minus

Cgl1108
succinyltransferase

1019091
NCgl1061
NCgl1061,
2,3,4,5-tetrahydropyridine-
1152370
1153263
minus

Cgl1106
2,6-dicarboxylate N-

succinyltransferase

1019042
NCgl1013
NCgl1013,
phosphoglycerate mutase
1107503
1108204
plus

Cgl1058

1018983
glyA
NCgl0954,
serine
1050624
1051928
plus

Cgl0996
hydroxymethyltransferase

1018979
NCgl0950
NCgl0950,
phospho-2-dehydro-3-
1046610
1047710
plus

Cgl0990
deoxyheptonate aldolase

1018968
NCgl0939
NCgl0939,
threonine dehydratase
1038718
1039650
minus

Cgl0978

1018964
eno
NCgl0935,
phosphopyruvate hydratase
1034949
1036226
plus

Cgl0974

1018934
NCgl0905
NCgl0905,
ribose-phosphate
997463
998440
minus

Cgl0942
pyrophosphokinase

1018929
NCgl0900
NCgl0900,
glyceraldehyde-3-phosphate
993174
994616
plus

Cgl0937
dehydrogenase

1018848
NCgl0819
NCgl0819,
hypothetical protein
910852
911157
minus

Cgl0853

1018824
gltA
NCgl0795,
type II citrate synthase
877838
879151
plus

Cgl0829

1018823
NCgl0794
NCgl0794,
phosphoserine
875982
877112
minus

Cgl0828
aminotransferase

1018809
NCgl0780
NCgl0780,
Aminotransferase
861592
862755
plus

Cgl0814

1018794
NCgl0765
NCgl0765,
fructose-1,6-bisphosphatase
841514
842296
minus

Cgl0799

1018759
NCgl0730
NCgl0730,
3-phosphoshikimate 1-
801187
802479
minus

Cgl0764
carboxyvinyltransferase

1018688
NCgl0659
NCgl0659,
pyruvate carboxylase
705211
708633
plus

Cgl0689

1018663
NCgl0634
NCgl0634,
monomeric isocitrate
677828
680044
minus

Cgl0664
dehydrogenase (NADP+)

In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to an on- or off-pathway heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 2.

In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to an on- or off-pathway heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 2. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 2.

In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to an on- or off-pathway heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 3.

TABLE 3

C. glutamican L-lysine Biosynthetic Pathway

C. Glutamicum

Symbol
Gene Name (EC #)
Gene
Position
Expression

Asd
aspartate-semialdehyde
Asd
270660 . . . 271694
+

dehydrogenase (EC: 1.2.1.11)

dapA
4-hydroxy-tetrahydrodipicolinate
dapA
Complement
+

synthase (EC: 4.3.3.7)

(2079278 . . . 2080183)

dapB
dihydrodipicolinate reductase
Cgl1973
complement
+

(EC: 1.17.1.8)

(2081188 . . . 2081934)

dapD
2,3,4,5-tetrahydropyridine-2-
dapD
complement
+

carboxylate N-

(1153838 . . . 1154731)

succinyltransferase

(EC: 2.3.1.117)

dapD
2,3,4,5-tetrahydropyridine-2-
dapD2
complement

carboxylate N-

(1156194 . . . 1157144)

succinyltransferase

(EC: 2.3.1.117)

cg0931
N-succinyldiaminopimelate
cg0931
863063 . . . 864226
+

aminotransferase (EC: 2.6.1.17)

dapE
succinyl-diaminopimelate
dapE
1157199 . . . 1158308
+

desuccinylase (EC: 3.5.1.18)

dapF
diaminopimelate epimerase
dapF
complement
+

(EC: 5.1.1.7)

(2021891 . . . 2022724)

lysA
diaminopimelate decarboxylase
lysA
1241397 . . . 1242734
+

(EC: 4.1.1.20)

ddh
diaminopimelate dehydrogenase
Ddh
complement
+

(EC: 1.4.1.16)

(2760062 . . . 2761024)

ask (lysC)
Aspartokinase Lysc Alpha And
lysC
269371 . . . 270636
+

Beta Subunits (EC: 2.7.2.4)

aspB
Aspartate Aminotransferase
aspB
256618 . . . 257898
+

(EC: 2.6.1.1)

PTS
Phosphotransferase System
ptsG
1424684 . . . 1426735
+

(PTS); Glucose-Specific Enzyme

II BC Component Of PTS

(EC: 2.7.1.69)

zwf
glucose-6-phosphate 1-
Zwf
1669327 . . . 1670871
+

dehydrogenase (EC: 1.1.1.49

1.1.1.363)

pgi
glucose-6-phosphate isomerase
Pgi
complement
+

(EC: 5.3.1.9)

(909227 . . . 910849)

Tkt
transketolase (EC: 2.2.1.1)
Tkt
1665870 . . . 1667972
+

fbp
6-phosphofructokinase 1
Cgl1250
1315046 . . . 1316086
+

(EC: 2.7.1.11)

ppc
phosphoenolpyruvate
ppc
complement
+

carboxylase (EC: 4.1.1.31)

(1678851 . . . 1681610)

pyc
pyruvate carboxylase
pyc
706684 . . . 710106
+

(EC: 6.4.1.1)

icd
isocitrate dehydrogenase
Icd
complement
−

(EC: 1.1.1.42)

(679301 . . . 681517)

pck
phosphoenolpyruvate
pck
complement
−

carboxykinase (GTP)

(3025365 . . . 3027197)

(EC: 4.1.1.32)

odx
Oxaloacetate decarboxylase
odx
AP017369.1:
−

(EC 4.1.1.3)

1508967 . . . 1509782

(from C. glutamicum

N24)

hom
homoserine kinase (EC: 2.7.1.39)
Cgl1184
1243855 . . . 1244784
−

homoserine dehydrogenase
Cgl1183
1242507 . . . 1243844
−

(EC: 1.1.1.3);

threonine synthase
Cgl2220
complement
−

(EC: 4.2.3.1)

(2353598 . . . 2355043)

In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to an on- or off-pathway heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 3.

In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to an on or off-pathway heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 3. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 3.

The present specification provides for a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence selected from the group consisting of SEQ ID NOs:1 to 8 functionally linked to any one of the on- or off-pathway heterologous target genes from Corynebacterium glutamicum ATCC 13032 provided in Table 4 or their Corynebacterium glutamicum equivalent thereof. Sequence start and end positions correspond to genomic nucleotide accession NC 003450.3. It will be understood by those of ordinary skill in the art that corresponding genes exist in other strains of C. glutamicum and may be readily identified from Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 4.

TABLE 4

C. glutamican L-methionine Biosynthetic Pathway

C. Glutamicum

Symbol
Gene Name (EC #)
Gene
Position

lysC
aspartate kinase
Cgl0251
269371 . . . 270636

[EC: 2.7.2.4]

aspartate-
Cgl0252
270660 . . . 271694

semialdehyde

dehydrogenase

[EC: 1.2.1.11]

dapA
4-hydroxy-
dapA
complement

tetrahydro-

(2079278 . . . 2080183)

dipicolinate

synthase

[EC: 4.3.3.7]

dapA
4-hydroxy-
Cgl2646
2815459 . . . 2816397

tetrahydro-

dipicolinate

synthase

[EC: 4.3.3.7]

dapB
4-hydroxy-
Cgl1973
complement

tetrahydro-

(2081188 . . . 2081934)

dipicolinate

reductase

[EC: 1.17.1.8]

dapD
2,3,4,5-
Cgl1106
complement

tetrahydro-

(1152370 . . . 1153263)

pyridine-2-

carboxylate N-

succinyltransferase

[EC: 2.3.1.117]

dapC
N-
Cgl0814
861592 . . . 862755

succinyldiamino-

pimelate

aminotransferase

[EC: 2.6.1.17]

dapE
succinyl-
Cgl1109
1155731 . . . 1156840

diaminopimelate

desuccinylase

[EC: 3.5.1.18]

dapF
diaminopimelate
dapF
complement

epimerase

(2051842 . . . 2052675)

[EC: 5.1.1.7]

lysA
diaminopimelate
Cgl1180
1239929 . . . 1241266

decarboxylase

[EC: 4.1.1.20]

In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to an on- or off-pathway heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 4.

In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to an on- or off-pathway heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 4. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 4.

The present specification provides for a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence selected from the group consisting of SEQ ID NOs:1 to 8 functionally linked to any one of the off-pathway heterologous target genes from Corynebacterium glutamicum ATCC 13032 provided in Table 5 or their Corynebacterium glutamicum equivalent thereof. Sequence start and end positions correspond to genomic nucleotide accession NC 003450.3. It will be understood by those of ordinary skill in the art that corresponding genes exist in other strains of C. glutamicum and may be readily identified from Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 5.

TABLE 5

C. glutamicum Off-Pathway Target Genes

C. glutamicum

Symbol
Gene Name (EC #)
Gene
Position

Putative
NCgl0019
21501 . . . 21872

uncharacterized

protein Cgl0020

Rhodanese-related
NCgl0054
complement

sulfurtransferases

(92527 . . . 93120)

ureR
Transcriptional
NCgl0082
complement

regulators

(128299 . . . 128814)

tyrA
Prephenate
NCgl0223
complement

dehydrogenase

(283783 . . . 284805)

[EC 1.3.1.12]

topA
DNA
NCgl0304
375048 . . . 378053

topoisomerase 1

[EC 5.99.1.2]

nusG
Transcription
NCgl0458
604434 . . . 605405

termination/

antitermination

protein NusG

rpoB
DNA-directed RNA
NCgl0471
619984 . . . 623463

polymerase subunit

beta (RNAP

subunit beta)

[EC 2.7.7.6]

Transcriptional
NCgl0601
758218 . . . 758801

regulators

rhlE
Superfamily II DNA
NCgl0737
complement

and RNA helicases

(925165 . . . 926439)

prfB
Peptide chain
NCgl0767
962986 . . . 964092

release factor 2

(RF-2)

purH
Bifunctional purine
NCgl0827
1042072 . . . 1043634

biosynthesis

protein PurH

[EC 2.1.2.3],

IMP cyclohydrolase

[EC 3.5.4.10]

Putative
NCgl0966
1184332 . . . 1185576

uncharacterized

protein Cgl1009

rho
Transcription
NCgl1152
1386927 . . . 1389359

termination

factor Rho

[EC 3.6.4.—]

Transcriptional
NCgl1261
complement

regulator

(1533093 . . . 1533800)

leuC
3-isopropylmalate
NCgl1262
1534054 . . . 1535493

dehydratase large

subunit

[EC 4.2.1.33]

ddl
D-alanine--
NCgl1267
1538769 . . . 1539844

D-alanine

ligase

[EC 6.3.2.4]

Predicted
NCgl1301
1571632 . . . 1572609

transcriptional

regulators

cmk
Cytidylate kinase
NCgl1372
1667477 . . . 1668187

(CK)

[EC 2.7.4.25]

ctaB
Protoheme IX
NCgl1511
complement

farnesyltransferase

(1826368 . . . 1827339)

[EC 2.5.1.—]

Transcriptional
NCgl2425
complement

regulators

(2640625 . . . 2641119)

tRNA-
NCgl2481
2699108 . . . 2700253

dihydrouridine

synthase

[EC 1.3.1.—]

Transcriptional
NCgl2527
complement

regulator

(2760930 . . . 2761629)

AraC-type
NCgl2587
2824273 . . . 2825286

DNA-binding

domain-containing

proteins

hspR
Predicted
NCgl2699
complement

transcriptional

(2955572 . . . 2956012)

regulators

dnaK
Chaperone protein
NCgl2702
complement

DnaK (HSP70)

(2958093 . . . 2959949)

Transcriptional
NCgl2802
3083687 . . . 3084724

regulator

mutM2
Formamido-
NCgl2898
3217037 . . . 3217852

pyrimidine-

DNA glycosylase

[EC 3.2.2.23]

Transcriptional
NCgl2921
complement

regulator

(3246164 . . . 3246940)

Uncharacterized
NCgl2982
3305877 . . . 3309221

membrane protein,

putative virulence

factor

In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to an off-pathway heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 5.

In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to an off-pathway heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 5. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 5.

In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to an off-pathway heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to an off-pathway heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to an off-pathway heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 10.

The present specification provides for a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence selected from a plurality of promoter polynucleotides comprising a promoter ladder. In some cases, the host cell is a component of a plurality of transformed host cells comprising the promoter ladder, e.g., wherein each cell of the plurality comprises a different promoter polynucleotide of the promoter ladder. In some cases, the promoter polynucleotides of the promoter ladder, in the same or different transformed host cells of the plurality, are operably linked to the same heterologous, e.g., ancillary, target gene. In some cases, the heterologous target gene is a shell 2, a shell 3, and/or a shell 4 heterologous target gene. In some cases, the heterologous target gene is a shell 3, and/or a shell 4 heterologous target gene. In some cases the heterologous target gene is a shell 4 heterologous target gene. In some cases, the heterologous target gene is a shell 2 heterolgous target gene. In some cases, the heterologous target gene is a shell 3 heterologous target gene. In some cases, the heterologous target gene is a heterologous target gene from Corynebacterium glutamicum, such as the heterologous target genes provided in Table 10, or optionally any one of the tables described herein. Sequence start and end positions in Table 10 correspond to genomic nucleotide accession NC_003450.3. It will be understood by those of ordinary skill in the art that corresponding genes exist in other strains of C. glutamicum and may be readily identified from the present disclosure.

In some cases, the promoter polynucleotides comprising the promoter ladder are selected from the group consisting of SEQ ID NOs:1 to 8 functionally linked to an off-pathway heterologous target gene, e.g., a shell 2, a shell 3, and/or a shell 4 heterologous target gene, an off-pathway heterologous target gene provided in Table 10, or optionally an off pathway target gene in any one of the tables described herein. In some cases the heterologous target gene is a shell 4 heterologous target gene.

In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to a shell 2, a shell 3, and/or a shell 4 heterologous target gene, e.g., a heterologous target gene recited in Table 10. In some cases the heterologous target gene is a shell 4 heterologous target gene. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a shell 2, a shell 3, and/or a shell 4 heterologous target gene, e.g., a heterologous target gene recited in Table 10. In some cases the heterologous target gene is a shell 4 heterologous target gene. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a shell 2, a shell 3, and/or a shell 4 heterologous target gene, e.g., a heterologous target gene recited in Table 10. In some cases the heterologous target gene is a shell 4 heterologous target gene. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a shell 2, a shell 3, and/or a shell 4 heterologous target gene, e.g., a heterologous target gene recited in Table 10. In some cases the heterologous target gene is a shell 4 heterologous target gene.

In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a shell 2, a shell 3, and/or a shell 4 heterologous target gene, e.g., a heterologous target gene recited in Table 10. In some cases the heterologous target gene is a shell 4 heterologous target gene. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a shell 2, a shell 3, and/or a shell 4 heterologous target gene, e.g., a heterologous target gene recited in Table 10. In some cases the heterologous target gene is a shell 4 heterologous target gene.

In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a shell 2, a shell 3, and/or a shell 4 heterologous target gene, e.g., a heterologous target gene recited in Table 10. In some cases the heterologous target gene is a shell 2 heterologous target gene. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a functionally linked to a shell 2, a shell 3, and/or a shell 4 heterologous target gene, e.g., a heterologous target gene recited in Table 10. In some cases the heterologous target gene is a shell 4 heterologous target gene.

In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to an off-pathway heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 10.

In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:1 functionally linked to an off-pathway heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:2 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:3 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:4 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:5 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:6 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:7 functionally linked to a heterologous target gene recited in Table 10. In an embodiment, the present specification provides for, and includes, a host cell transformed with a recombinant vector comprising a promoter polynucleotide sequence of SEQ ID NO:8 functionally linked to a heterologous target gene recited in Table 10.

As used herein, a host cell refers to an organisms described below in the section entitled ‘Expression’ that have been transformed with one or more of the promoter cassettes. As will be apparent to one of ordinary skill in the art, a host cell may comprise one or more promoter cassettes as described herein.

In some embodiments, the target gene is associated with a biosynthetic pathway producing a biomolecule selected from: amino acids, organic acids, flavors and fragrances, biofuels, proteins and enzymes, polymers/monomers and other biomaterials, lipids, nucleic acids, small molecule therapeutics, protein therapeutics, fine chemicals, and nutraceuticals.

In some embodiments the target gene is associated with a biosynthetic pathway producing a secondary metabolite selected from: antibiotics, alkaloids, terpenoids, and polyketides. In some embodiments the target gene is associated with a metabolic pathway producing a primary metabolite selected from: alcohols, amino acids, nucleotides, antioxidants, organic acids, polyols, vitamins, and lipids/fatty acids. In some embodiments the target gene is associated with a biosynthetic pathway producing a macromolecule selected from: proteins, nucleic acids, and polymers

In addition it may be advantageous for the production of L-amino acids to enhance, in particular to overexpress one or more enzymes of the respective biosynthesis pathway, glycolysis, anaplerosis, citric acid cycle, pentose phosphate cycle, amino acid export and optionally regulatory proteins.

Thus for example, for the production of L-amino acids, it may be advantageous for one or more genes selected from the following group to be enhanced, in particular overexpressed: the gene dapA coding for dihydrodipicolinate synthase (EP-B 0 197 335); the gene eno coding for enolase (DE: 19947791.4); the gene gap coding for glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086); the gene tpi coding for triosephosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086); the gene pgk coding for 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086); the gene zwf coding for glucose-6-phosphate dehydrogenase (JP-A-09224661); the gene pyc coding for pyruvate carboxylase (DE-A-198 31 609; Eikmanns (1992), Journal of Bacteriology 174:6076-6086); the gene mqo coding for malate-quinone-oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)); the gene lysC coding for a feedback-resistant aspartate kinase (Accession No. P26512); the gene lysE coding for lysine export (DE-A-195 48 222); the gene hom coding for homoserine dehydrogenase (EP-A 0131171); the gene ilvA coding for threonine dehydratase (Mockel et al., Journal of Bacteriology (1992) 8065-8072)) or the allele ilvA (Fbr) coding for a feedback-resistant threonine dehydratase (Mockel et al., (1994) Molecular Microbiology 13: 833-842); the gene ilvBN coding for acetohydroxy acid synthase (EP-B 0356739); the gene ilvD coding for dihydroxy acid dehydratase (Sahm and Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979); and the gene zwa1 coding for the Zwa1 protein (DE: 19959328.0, DSM 13115).

Furthermore it may be advantageous for the production of L-amino acids also to attenuate, in particular to reduce, the expression of one or more genes selected from the group: the gene pck coding for phosphoenol pyruvate carboxykinase (DE 199 50 409.1; DSM 13047); the gene pgi coding for glucose-6-phosphate isomerase (U.S. Pat. No. 6,586,214; DSM 12969); the gene poxB coding for pyruvate oxidase (DE: 1995 1975.7; DSM 13114); and the gene zwa2 coding for the Zwa2 protein (DE: 19959327.2, DSM 13113).

In addition, it may furthermore be advantageous, for the production of amino acids, in particular L-lysine, to eliminate undesirable side reactions, (Nakayama: “Breeding of Amino Acid Producing Micro-organisms”, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, U K, 1982).

The promoter according to the disclosure can thus be used in each case for overexpressing or underexpressing the target gene in C. glutamicum.

Methods of Identifying Ancillary Target Genes for Optimizing Production of Target Biomolecules

Described herein are methods for screening for and/or identifying ancillary target genes for modulation of expression and/or activity to improve target biomolecule production. In some cases, the heterologous ancillary target genes are shell 2, shell 3, and/or shell 4 target genes. In some cases, the heterologous ancillary target genes are shell 3 and/or shell 4 target genes. In some cases, the heterologous ancillary target genes are shell 4 target genes. Typically the methods involve screening a library of transformed host cells, wherein individual transformed host cells of the library comprise a different [promoter polynucleotide: operably linked heterologous ancillary target gene] combination as compared to other transformed cells of the library. Such combinations can then be identified from the library that improve target biomolecule production and used for manufacture of target biomolecule or further optimized.

Thus the methods can include one or more steps of providing such a library, and/or screening such a library, and/or identifying transformants exhibiting improved target molecule production, and/or isolating such improved transformants, and/or storing or expanding such improved transformants. In some embodiments, the promoter polynucleotides comprise a promoter ladder.

Generally, transformed host cells of the library further comprise an on-pathway modification. In some cases, the on-pathway modification is the same for all, essentially all, substantially all, or a majority of the transformed cells of the library. For example, for lysine production, all, essentially all, substantially all, or a majority of the transformed cells of the library can comprise a promoter polynucleotide operably linked to the on-pathway heterologous target gene lysA and/or one or more other promoter polynucleotide(s) operably linked to on-pathway heterologous target gene(s). In some cases, the transformed host cells comprise a wild-type strain background such that endogenous on-pathway target genes are operably linked to their corresponding endogenous promoters.

The library of transformed cells can comprise a promoter ladder, wherein the individual promoter polynucleotides of the promoter ladder are in different cells of the library. In general, different promoter polynucleotides of the promoter ladder are operably linked to the same heterologous ancillary target gene in the different transformed cells. As an example, for a library comprising a promoter ladder having eight different promoter polynucleotides and interrogating a single heterologous ancillary target gene, the minimum library size is eight cells, one cell containing each possible [promoter polynucleotide: operably linked heterologous ancillary target gene] combination, or nine cells where one cell is a control cell without a promoter polynucleotide of the promoter ladder. One of skill the in art can appreciate that the library of transformed host cells can contain a plurality (e.g., >10; >100; >1,000; 10-1,000; 10-10,000; or 100-100,000) of redundant copies of the minimal cellular set, of the library or a subset thereof. The library can further comprise an additional set of cells for each interrogated heterologous ancillary target gene, such that each interrogated heterologous ancillary target gene is operably linked to each of the different promoter polynucleotides of the promoter ladder in a different cell. This provides a set of cells, where each cell in the library is an experiment interrogating a different [promoter polynucleotide: operably linked heterologous ancillary target gene] combination.

The library can be provided by a number of techniques available to one of skill in the art. For example, a plurality of host cells having a selected background (e.g., modified for lysA overexpression) can be transformed with a library of recombinant vectors under conditions such that substantially all transformants are singly modified to contain a single [promoter polynucleotide operably linked heterologous ancillary target gene] combination. The recombinant vectors can be integrating vectors, such that the providing comprises engineering the genome of the host cell. The transformants can be isolated, stored, and/or expanded, and optionally assayed for target molecule production. Exemplary isolating methods include without limitation limiting dilution, plating, streaking, and/or colony picking. Exemplary storage methods include without limitation cryopreservation or sporulation. For example, transformants can be isolated, mixed with a suitable cryoprotectant (e.g., glycerol), cryogenically frozen under conditions suitable to limit ice crystal formation, and stored.

Moreover, the interrogated heterologous ancillary target genes can be assayed in plurality of (e.g., two or more) different on-pathway modification backgrounds. The assay of different on-pathway backgrounds can be performed simultaneously, e.g., in parallel, or sequentially. For example, the library of transformed host cells for increasing production of lysine can comprise a first sub-library of transformed host cells having a lysA overexpression modification and interrogating a plurality of [promoter polynucleotide operably linked heterologous target gene] combinations; and a second sub-library that differs from the first sub-library by having a different, or additional, on-pathway modification. Similarly, the library can comprise, or further comprise an off-pathway modification background and interrogating a plurality of [promoter polynucleotide operably linked heterologous target gene] combinations and/or interrogating a plurality of [promoter polynucleotide operably linked heterologous ancillary target gene] combinations. As an example, a library of transformed host cells for increasing production of lysine can comprise transformed host cells having a background comprising: an on-pathway lysA overexpression modification; an off-pathway pgi overexpression modification; and various [promoter polynucleotide operably linked heterologous ancillary target gene] combinations.

In some embodiments, the method includes identifying a host cell from the plurality of host cells that exhibits increased production of the target biomolecule. In some cases, the identifying step includes a reproducibility filter to identify host cells, and the underlying [promoter polynucleotide operably linked heterologous ancillary target gene] combinations that reproducibly exhibit increased production of the target biomolecule. For example, the identifying step can assay redundant copies of each [promoter polynucleotide operably linked heterologous ancillary target gene] combination and identify combinations that exhibit reproducibly improved target biomolecule production in all, substantially all, or a majority of the redundant copies. As another example, a statistical filter can be applied to exclude combinations that do not meet a selected level of statistical significance (e.g., p<0.05, 0.01, 0.005, or 0.001).

In some embodiments, the method can comprise an iterative method of providing a library. For example, a library can be provided, cultured, and one or more host cells exhibiting increased production of target biomolecule can comprise the background strain for a second round of library generation and screening. Thus, in some embodiments, a subsequent iteration creates a new host cell library comprising individual host cells harboring unique genetic variations that are a combination of genetic variation selected from amongst at least two individual host cells of a preceding host cell library. Iterations can be performed multiple times until a resulting host cell has acquired a selected level of target biomolecule production improvement; until further rounds of providing and screening a library exhibit diminishing improvement; or until improvement pleateus. In an embodiment, at least one round interrogates heterologous ancillary target genes. Additionally or alternatively, on-pathway genes can be interrogated in earlier or later rounds of library generation and screening, optionally in combination with further interrogation of heterologous ancillary target genes.

Linkers

The target gene is positioned downstream of the promoter polynucleotide according to the invention, i.e. at the 3′ end, such that both polynucleotides are functionally linked to one another either directly or by means of a linker oligonucleotide or linker polynucleotide. Preference is given to the promoter and the target gene being functionally linked to one another by means of a linker oligonucleotide or linker polynucleotide. Said linker oligonucleotide or linker polynucleotide consists of deoxyribonucleotides.

In this context, the expression “functionally linked to one another directly” means that the nucleotide at the 3′ end of the promoter polynucleotide, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, is linked directly to the first nucleotide of the start codon of a target gene. This results in “leaderless” mRNAs which start immediately with the 5′-terminal AUG start codon and therefore do not have any other translation initiation signals.

In this context, the expression “functionally linked to one another by means of a linker oligonucleotide” means that the nucleotide at the 3′ end of the promoter polynucleotide, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, is linked by an oligonucleotide of 1, 2, 3, 4 or 5 nucleotides in length to the first nucleotide of the start codon of a target gene.

In this context, the expression “functionally linked to one another by means of a linker polynucleotide” means that the nucleotide at the 3′ end of the promoter polynucleotide, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, is linked by a polynucleotide of from 6 to no more than 600 nucleotides in length to the first nucleotide of the start codon of a target gene.

In this context, the expression “functionally linked to one another” means that the target gene is bound to the promoter polynucleotide according to the invention in such a way that transcription of the target gene and translation of the synthesized RNA are ensured.

Depending on the technical requirement, the linker polynucleotide is:

6-600, 6-500, 6-400, 6-300, 6-200, 6-180, 6-160, 6-140, 6-120, 6-100, 6-80, 6-60, 6-50, 6-40, 6-30, 6-28, 6-27, 6-26, or 6-25; or

8-600, 8-500, 8-400, 8-300, 8-200, 8-180, 8-160, 8-140, 8-120, 8-100, 8-80, 8-60, 8-50, 8-40, 8-30, 8-28, 8-27, 8-26, or 8-25; or

10-600, 10-500, 10-400, 10-300, 10-200, 10-180, 10-160, 10-140, 10-120, 10-100, 10-80, 10-60, 10-50, 10-40, 10-30, 10-28, 10-27, 10-26, or 10-25; or

12-600, 12-500, 12-400, 12-300, 12-200, 12-180, 12-160, 12-140, 12-120, 12-100, 12-80, 12-60, 12-50, 12-40, 12-30, 12-28, 12-27, 12-26, or 12-25; or

14-600, 14-500, 14-400, 14-300, 14-200, 14-180, 14-160, 14-140, 14-120, 14-100, 14-80, 14-60, 14-50, 14-40, 14-30, 14-28, 14-27, 14-26, or 14-20; or

16-600, 16-500, 16-400, 16-300, 16-200, 16-180, 16-160, 16-140, 16-120, 16-100, 16-80, 16-60, 16-50, 16-40, 16-30, 16-28, 16-27, 16-26, or 16-25; or

18-600, 18-500, 18-400, 18-300, 18-200, 18-180, 18-160, 18-140, 18-120, 18-100, 18-80, 18-60, 18-50, 18-40, 18-30, 18-28, 18-27, 18-26, or 18-25; or

20-600, 20-500, 20-400, 20-300, 20-200, 20-180, 20-160, 20-140, 20-120, 20-100, 20-80, 20-60, 20-50, 20-40, 20-30, 20-28, 20-27, 20-26, or 20-25 nucleotides in length.

In particularly preferred embodiments, the linker polynucleotide is 20, 21, 22, 23, 24, or 25 nucleotides in length because this produces preferably functional constructs.

The disclosure further relates accordingly to an isolated promoter polynucleotide, essentially consisting of a promoter polynucleotide, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, which, via the nucleotide at its 3′ end, is functionally linked, directly or by means of a linker polynucleotide which ensures translation of RNA, to a target gene which contains at its 5′ end an ATG or GTG start codon and codes for one or more off-pathway polypeptide(s). Preference is given to the promoter and target gene being functionally linked to one another by means of a linker polynucleotide.

The disclosure furthermore also relates to an isolated polynucleotide, essentially consisting of a promoter polynucleotide, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, which, via the nucleotide at its 3′ end, is functionally linked to a linker oligonucleotide.

In addition, the disclosure furthermore relates to an isolated polynucleotide, essentially consisting of a promoter polynucleotide, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, which, via the nucleotide at its 3′ end, is functionally linked to a linker polynucleotide which ensures translation of RNA.

In this context, the term “essentially” means that a polynucleotide of no more than 1,000, no more than 800, no more than 700, no more than 600, no more than 500, or no more than 400 nucleotides in length has been added to the 5′ end of the promoter polynucleotide, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, and a polynucleotide of no more than 1,000, no more than 800, no more than 700, no more than 600, no more than 500, or no more than 400 nucleotides in length has been added to the 3′ of the target gene, or a polynucleotide of no more than 15,000, no more than 10,000, no more than 7,500, no more than 5,000, no more than 2,500, no more than 1,000, no more than 800, no more than 700, no more than 600, no more than 500, or no more than 400 nucleotides in length has been added to the 3′ end of the linker oligo- or polynucleotide.

Any useful combination of the features from the preceding three lists of polynucleotides is in accordance with the invention here. “Useful combination” means, for example, a combination of features which results in an efficient recombination being carried out. The use of additions of the same length flanking a DNA region to be replaced facilitates the transfer of the region by homologous recombination in the experimental procedure. Relatively long flanking homologous regions are advantageous for efficient recombination between circular DNA molecules but cloning of the replacement vector is made more difficult with increasing length of the flanks (Wang et al., Molecular Biotechnology 32:43-53 (2006)).

In addition, the flank at the 3′ end of the linker oligo- or polynucleotide increases in length to no more than 15,000 nucleotides when the 3′ end is functionally linked to a target gene which contains at its 5′ end an ATG or GTG start codon and codes for one or more polypeptide(s).

These particularly preferred embodiments of the linker polynucleotide ensure translation of RNA in an advantageous manner.

To facilitate chemical linking between the promoter polynucleotide, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, the linker polynucleotide which ensures translation of RNA, and the target gene coding for one or more polypeptide(s), which has an ATG or GTG start codon at its 5′ end, functional nucleotide sequences required for cloning may be incorporated into said polynucleotides at their 5′ and 3′ ends and are at least partially retained even after said cloning.

The term “functional nucleotide sequence required for cloning” here represents any REII (type II restriction endonuclease) cleavage site present, whose sequence normally consists of from 4 to 8 nucleotides.

In addition, it should be mentioned here that site-specific mutagenesis by means of mutagenesis primers or a de novo gene synthesis (e.g. by GENEART AG (Regensburg, Germany)) of the nucleotide sequences to remove cleavage sites for restriction endonucleases may introduce silent mutations into the sequence in order to enable said cleavage sites to be used advantageously for subsequent cloning steps.

The polynucleotide resulting from the promoter according to the invention being functionally linked to the linker polynucleotide which ensures translation of RNA is also referred to as expression unit herein below.

Expression

The disclosure furthermore relates to the use of the promoter according to the invention or of the expression unit according to the invention for expressing target genes or polynucleotides in microorganisms. The promoter according to the invention or the expression unit according to the invention ensures transcription and translation of the synthesized RNA, preferably mRNA, into a polypeptide. As used herein, the term “host cell” refers to a transformed cell of a microorganism.

The present disclosure, provides for, and includes, transformed host cells comprising the recombinant nucleic acids and recombinant vectors described in detail above. The present disclosure further provides for, and includes, host cells transformed with two recombinant nucleic acids. In an embodiment, the host cells are transformed with three recombinant nucleic acids. As provided above, the nucleic acids may be selected from biosynthetic pathways based on the overall effect on the yield of the desired product. There is no practical limit the number of recombinant nucleic acids that may be incorporated into the host cells of the present specification. Expression is preferably carried out in microorganisms of the genus Corynebacterium. Preference is given to strains within the genus Corynebacterium which are based on the following species: C. efficiens, with the deposited type strain being DSM44549; C. glutamicum, with the deposited type strain being ATCC13032; and C. ammoniagenes, with the deposited type strain being ATCC6871. Very particular preference is given to the species C. glutamicum. In this way it is possible to express polynucleotides that code for polypeptides having a property, preferably enzyme activity, which are not present or detectable in the corresponding host. Thus, for example, Yukawa et al. describe expression of Escherichia coli genes for utilizing D-xylose in C. glutamicum R under the control of the constitutive Ptrc promoter (Applied Microbiology and Biotechnology 81, 691-699 (2008)).

The present specification provides for, and includes host cells such as C. glutamicum having two or more genes of a biosynthetic pathway under the control of the promoter polynucleotide sequences described above. In various embodiments, one or more target genes (e.g., ancillary target genes, and/or shell 2, and/or shell 3, and/or 4 target genes) are placed under the control of a promoter polynucleotide sequence having as sequence of SEQ ID NOs:1 to 8 as described above. In other embodiments, one or more target genes are placed under the control of a promoter polynucleotide sequence having as sequence of SEQ ID NOs:1, 5 or 7 as described above.

In certain embodiments according to the present specification, C. glutamicum host cells have two target genes under the control of the promoters having sequences of SEQ ID NOs:1 to 8. In certain other embodiments according to the present specification, C. glutamicum host cells have two target genes under the control of the promoters having sequences of SEQ ID NOs:1, 5 or 7. Using homologous recombination, the promoters of the present disclosure replace the endogenous promoter and endogenous sequence to prepare a promoter functionally linked to a heterologous gene. One of ordinary skill in the art would recognize that the recombination results in a replacement of the endogenous promoter while retaining the gene in its native locus. Specific non-limiting examples are illustrated below in Table 8. Multiple promoter-heterologous target pairs (e.g., promoter cassettes) can be readily incorporated into the genome of a host cell. In an embodiment, the promoter cassettes can be incorporated into host cells sequentially. In certain embodiments, the recombinant vectors of the present disclosure provide for two or more different promoter cassettes in a single construct. The present specification provides no practical limit to the number of promoter replacements that can be developed using the described methods.

Also described herein is a plurality of host cells comprising a promoter ladder, wherein one cell of the plurality comprises a first promoter polynucleotide operably linked to a heterologous target gene, e.g., an ancillary target gene, a shell 2 target gene, a shell 3 target gene, or a shell 4 target gene, and a second cell of the plurality comprises a second promoter polynucleotide operably linked to the same heterologous target gene, wherein the first and second promoter polynucleotides are different promoter polynucleotides of the promoter ladder.

In some cases, the plurality of host cells further comprise a third cell of the plurality comprising a third promoter polynucleotide operably linked to the same heterologous target gene, wherein the third promoter polynucleotide is a promoter polynucleotide of the promoter ladder that is different from the first and second promoter polynucleotides. In some cases, the plurality of host cells further comprise a fourth cell of the plurality comprising a fourth promoter polynucleotide operably linked to the same heterologous target gene, wherein the fourth promoter polynucleotide is a promoter polynucleotide of the promoter ladder that is different from the first, second, and third promoter polynucleotides. In some cases, the plurality of host cells further comprise a fifth cell of the plurality comprising a fifth promoter polynucleotide operably linked to the same heterologous target gene, wherein the fifth promoter polynucleotide is a promoter polynucleotide of the promoter ladder that is different from the first, second, third, and fourth promoter polynucleotides. In some cases, the plurality of host cells further comprise a sixth cell of the plurality comprising a sixth promoter polynucleotide operably linked to the same heterologous target gene, wherein the sixth promoter polynucleotide is a promoter polynucleotide of the promoter ladder that is different from the first, second, third, fourth, and fifth promoter polynucleotides. In some cases, the plurality of host cells further comprise a seventh cell of the plurality comprising a seventh promoter polynucleotide operably linked to the same heterologous target gene, wherein the seventh promoter polynucleotide is a promoter polynucleotide of the promoter ladder that is different from the first, second, third, fourth, fifth, and sixth promoter polynucleotides. In some cases, the plurality of host cells further comprise an eighth cell of the plurality comprising an eighth promoter polynucleotide operably linked to the same heterologous target gene, wherein the eighth promoter polynucleotide is a promoter polynucleotide of the promoter ladder that is different from the first, second, third, fourth, fifth, sixth, and seventh promoter polynucleotides.

In some cases each of the first, second, third, fourth, fifth, sixth, seventh, and/or eighth promoter polynucleotide of the promoter ladder is selected from SEQ ID NO:1-8. In some cases, the promoter polynucleotides of the promoter ladder are selected from SEQ ID NO:1, 5, and 7. The number of cells in the plurality can comprise at least about 1×10⁵, 1×10⁶, or 1×10⁷cells.

In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007-lysA and Pcg3121-pgi. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg1860-pyc and Pcg0007-zwf. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007-lysA and Pcg0007-zwf. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3121-pck and Pcg0007-zwf. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_39-ppc and Pcg0007-zwf. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3121-pck and Pcg3121-pgi. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-ddh and Pcg0007-zwf. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_265-dapB and Pcg0007-zwf. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007-zwf and Pcg3121-pgi. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-ddh and Pcg3121-pgi. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3121-pgi and Pcg1860-pyc. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg1860-pyc and Pcg0007_265-dapB. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg1860-pyc and Pcg0007-lysA. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg1860-asd and Pcg0007-zwf. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_265-dapB and Pcg3121-pgi. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg1860-pyc and Pcg1860-asd. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-aspB and Pcg1860-pyc. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-fbp and Pcg1860-pyc. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-ddh and Pcg3381-fbp. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0755-ptsG and Pcg3121-pgi. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg1860-pyc and Pcg3121-pck. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg1860-asd and Pcg3121-pgi. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg1860-asd and Pcg33 81-fbp. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_39-lysE and Pcg3381-fbp. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-fbp and Pcg0007-lysA. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_39-lysE and Pcg1860-pyc. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3121-pgi and Pcg3381-fbp. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3121-pck and Pcg0007-lysA. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007-lysA and Pcg0007_265-dapB. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_265-dapB and Pcg1860-asd. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3121-pgi and Pcg0007_265-dapD. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007-lysA and Pcg3381-ddh. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3121-pck and Pcg1860-asd. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007-lysA and Pcg1860-asd. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3121-pck and Pcg0007_265-dapB. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-ddh and Pcg1860-asd. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_39-ppc and Pcg1860-asd. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_39-ppc and Pcg0007-lysA. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-ddh and Pcg0007_265-dapB. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_265-dapB and Pcg3381-fbp. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_39-ppc and Pcg0007_265-dapB. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-aspB and Pcg3121-pck. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_265-dapB and Pcg0007_265-dapD. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_39-lysE and Pcg3381-aspB. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007_39-lysE and Pcg0007_265-dapD. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-aspB and Pcg0007_265-dapB. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg1860-asd and Pcg0007_265-dapD. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-aspB and Pcg0007-lysA. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg3381-aspB and Pcg3381-ddh. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0755-ptsG and Pcg1860-pyc. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0755-ptsG and Pcg3381-fbp. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0007-zwf and Pcg3381-fbp. In an embodiment the host cell is a transgenic C. glutamicum host cell comprising the promoter cassettes Pcg0755-ptsG and Pcg0007_265-dapD.

The present disclosure provides for, and includes, host cells having three or more promoter cassettes as described above. In an embodiment, the host cell includes the Pcg0007_39-zwf, Pcg0007_39-lysA and Pcg1860-pyc promoter cassettes. In an embodiment, the host cell is a C. glutamicum host cell.

In an embodiment, the host cell includes any one of the foregoing promoter cassettes, and/or includes pcg0007_39-dnak; pcg0007_39-cg0074; pcg3121-cg0074; pcg1860-rhle_609; pcg3121-cg1144; pcg1860-rhle_609; pcg0007_39-cg2899_2194; pcg0007_39-cg1486; pcg0007_39-cg2766; pcg0007_39-cmk; pcg0007_39-rpob_383; pcg0007_39-ddl; pcg0007_39-cg0027; pcg0007_39-ddl; pcg0007_39-rpob_383; pcg0007_39-rpob_383; pcg0007_39-cg0027; pcg1860-cg1144; pcg0007_39-cg0725; pcg0007_39-cg0027; pcg0007_39-cg1527; pcg0007_39-ddl; pcg0007_39-rpob_383; pcg0007_39-cg0725; pcg0007_39-cg0725; pcg0007_39-ddl; pcg0007_39-cg0725; pcg0007_39-cg2766; pcg0007_39-cg0725; pcg0007_39-hspr; pcg0007_39-cg3352; pcg0007_39-cg2899_2194; pcg0007_39-cg2766; pcg0007_39-cg2766; pcg0007_39-cg2965; pcg0007_39-rpob_383; pcg0007_39-cg2766; pcg0007_39-cg2766; pcg0007_39-cg2899_2194; pcg0007_39-cg0074; pcg3121-cg0074; pcg0007_39-cg2766; pcg3121-cg1144; pcg0007_39-cg2766; pcg0007_39-cg2766; pcg0007_39-cg2899; pcg0007_39-rho; pcg0007_39-cg2766; pcg0007_39-cg2766; pcg0007_39-cg0725; pcg1860-cg1144; pcg0007_39-cg2766 pcg0007_39-cg2766; pcg0007_39-urer; pcg0007_39-nusg; pcg3121-mutm2_2522; pcg0007_39-ddl; pcg1860-cg1144; pcg0007_39-cg2899; pcg0007_39-cg2965; pcg0007_39-ddl; pcg3121-mutm2_2522; pcg0007_39-cg2766; pcg0007_39-cg2766; pcg0007_39-cg2766; pcg0007_39-cg2766; pcg0007_39-cg2766; pcg0007_39-cg2766; pcg0007_39-tyra; pcg0007_39-cg1486; pcg0007_39-cg2899; pcg0007_39-cg0027; pcg0007_39-ncg11511; pcg0007_39-ncg11262; pcg0007_39-cg3419; pcg0007_39-cg1486; pcg0007_39-cg3210; pcg0007_39-cg1486; pcg0007_39-cg1486; pcg0007_39-cg1486; pcg0007_39-cg1486; pcg0007_39-ncg10767; pcg0007_39-ncg12481; pcg0007_39-tyra; pcg0007_39-cg1486; pcg0007_39-ncg11511; pcg0007_39-ncg10827; pcg0007_39-tyra; pcg0007_39-cg1486; pcg0007_39-ncg11262; pcg0007_39-cg1486; pcg0007_39-ncg11262; pcg0007_39-ncg11262; pcg0007_39-ncg10767; pcg0007_39-ncg10304; pcg0007_39-ncg11511; pcg0007_39-ncg10767; pcg0007_39-ncg11262; pcg0007_39-ncg11511; pcg0007_39-ncg10767; pcg0007_39-ncg10767; pcg0007_39-ncg11262; pcg0007_39-cg1486; pcg0007_39-ncg11262; pcg0007_39-ncg10304; pcg0007_39-ncg11262; pcg0007_39-ncg10767; pcg0007_39-ncg11262, or a combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all thereof.

The promoter according to the invention or the expression unit according to the invention is furthermore used for improving the performance characteristics of microorganisms, which can include, for example, yield, titer, productivity, by-product elimination, tolerance to process excursions, optimal growth temperature and growth rate. In some embodiments, the promoter according to the invention or the expression unit according to the invention is used for up-regulating a target gene in a microorganism (overexpression). Overexpression generally means an increase in the intracellular concentration or activity of a ribonucleic acid, a protein (polypeptide) or an enzyme in comparison with the starting strain (parent strain) or wild-type strain, if the latter is the starting strain. In some embodiments, the promoter according to the invention or the expression unit according to the invention is used for down-regulating a target gene in a microorganism (underexpression). Underexpression generally means an decrease in the intracellular concentration or activity of a ribonucleic acid, a protein (polypeptide) or an enzyme in comparison with the starting strain (parent strain) or wild-type strain, if the latter is the starting strain. In some embodiments, a combination of promoters and/or expression units according to the invention are used for regulating expression of more than one target gene in a microorganism, wherein each target gene is either up-regulated or down-regulated. In some embodiments the target genes up- or down-regulated by the combination of promoters and/or expression units are part of the same metabolic pathway. In some embodiments the target genes up- or down-regulated by the combination of promoters and/or expression units are not part of the same metabolic pathway.

The promoters described herein can be used in combination with other methods very well-known in the art for attenuating (reducing or eliminating) the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene, or allele, which codes for a corresponding enzyme with a low activity, or inactivates the corresponding gene or enzyme (protein), and optionally combining these measures.

The reduction in gene expression can take place by suitable culturing or by genetic modification (mutation) of the signal structures of gene expression. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. The expert can find information on this e.g. in the patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al. (Microbiology 142: 1297 (1996)), Vašicová et al. (Journal of Bacteriology 181: 6188 (1999)) and in known textbooks of genetics and molecular biology, such as e.g. the textbook by Knippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or that by Winnacker (“Gene und Klone [Genes and Clones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

Mutations which lead to a change or reduction in the catalytic properties of enzyme proteins are known from the prior art; examples which may be mentioned are the works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms [Threonine dehydratase from Corynebacterium glutamicum: Cancelling the allosteric regulation and structure of the enzyme]”, Reports from the Jülich Research Centre, Jül-2906, ISSN09442952, Jülich, Germany, 1994). Comprehensive descriptions can be found in known textbooks of genetics and molecular biology, such as e.g. that by Hagemann (“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag, Stuttgart, 1986).

Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, missense mutations or nonsense mutations are referred to. Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, as a consequence of which incorrect amino acids are incorporated or translation is interrupted prematurely. Deletions of several codons typically lead to a complete loss of the enzyme activity. Instructions on generation of such mutations are prior art and can be found in known textbooks of genetics and molecular biology, such as e.g. the textbook by Knippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that by Winnacker (“Gene und Klone [Genes and Clones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann (“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag, Stuttgart, 1986). A common method of mutating genes of C. glutamicum is the method of gene disruption and gene replacement described by Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991)).

In the method of gene disruption a central part of the coding region of the gene of interest is cloned in a plasmid vector which can replicate in a host (typically E. coli), but not in C. glutamicum. Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schafer et al., Gene 145, 69-73 (1994)), pK18mobsacB or pK19mobsacB (Jager et al., Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516). The plasmid vector which contains the central part of the coding region of the gene is then transferred into the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation is described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a “cross-over” event, the coding region of the gene in question is interrupted by the vector sequence and two incomplete alleles are obtained, one lacking the 3′ end and one lacking the 5′ end. This method has been used, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to eliminate the recA gene of C. glutamicum.

In the method of gene replacement, a mutation, such as e.g. a deletion, insertion or base exchange, is established in vitro in the gene of interest. The allele prepared is in turn cloned in a vector which is not replicative for C. glutamicum and this is then transferred into the desired host of C. glutamicum by transformation or conjugation. After homologous recombination by means of a first “cross-over” event which effects integration and a suitable second “cross-over” event which effects excision in the target gene or in the target sequence, the incorporation of the mutation or of the allele is achieved. This method was used, for example, by Peters-Wendisch (Microbiology 144, 915-927 (1998)) to eliminate the pyc gene of C. glutamicum by a deletion.

The promoters described herein can be used in combination with other methods very well-known in the art for raising (enhancing) the intracellular activity of one or more enzymes in a microorganism that are coded by the corresponding DNA, by for example increasing the number of copies of the gene or genes, using a strong promoter, or using a gene that codes for a corresponding enzyme having a high activity, and optionally combining these measures.

In order to achieve an overexpression the number of copies of the corresponding genes can be increased, or alternatively the promoter and regulation region or the ribosome binding site located upstream of the structure gene can be mutated. Expression cassettes that are incorporated upstream of the structure gene act in the same way. By means of inducible promoters it is in addition possible to increase the expression in the course of the enzymatic amino acid production. The expression is similarly improved by measures aimed at prolonging the lifetime of the m-RNA. Furthermore, the enzyme activity is also enhanced by preventing the degradation of the enzyme protein. The genes or gene constructs may either be present in plasmids having different numbers of copies, or may be integrated and amplified in the chromosome. Alternatively, an overexpression of the relevant genes may furthermore be achieved by altering the composition of the media and the culture conditions.

The person skilled in the art can find details on the above in, inter alia, Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European Patent Specification 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in Patent Application WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in Japanese laid open Specification JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in known textbooks on genetics and molecular biology.

Genes may be overexpressed for example by means of episomal plasmids. Suitable plasmids are those that are replicated in coryneform bacteria. Numerous known plasmid vectors, such as for example pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such as for example those based on pCG4 (U.S. Pat. No. 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (U.S. Pat. No. 5,158,891) may be used in a similar way.

Furthermore, also suitable are those plasmid vectors with the aid of which the process of gene amplification by integration in the chromosome can be employed, such as has been described for example by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for the duplication and amplification of the hom-thrB operon. In this method the complete gene is cloned into a plasmid vector that can replicate in a host (typically E. coli) but not in C. glutamicum. Suitable vectors are for example pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19 mob (Schafer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega Corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Sprat et al., 1986, Gene 41: 337-342). The plasmid vector that contains the gene to be amplified is then transferred by conjugation or transformation into the desired strain of C. glutamicum. The method of conjugation is described for example in Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Transformation methods are described for example in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a crossover event, the resulting strain contains at least two copies of the relevant gene.

Methods of regulating, i.e., either increasing or decreasing, gene expression include recombinant methods in which a microorganism is produced using a DNA molecule provided in vitro. Such DNA molecules comprise, for example, promoters, expression cassettes, genes, alleles, coding regions, etc. They are introduced into the desired microorganisms by methods of transformation, conjugation, transduction or similar methods.

In the case of the present disclosure, the promoters are preferably a polynucleotide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, and the expression cassettes are preferably a polynucleotide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 which, via the nucleotide at its 3′ end, are functionally linked to a linker polynucleotide which ensures translation of RNA.

The measures of overexpression using the promoter according to the invention or the expression unit according to the invention increase the activity or concentration of the corresponding polypeptide usually by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, preferably by no more than 1,000%, 2,000%, 4,000%, 10,000% or 20,000%, based on the activity or concentration of said polypeptide in the strain prior to the measure resulting in overexpression.

The extent of expression or overexpression may be established by measuring the amount of mRNA transcribed from the gene, by determining the amount of polypeptide and by determining enzyme activity.

The amount of mRNA may be determined inter alia by using the methods of “Northern Blotting” and of quantitative RT-PCR. Quantitative RT-PCR involves reverse transcription which precedes the polymerase chain reaction. For this, the LightCycler™ System from Roche Diagnostics (Boehringer Mannheim GmbH, Roche Molecular Biochemicals, Mannheim, Germany) may be used, as described in Jungwirth et al. (FEMS Microbiology Letters 281, 190-197 (2008)), for example. The concentration of the protein may be determined via 1- and 2-dimensional protein gel fractionation and subsequent optical identification of the protein concentration using appropriate evaluation software in the gel. A customary method of preparing protein gels for coryneform bacteria and of identifying said proteins is the procedure described by Hermann et al. (Electrophoresis, 22:1712-23 (2001)). The protein concentration may likewise be determined by Western-Blot hybridization using an antibody specific for the protein to be detected (Sambrook et al., Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and subsequent optical evaluation using appropriate software for concentration determination (Lohaus and Meyer (1998) Biospektrum 5:32-39; Lottspeich, Angewandte Chemie 321: 2630-2647 (1999)). The statistical significance of the data collected is determined by means of a T test (Gosset, Biometrika 6(1): 1-25 (1908)).

The measure of overexpressing target genes using the promoter according to the invention may be combined in a suitable manner with further overexpression measures. Overexpression is achieved by a multiplicity of methods available in the prior art. These include increasing the copy number in addition to modifying the nucleotide sequences which direct or control expression of the gene. The copy number may be increased by means of plasmids which replicate in the cytoplasm of the microorganism. To this end, an abundance of plasmids are described in the prior art for very different groups of microorganisms, which plasmids can be used for setting the desired increase in the copy number of the gene. Plasmids suitable for the genus Corynebacterium are described, for example, in Tauch et al. (Journal of Biotechnology 104 (1-3), 27-40, (2003)), and in Stansen et al. (Applied and Environmental Microbiology 71, 5920-5928 (2005)).

The copy number may furthermore be increased by at least one (1) copy by introducing further copies into the chromosome of the microorganism. Methods suitable for the genus Corynebacterium are described, for example, in the patents WO 03/014330, WO 03/040373 and WO 04/069996.

Gene expression may furthermore be increased by positioning a plurality of promoters upstream of the target gene or functionally linking them to the gene to be expressed and achieving increased expression in this way. Examples of this are described in the patent WO 2006/069711.

Transcription of a gene is controlled, where appropriate, by proteins which suppress (repressor proteins) or promote (activator proteins) transcription. Accordingly, overexpression can likewise be achieved by increasing the expression of activator proteins or reducing or switching off the expression of repressor proteins or else eliminating the binding sites of the repressor proteins. The rate of elongation is influenced by the codon usage, it being possible to enhance translation by utilizing codons for transfer RNAs (tRNAs) which are frequent in the starting strain. Moreover, replacing a start codon with the ATG codon most frequent in many microorganisms (77% in E. coli) may considerably improve translation, since, at the RNA level, the AUG codon is two to three times more effective than the codons GUG and UUG, for example (Khudyakov et al., FEBS Letters 232(2):369-71(1988); Reddy et al., Proceedings of the National Academy of Sciences of the USA 82(17):5656-60 (1985)). It is also possible to optimize the sequences surrounding the start codon because synergistic effects between the start codon and the flanking regions have been described (Stenström et al., Gene 273(2):259-65 (2001); Hui et al., EMBO Journal 3(3):623-9 (1984)).

Instructions for handling DNA, digestion and ligation of DNA, transformation and selection of transformants can be found inter alia in the known manual by Sambrook et al. “Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989).

The disclosure also relates to vectors comprising the polynucleotides according to the invention.

Kirchner and Tauch (Journal of Biotechnology 104:287-299 (2003)) describe a selection of vectors to be used in C. glutamicum.

Homologous recombination using the vectors according to the invention allows DNA segments on the chromosome to be replaced with polynucleotides according to the invention which are transported into the cell by the vector. For efficient recombination between the circular DNA molecule of the vector and the target DNA on the chromosome, the DNA region to be replaced with the polynucleotide according to the invention is provided at the ends with nucleotide sequences homologous to the target site which determine the site of integration of the vector and of replacement of the DNA.

Thus the promoter polynucleotide according to the invention may: 1) be replaced with the native promoter at the native gene locus of the target gene in the chromosome; or 2) be integrated with the target gene at the native gene locus of the latter or at another gene locus.

“Replacement of the native promoter at the native gene locus of the target gene” means the fact that the naturally occurring promoter of the gene which usually is naturally present by way of a single copy at its gene locus in the corresponding wild type or corresponding starting organism in the form of its nucleotide sequence is replaced. “Another gene locus” means a gene locus whose nucleotide sequence is different from the sequence of the target gene. Said other gene locus or the nucleotide sequence at said other gene locus is preferably located within the chromosome and normally is not essential for growth and for production of the desired chemical compounds. It is furthermore possible to use intergenic regions within the chromosome, i.e. nucleotide sequences without coding function.

Expression or overexpression is preferably carried out in microorganisms of the genus Corynebacterium. Within the genus Corynebacterium, preference is given to strains based on the following species: C. efficiens, with the deposited type strain being DSM44549, C. glutamicum, with the deposited type strain being ATCC13032, and C. ammoniagenes, with the deposited type strain being ATCC6871. Very particular preference is given to the species C. glutamicum.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are in particular the known wild-type strains: Corynebacterium glutamicum ATCC13032, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium melassecola ATCC17965, Corynebacterium thermoaminogenes FERM BP-1539, Brevibacterium flavum ATCC14067, Brevibacterium lactofermentum ATCC13869, and Brevibacterium divaricatum ATCC14020; and L-amino acid-producing mutants, or strains, prepared therefrom, such as, for example, the L-lysine-producing strains: Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum FERM-P 6464, Corynebacterium glutamicum DM58-1, Corynebacterium glutamicum DG52-5, Corynebacterium glutamicum DSM5714, and Corynebacterium glutamicum DSM12866.

The term “Micrococcus glutamicus” has also been in use for C. glutamicum. Some representatives of the species C. efficiens have also been referred to as C. thermoaminogenes in the prior art, such as the strain FERM BP-1539, for example.

The microorganisms or strains (starting strains) employed for the expression or overexpression measures according to the invention preferably already possess the ability to secrete a desired fine chemical into the surrounding nutrient medium and accumulate there. The expression “to produce” is also used for this herein below. More specifically, the strains employed for the overexpression measures possess the ability to accumulate the desired fine chemical in concentrations of at least 0.10 g/L, at least 0.25 g/L, at least 0.5 g/L, at least 1.0 g/L, at least 1.5 g/L, at least 2.0 g/L, at least 4.0 g/L, or at least 10.0 g/L in no more than 120 hours, no more than 96 hours, no more than 48 hours, no more than 36 hours, no more than 24 hours, or no more than 12 hours in the cell or in the nutrient medium. The starting strains are preferably strains prepared by mutagenesis and selection, by recombinant DNA technologies or by a combination of both methods.

A person skilled in the art understands that a microorganism suitable for the measures of the invention may also be obtained by firstly employing the promoter according to the invention, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 for overexpression or underexpression of the target genes in a wild strain such as, for example, the C. glutamicum type strain ATCC 13032 or the strain ATCC 14067, and then, by means of further genetic measures described in the prior art, causing the microorganism to produce the desired fine chemical(s).

The term “biomolecules” means with regard to the measures of the invention amino acids, organic acids, vitamins, nucleosides and nucleotides. Particular preference is given to proteinogenic amino acids, non-proteinogenic amino acids, macromolecules, and organic acids.

“Proteinogenic amino acids” mean the amino acids which occur in natural proteins, i.e. in proteins of microorganisms, plants, animals and humans. They serve as structural units for proteins in which they are linked to one another via peptide bonds.

Where L-amino acids or amino acids are mentioned hereinbelow, they are to be understood as meaning one or more amino acids, including their salts, selected from the group L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine. L-lysine is especially preferred. L-Amino acids, in particular lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition. There is therefore a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.

The terms protein and polypeptide are interchangeable.

The present disclosure provides a microorganism which produces a fine chemical, said microorganism having increased expression of one or more genes in comparison to the particular starting strain by using a promoter of a promoter ladder, such as a promoter selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

Fermentative Preparation

The present disclosure furthermore provides a process for fermentative preparation of a fine chemical, comprising the steps of:

a) culturing the above-described microorganism according to the present disclosure in a suitable medium, resulting in a fermentation broth; and

b) concentrating the fine chemical in the fermentation broth of a) and/or in the cells of the microorganism.

Preference is given here to obtaining from the fine chemical-containing fermentation broth the fine chemical or a liquid or solid fine chemical-containing product. The microorganisms produced may be cultured continuously—as described, for example, in WO 05/021772—or discontinuously in a batch process (batch cultivation) or in a fed-batch or repeated fed-batch process for the purpose of producing the desired organic-chemical compound. A summary of a general nature about known cultivation methods is available in the textbook by Chmiel (Bioprozeßtechnik. 1: Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium or fermentation medium to be used must in a suitable manner satisfy the demands of the respective strains. Descriptions of culture media for various microorganisms are present in the “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms culture medium and fermentation medium are interchangeable.

It is possible to use, as carbon source, sugars and carbohydrates such as, for example, glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from sugar beet or sugar cane processing, starch, starch hydrolysate, and cellulose; oils and fats such as, for example, soybean oil, sunflower oil, groundnut oil and coconut fat; fatty acids such as, for example, palmitic acid, stearic acid, and linoleic acid; alcohols such as, for example, glycerol, methanol, and ethanol; and organic acids such as, for example, acetic acid or lactic acid.

It is possible to use, as nitrogen source, organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour, and urea; or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The nitrogen sources can be used individually or as a mixture.

It is possible to use, as phosphorus source, phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts.

The culture medium may additionally comprise salts, for example in the form of chlorides or sulfates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth factors such as amino acids, for example homoserine and vitamins, for example thiamine, biotin or pantothenic acid, may be employed in addition to the abovementioned substances.

Said starting materials may be added to the culture in the form of a single batch or be fed in during the cultivation in a suitable manner.

The pH of the culture can be controlled by employing basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or aqueous ammonia; or acidic compounds such as phosphoric acid or sulfuric acid in a suitable manner. The pH is generally adjusted to a value of from 6.0 to 8.5, preferably 6.5 to 8. To control foaming, it is possible to employ antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids, it is possible to add to the medium suitable selective substances such as, for example, antibiotics. The fermentation is preferably carried out under aerobic conditions. In order to maintain these conditions, oxygen or oxygen-containing gas mixtures such as, for example, air are introduced into the culture. It is likewise possible to use liquids enriched with hydrogen peroxide. The fermentation is carried out, where appropriate, at elevated pressure, for example at an elevated pressure of from 0.03 to 0.2 MPa. The temperature of the culture is normally from 20° C. to 45° C. and preferably from 25° C. to 40° C., particularly preferably from 30° C. to 37° C. In batch or fed-batch processes, the cultivation is preferably continued until an amount of the desired organic-chemical compound sufficient for being recovered has formed. This aim is normally achieved within 10 hours to 160 hours. In continuous processes, longer cultivation times are possible. The activity of the microorganisms results in a concentration (accumulation) of the organic-chemical compound in the fermentation medium and/or in the cells of said microorganisms.

Examples of suitable fermentation media can be found inter alia in the U.S. Pat. Nos. 5,770,409, 5,990,350, 5,275,940, WO 2007/012078, U.S. Pat. No. 5,827,698, WO 2009/043803, U.S. Pat. Nos. 5,756,345 and 7,138,266.

Analysis of L-amino acids to determine the concentration at one or more time(s) during the fermentation can take place by separating the L-amino acids by means of ion exchange chromatography, preferably cation exchange chromatography, with subsequent post-column derivatization using ninhydrin, as described in Spackman et al. (Analytical Chemistry 30:1190-1206 (1958)). It is also possible to employ ortho-phthaldialdehyde rather than ninhydrin for post-column derivatization. An overview article on ion exchange chromatography can be found in Pickering (LC-GC Magazine of Chromatographic Science) 7(6), 484-487 (1989)).

It is likewise possible to carry out a pre-column derivatization, for example using ortho-phthaldialdehyde or phenyl isothiocyanate, and to fractionate the resulting amino acid derivatives by reversed-phase (RP) chromatography, preferably in the form of high-performance liquid chromatography (HPLC). A method of this type is described, for example, in Lindroth et al. (Analytical Chemistry 51:1167-1174 (1979)).

Detection is carried out photometrically (absorption, fluorescence).

A review regarding amino acid analysis can be found inter alia in the textbook “Bioanalytik” from Lottspeich and Zorbas (Spektrum Akademischer Verlag, Heidelberg, Germany 1998).

Determination of the concentration of α-ketoacids at one or more time point(s) in the course of the fermentation may be carried out by separating the ketoacids and other secreted products by means of ion exchange chromatography, preferably cation exchange chromatography, on a sulfonated styrene-divinylbenzene polymer in the H+ form, for example by means of 0.025 M sulfuric acid with subsequent UV detection at 215 nm (alternatively also at 230 or 275 nm). Preferably, a REZEK RFQ-Fast Fruit H+ column (Phenomenex) may be employed, but other suppliers for the separating phase (e.g. Aminex from BioRad) are feasible. Similar separations are described in application examples by the suppliers.

The performance of the processes or fermentation processes containing the promoter variants according to the invention, in terms of one or more of the parameters selected from the group of concentration (compound formed per unit volume), yield (compound formed per unit carbon source consumed), formation (compound formed per unit volume and time) and specific formation (compound formed per unit dry cell matter or dry biomass and time or compound formed per unit cellular protein and time) or else other process parameters and combinations thereof, is increased by at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% based on processes or fermentation processes using microorganisms not containing the promoter variants according to the invention. This is considered to be very worthwhile in terms of a large-scale industrial process.

The fermentation measures result in a fermentation broth which contains the desired fine chemical, preferably amino acids, organic acids, vitamins, nucleosides or nucleotides.

A product containing the fine chemical is then provided or produced or recovered in liquid or solid form.

A fermentation broth means a fermentation medium or nutrient medium in which a microorganism has been cultivated for a certain time and at a certain temperature. The fermentation medium or the media employed during fermentation comprise(s) all the substances or components which ensure production of the desired compound and typically propagation and viability.

When the fermentation is complete, the resulting fermentation broth accordingly comprises:

a) the biomass (cell mass) of the microorganism, said biomass having been produced due to propagation of the cells of said microorganism;

b) the desired fine chemical formed during the fermentation;

c) the organic byproducts possibly formed during the fermentation; and

d) the constituents of the fermentation medium employed or of the starting materials, such as, for example, vitamins such as biotin or salts such as magnesium sulfate, which have not been consumed in the fermentation.

The organic byproducts include substances which are produced by the microorganisms employed in the fermentation in addition to the particular desired compound and are optionally secreted.

The fermentation broth is removed from the culture vessel or fermentation tank, collected where appropriate, and used for providing a product containing the fine chemical in liquid or solid form. The expression “recovering the fine chemical-containing product” is also used for this. In the simplest case, the fine chemical-containing fermentation broth itself, which has been removed from the fermentation tank, constitutes the recovered product.

One or more of the measures selected from the group consisting of

a) partial (>0% to <80%) to complete (100%) or virtually complete (≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%) removal of the water;

b) partial (>0% to <80%) to complete (100%) or virtually complete (≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%) removal of the biomass, the latter being optionally inactivated before removal;

c) partial (>0% to <80%) to complete (100%) or virtually complete (≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥99.3%, or ≥99.7%) removal of the organic byproducts formed during fermentation; and

d) partial (>0%) to complete (100%) or virtually complete (≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥99.3%, or ≥99.7%) removal of the constituents of the fermentation medium employed or of the starting materials, which have not been consumed in the fermentation, from the fermentation broth achieves concentration or purification of the desired organic-chemical compound. Products having a desired content of said compound are isolated in this way.

The partial (>0% to <80%) to complete (100%) or virtually complete (≥80% to <100%) removal of the water (measure a)) is also referred to as drying.

In one variant of the process, complete or virtually complete removal of the water, of the biomass, of the organic byproducts and of the unconsumed constituents of the fermentation medium employed results in pure (≥80% by weight, ≥90% by weight) or high-purity (≥95% by weight, ≥97% by weight, or ≥99% by weight) product forms of the desired organic-chemical compound. An abundance of technical instructions for measures a), b), c) and d) are available in the prior art.

Depending on requirements, the biomass can be removed wholly or partly from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decantation or a combination thereof, or be left completely therein. Where appropriate, the biomass or the biomass-containing fermentation broth is inactivated during a suitable process step, for example by thermal treatment (heating) or by addition of acid.

In one procedure, the biomass is completely or virtually completely removed so that no (0%) or at most 30%, at most 20%, at most 10%, at most 5%, at most 1% or at most 0.1% biomass remains in the prepared product. In a further procedure, the biomass is not removed, or is removed only in small proportions, so that all (100%) or more than 70%, 80%, 90%, 95%, 99% or 99.9% biomass remains in the product prepared. In one process according to the invention, accordingly, the biomass is removed in proportions of from ≥0% to ≤100%.

Finally, the fermentation broth obtained after the fermentation can be adjusted, before or after the complete or partial removal of the biomass, to an acidic pH with an inorganic acid such as, for example, hydrochloric acid, sulfuric acid, or phosphoric acid; or organic acid such as, for example, propionic acid, so as to improve the handling properties of the final product (GB 1,439,728 or EP 1 331220). It is likewise possible to acidify the fermentation broth with the complete content of biomass. Finally, the broth can also be stabilized by adding sodium bisulfite (NaHCO₃, GB 1,439,728) or another salt, for example ammonium, alkali metal, or alkaline earth metal salt of sulfurous acid.

During the removal of the biomass, any organic or inorganic solids present in the fermentation broth are partially or completely removed. The organic byproducts dissolved in the fermentation broth, and the dissolved unconsumed constituents of the fermentation medium (starting materials), remain at least partly (>0%), preferably to an extent of at least 25%, particularly preferably to an extent of at least 50% and very particularly preferably to an extent of at least 75% in the product. Where appropriate, they also remain completely (100%) or virtually completely, meaning >95% or >98% or >99%, in the product. If a product in this sense comprises at least part of the constituents of the fermentation broth, this is also described by the term “product based on fermentation broth”.

Subsequently, water is removed from the broth, or said broth is thickened or concentrated, by known methods such as, for example, using a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration. This concentrated fermentation broth can then be worked up to free-flowing products, in particular to a fine powder or preferably coarse granules, by methods of freeze drying, spray drying, spray granulation or by other processes such as in the circulating fluidized bed, as described for example according to PCT/EP2004/006655. A desired product is isolated where appropriate from the resulting granules by screening or dust removal. It is likewise possible to dry the fermentation broth directly, i.e. without previous concentration by spray drying or spray granulation.

“Free-flowing” means powders which, from a series of glass orifice vessels with orifices of different sizes, flow unimpeded at least out of the vessel with a 5 mm orifice (Klein: Seifen, Öle, Fette, Wachse 94, 12 (1968)).

“Fine” means a powder predominantly (>50%) having a particle size of diameter from 20 to 200 μm.

“Coarse” means a product predominantly (>50%) of a particle size of diameter from 200 to 2000 μm.

The particle size determination can be carried out by methods of laser diffraction spectrometry. Corresponding methods are described in the textbook “TeilchengroBenmessung in der Laborpraxis” by R. H. Müller and R. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) or in the text book “Introduction to Particle Technology” by M. Rhodes, published by Wiley &Sons (1998).

The free-flowing, fine powder can in turn be converted by suitable compaction or granulation processes into a coarse, very free-flowing, storable and substantially dust-free product.

The term “dust-free” means that the product comprises only small proportions (<5%) of particle sizes below 100 μm in diameter.

“Storable” in the sense of this invention means a product which can be stored for at least one (1) year or longer, preferably at least 1.5 years or longer, particularly preferably two (2) years or longer, in a dry and cool environment without any substantial loss of the respective organic-chemical compound occurring. “Substantial loss” means a loss of >5%.

It is advantageous to employ during the granulation or compaction the usual organic or inorganic auxiliaries or carriers such as starch, gelatin, cellulose derivatives or similar substances, as normally used in the processing of food products or feeds as binders, gelling agents or thickeners, or further substances such as, for example, silicas, silicates (EP0743016A) and stearates.

It is further advantageous to treat the surface of the resulting granules with oils or fats as described in WO04/054381. Oils which can be used are mineral oils, vegetable oils or mixtures of vegetable oils. Examples of such oils are soybean oil, olive oil, soybean oil/lecithin mixtures. In the same way, silicone oils, polyethylene glycols or hydroxyethylcellulose are also suitable. Treatment of the surfaces of the granules with said oils achieves an increased abrasion resistance of the product and a reduction in the dust content. The oil content in the product is 0.02 to 2.0% by weight, preferably 0.02 to 1.0% by weight, and very particularly preferably 0.2 to 1.0% by weight, based on the total amount of the feed additive.

Preferred products have a proportion of ≥97% by weight with a particle size of from 100 to 1800 μm or a proportion of ≥95% by weight with a particle size of diameter 300 to 1800 μm. The proportion of dust, i.e. particles with a particle size<100 μm, is preferably >0 to 1% by weight, particularly preferably not exceeding 0.5% by weight.

However, alternatively, the product may also be absorbed on an organic or inorganic carrier known and customary in the processing of feeds, such as, for example, silicas, silicates, meals, brans, flours, starches, sugars or others, and/or be mixed and stabilized with customary thickeners or binders. Examples of use and processes therefor are described in the literature (Die Mühle+Mischfuttertechnik 132 (1995) 49, page 817).

The following examples are provided for purposes of illustration, not limitation.

EXAMPLES
Example 1: Application of Candidate Promoters to the L-Lysine Biosynthetic Pathway

The promoters of the present disclosure are useful for improved processes for the production of biomolecules in host cells. An example of the application and use of the promotor of the present disclosure is directed to the production of the amino acid L-lysine.

FIG. 1 presents the biosynthetic pathway for the production of L-lysine and includes the genes pck, odx, icd, and hom (e.g., the homoserine/threonine synthase pathway), that divert intermediates from the pathway leading to reductions in overall L-lysine yield. The symbols, gene names, Enzyme Commission number (EC number), and map position in C. glutamicum strain ATCC 13032 are provided in Table 3.

Recombinant vectors comprising a promoter of SEQ ID NOs: 1 to 8 functionally linked to a target gene as provided in Table 3 are cloned into Corynebacterium cloning vectors using yeast homologous recombination cloning techniques to assemble a vector in which each promoter was flanked by direct repeat regions to provide for homologous recombination in Corynebacterium glutamicum at the target gene locus. Upon recombination, the endogenous promoter is replaced by the promoter of SEQ ID NOs: 1 to 8 functionally linked to the respective target gene in the endogenous C. glutamicum locus. A variety of targeting vectors comprising the promoter and functionally linked target gene included a range of homology direct repeat arm lengths ranging from 0.5 Kb, 1 Kb, 2 Kb, and 5 Kb. Each DNA insert was produced by PCR amplification of homologous regions using commercially sourced oligos and the host strain genomic DNA described above as template. The promoter to be introduced into the genome was encoded in the oligo tails. PCR fragments were assembled into the vector backbone using homologous recombination in yeast.

Vectors are initially transformed into E. coli using standard heat shock transformation techniques and correctly assembled clones are identified and validated. Transformed E. coli bacteria are tested for assembly success. Four colonies from each E. coli transformation plate are cultured and tested for correct assembly via PCR. Vectors are amplified in the E. coli hosts to provide vector DNA for Corynebacterium transformation.

Validated clones are transformed into Corynebacterium glutamicum host cells via electroporation. For each transformation, the number of Colony Forming Units (CFUs) per μg of DNA is determined as a function of the insert size. Corynebacterium genome integration is analyzed as a function of homology arm length. Shorter arms had a lower efficiency.

Cultures of Corynebacterium identified as having successful integrations of the insert cassette are cultured on media containing 5% sucrose to counter select for loop outs of the sacb selection gene. Sucrose resistance frequency for various homology direct repeat arms do not vary significantly with arm length. These results suggest that loopout efficiencies remain steady across homology arm lengths of 0.5 kb to 5 kb.

In order to further validate loop out events, colonies exhibiting sucrose resistance are cultured and analyzed via sequencing. The results for the sequencing of the insert genomic regions are summarized below in Table 6.

TABLE 6

Loop-out Validation Frequency

Outcome
Frequency (sampling error 95% confidence)

Successful
13%
(9%/20%)

Loop out

Loop Still
42%
(34%/50%)

present

Mixed read
44%
(36%/52%)

Sequencing results show a 10-20% efficiency in loop outs. Not to be limited by any particular theory, loop-out may be dependent on insert sequence. Even if correct, picking 10-20 sucrose-resistant colonies leads to high success rates.

Upon integration, the recombinant vectors replace the endogenous promoter sequences with a promoter selected from the group consisting of Pcg1860 (SEQ ID NO:2), Pcg0007 (SEQ ID NO:3), Pcg0755 (SEQ ID NO:4), Pcg0007_lib_265 (SEQ ID NO:5), Pcg3381 (SEQ ID NO:6), Pcg007_lib_119 (SEQ ID NO:7), and Pcg3121 (SEQ ID NO:8). The resulting recombinant strains is provided in the following list:

Pcg1860-asd; Pcg0755-asd; Pcg0007_119-asd; Pcg3121-asd; Pcg0007_265-asd; Pcg3381-asd; Pcg1860-ask; Pcg0755-ask; Pcg3121-ask; Pcg0007_119-ask; Pcg0007_265-ask; Pcg3381-ask; Pcg3381-aspB; Pcg0007_119-aspB; Pcg0007_119-cg0931; Pcg1860-cg0931; Pcg0007_265-cg0931; Pcg0007_39-cg0931; Pcg0755-cg0931; Pcg0007-cg0931; Pcg0007-dapA; Pcg3381-dapA; Pcg0007_265-dapA; Pcg0007_119-dapA; Pcg0007_265-dapB; Pcg0755-dapB; Pcg0007-dapB; Pcg3381-dapB; Pcg1860-dapB Pcg3121-dapB; Pcg0007_119-dapB; Pcg0007_265-dapD; Pcg0007_119-dapD; Pcg3381-dapD; Pcg0007_39-dapD; Pcg3121-dapD; Pcg0007-dapD; Pcg1860-dapD; Pcg0755-dapD; Pcg3381-dapE; Pcg3121-dapE; Pcg0755-dapE; Pcg0007_119-dapE; Pcg1860-dapE; Pcg0007_39-dapE; Pcg0007_265-dapF; Pcg3381-dapF; Pcg0007_119-dapF; Pcg0007-dapF; Pcg1860-dapF; Pcg0007_39-dapF; Pcg3381-ddh; Pcg3121-ddh; Pcg0007_119-ddh; Pcg0007_39-ddh; Pcg1860-ddh; Pcg0007_265-ddh; Pcg0755-ddh; Pcg0007-ddh; Pcg3381-fbp; Pcg0007_119-fbp; Pcg1860-fbp; Pcg0007-fbp; Pcg3121-fbp; Pcg0755-fbp; Pcg0755-hom; Pcg3381-hom; Pcg1860-hom; Pcg3121-hom; Pcg0007_119-icd; Pcg3121-icd; Pcg3381-icd; Pcg1860-icd; Pcg0007_39-icd; Pcg0007-icd; Pcg0007_265-icd; Pcg0007-lysA; Pcg0007_39-lysA; Pcg3121-lysA; Pcg0007_265-lysA; Pcg0007_119-lysA; Pcg3381-lysA; Pcg0007_39-lysE; Pcg0007-lysE; Pcg0007_265-lysE; Pcg3121-lysE; Pcg3381-lysE; Pcg0007_119-lysE; Pcg3381-odx; Pcg0007_265-odx; Pcg0755-odx; Pcg0007-odx; Pcg1860-odx; Pcg0007_39-odx; Pcg0007_119-odx; Pcg3121-odx; Pcg3121-pck; Pcg3381-pck; Pcg0007_119-pck; Pcg0007_265-pck; Pcg0755-pck; Pcg0007_39-pck; Pcg0007-pck; Pcg1860-pck; Pcg3121-pgi; Pcg0007_119-pgi; Pcg3381-pgi; Pcg0007_265-pgi; Pcg1860-pgi; Pcg0007-pgi; Pcg0007_39-ppc; Pcg0007_265-ppc; Pcg0755-ppc; Pcg3381-ppc; Pcg0007_119-ppc; Pcg1860-ppc; Pcg3121-ppc; Pcg0755-ptsG; Pcg1860-ptsG; Pcg0007_39-ptsG; Pcg3381-ptsG; Pcg0007_119-ptsG; Pcg3121-ptsG; Pcg1860-pyc; Pcg0755-pyc; Pcg0007_39-pyc; Pcg0007_265-pyc; Pcg0007-pyc; Pcg3381-pyc; Pcg0007_119-pyc; Pcg3121-pyc; Pcg3121-tkt; Pcg0007_119-tkt; Pcg0755-tkt; Pcg0007-tkt; Pcg3381-tkt; Pcg0007_265-tkt; Pcg0007-zwf; Pcg0755-zwf; Pcg0007_265-zwf; and Pcg1860-zwf; Pcg3121-zwf.

Multiple single colonies are picked, inoculated and grown as a small scale culture. Each newly created strain comprising a test promoter is tested for lysine yield in small scale cultures designed to assess product titer performance. Small scale cultures are conducted using media from industrial scale cultures. Product titer is optically measured at carbon exhaustion (i.e., representative of single batch yield) with a standard colorimetric assay. Briefly, a concentrated assay mixture is prepared and is added to fermentation samples such that final concentrations of reagents are 160 mM sodium phosphate buffer, 0.2 mM Amplex Red, 0.2 U/mL Horseradish Peroxidase and 0.005 U/mL of lysine oxidase. Reactions proceed to completion and optical density is measured using a Tecan M1000 plate spectrophotometer at a 560 nm wavelength.

In some cases, the yield of L-lysine is increased by over 24% (e.g., recombinant strain 7000007840) over the non-engineered strain. In other embodiments, the yield of L-lysine is decreased by nearly 90% (e.g., recombinant strain 700000773). Replacement of the promoter for the pgi and zwf results in greater than 10% improvements to L-lysine production.

Notably, the production of L-lysine is not a simple dependence on incorporating the most active promoters. Lysine yield is maximized by a relatively weak promoter (e.g., pgi having relative promoter expression of 1, 7×, or 48×, or dapB at a relative promoter strength of 7×) or maximized by intermediate expression (e.g., lysA at having a relative promoter expression of 454×). In certain cases, expression is maximal when the relative promoter strength is maximized (e.g., ppc). The location of the gene in the genetic pathway does not reliably predict the relative increase or decrease in L-lysine yield or the optimal promoter strength. For example, high level expression of cg0931 results in improved yield while higher levels of dapD result in no improvement or decreased yield.

Example 2: Engineering the L-Lysine Biosynthetic Pathway

The yield of L-lysine is modified by swapping pairs of promoters for target genes. The constructs of Example 1 are used to prepare recombinant organsims as follows:

The combination of Pcg0007-lysA and Pcg3121-pgi provide for the highest yields of L-lysine.

TABLE 7

Paired Promoter Swapping of Target Genes in the L-lysine biosynthetic pathway

Mean Yield

Strain ID
Number
PRO Swap 1
PRO Swap 2
(A₅₆₀)
Std Dev

7000008489
4
Pcg0007-lysA
Pcg3121-pgi
1.17333
0.020121

7000008530
8
Pcg1860-pyc
Pcg0007-zwf
1.13144
0.030023

7000008491
7
Pcg0007-lysA
Pcg0007-zwf
1.09836
0.028609

7000008504
8
Pcg3121-pck
Pcg0007-zwf
1.09832
0.021939

7000008517
8
Pcg0007_39-ppc
Pcg0007-zwf
1.09502
0.030777

7000008502
4
Pcg3121-pck
Pcg3121-pgi
1.09366
0.075854

7000008478
4
Pcg3381-ddh
Pcg0007-zwf
1.08893
0.025505

7000008465
4
Pcg0007_265-dapB
Pcg0007-zwf
1.08617
0.025231

7000008535
8
Pcg0007-zwf
Pcg3121-pgi
1.06261
0.019757

7000008476
6
Pcg3381-ddh
Pcg3121-pgi
1.04808
0.084307

7000008510
8
Pcg3121-pgi
Pcg1860-pyc
1.04112
0.021087

7000008525
8
Pcg1860-pyc
Pcg0007_265-dapB
1.0319
0.034045

7000008527
8
Pcg1860-pyc
Pcg0007-lysA
1.02278
0.043549

7000008452
5
Pcg1860-asd
Pcg0007-zwf
1.02029
0.051663

7000008463
4
Pcg0007_265-dapB
Pcg3121-pgi
1.00511
0.031604

7000008524
8
Pcg1860-pyc
Pcg1860-asd
1.00092
0.026355

7000008458
4
Pcg3381-aspB
Pcg1860-pyc
1.00043
0.020083

7000008484
8
Pcg3381-fbp
Pcg1860-pyc
0.99686
0.061364

7000008474
8
Pcg3381-ddh
Pcg3381-fbp
0.99628
0.019733

7000008522
8
Pcg0755-ptsG
Pcg3121-pgi
0.99298
0.066021

7000008528
8
Pcg1860-pyc
Pcg3121-pck
0.99129
0.021561

7000008450
4
Pcg1860-asd
Pcg3121-pgi
0.98262
0.003107

7000008448
8
Pcg1860-asd
Pcg3381-fbp
0.97814
0.022285

7000008494
8
Pcg0007_39-lysE
Pcg3381-fbp
0.97407
0.027018

7000008481
8
Pcg3381-fbp
Pcg0007-lysA
0.9694
0.029315

7000008497
8
Pcg0007_39-lysE
Pcg1860-pyc
0.9678
0.028569

7000008507
8
Pcg3121-pgi
Pcg3381-fbp
0.96358
0.035078

7000008501
8
Pcg3121-pck
Pcg0007-lysA
0.96144
0.018665

7000008486
8
Pcg0007-lysA
Pcg0007_265-dapB
0.94523
0.017578

7000008459
8
Pcg0007_265-dapB
Pcg1860-asd
0.94462
0.023847

7000008506
2
Pcg3121-pgi
Pcg0007_265-dapD
0.94345
0.014014

7000008487
8
Pcg0007-lysA
Pcg3381-ddh
0.94249
0.009684

7000008498
8
Pcg3121-pck
Pcg1860-asd
0.94154
0.016802

7000008485
8
Pcg0007-lysA
Pcg1860-asd
0.94135
0.013578

7000008499
8
Pcg3121-pck
Pcg0007_265-dapB
0.93805
0.013317

7000008472
8
Pcg3381-ddh
Pcg1860-asd
0.93716
0.012472

7000008511
8
Pcg0007_39-ppc
Pcg1860-asd
0.93673
0.015697

7000008514
8
Pcg0007_39-ppc
Pcg0007-lysA
0.93668
0.027204

7000008473
8
Pcg3381-ddh
Pcg0007_265-dapB
0.93582
0.030377

7000008461
7
Pcg0007_265-dapB
Pcg3381-fbp
0.93498
0.037862

7000008512
8
Pcg0007_39-ppc
Pcg0007_265-dapB
0.93033
0.017521

7000008456
8
Pcg3381-aspB
Pcg3121-pck
0.92544
0.020075

7000008460
8
Pcg0007_265-dapB
Pcg0007_265-dapD
0.91723
0.009508

7000008492
8
Pcg0007_39-lysE
Pcg3381-aspB
0.91165
0.012988

7000008493
8
Pcg0007_39-lysE
Pcg0007_265-dapD
0.90609
0.031968

7000008453
8
Pcg3381-aspB
Pcg0007_265-dapB
0.90338
0.013228

7000008447
8
Pcg1860-asd
Pcg0007_265-dapD
0.89886
0.028896

7000008455
8
Pcg3381-aspB
Pcg0007-lysA
0.89531
0.027108

7000008454
6
Pcg3381-aspB
Pcg3381-ddh
0.87816
0.025807

7000008523
8
Pcg0755-ptsG
Pcg1860-pyc
0.87693
0.030322

7000008520
8
Pcg0755-ptsG
Pcg3381-fbp
0.87656
0.018452

7000008533
4
Pcg0007-zwf
Pcg3381-fbp
0.84584
0.017012

7000008519
8
Pcg0755-ptsG
Pcg0007_265-dapD
0.84196
0.025747

Example 3: Engineering the L-Lysine Biosynthetic Pathway with Promoters Operably Linked to Off-Pathway Genes

The yield of L-lysine is modified by including a second promoter polynucleotide sequence functionally linked to an off-pathway second heterologous target gene. The heterologous target genes are selected from ncg10009, ncg10019, ncg10054, ncg10082, ncg10142, ncg10223, ncg10241, ncg10242, ncg10304, ncg10306, ncg10356, ncg10398, ncg10408, ncg10424, ncg10425, ncg10427, ncg10439, ncg10458, ncg10471, ncg10531, ncg10546, ncg10564, ncg10573, ncg10578, ncg10581, ncg10598, ncg10600, ncg10601, ncg10641, ncg10663, ncg10668, ncg10737, ncg10767, ncg10813, ncg10823, ncg10827, ncg10853, ncg10874, ncg10877, ncg10905, ncg10916, ncg10966, ncg11065, ncg11124, ncg11137, ncg11152, ncg11187, ncg11196, ncg11202, ncg11203, ncg11208, ncg11261, ncg11262, ncg11267, ncg11301, ncg11320, ncg11322, ncg11364, ncg11366, ncg11371, ncg11372, ncg11457, ncg11484, ncg11500, ncg11503, ncg11508, ncg11511, ncg11545, ncg11550, ncg11583, ncg11607, ncg11855, ncg11858, ncg11880, ncg11886, ncg11900, ncg11905, ncg11911, ncg11928, ncg11948, ncg11961, ncg12001, ncg12002, ncg12019, ncg12048, ncg12077, ncg12104, ncg12147, ncg12153, ncg12190, ncg12204, ncg12210, ncg12211, ncg12247, ncg12250, ncg12274, ncg12286, ncg12287, ncg12298, ncg12327, ncg12399, ncg12425, ncg12440, ncg12441, ncg12446, ncg12449, ncg12472, ncg12473, ncg12481, ncg12491, ncg12505, ncg12527, ncg12535, ncg12538, ncg12559, ncg12567, ncg12569, ncg12576, ncg12587, ncg12614, ncg12669, ncg12684, ncg12699, ncg12702, ncg12755, ncg12789, ncg12790, ncg12802, ncg12827, ncg12886, ncg12898, ncg12901, ncg12905, ncg12921, ncg12929, ncg12930, ncg12931, ncg12982, and ncg12984.

Constructs containing a promoter identified herein linked to sequences homologous to a portion of the heterologous off-pathway genes identified above are used to prepare recombinant host cell organisms as provided in Tables 8 and 9. Upon integration, the recombinant vectors replace the endogenous promoter sequences with a promoter selected from the group consisting of Pcg1860 (SEQ ID NO:2), Pcg0007 (SEQ ID NO:3), Pcg0755 (SEQ ID NO:4), Pcg0007_lib_265 (SEQ ID NO:5), Pcg3381 (SEQ ID NO:6), Pcg007_lib_119 (SEQ ID NO:7), and Pcg3121 (SEQ ID NO:8). A list of the resulting recombinant strains is provided below in Table 8.

Multiple single colonies (N in Table 8) are picked, inoculated and grown as a small scale culture. Each newly created strain comprising a test promoter is tested for lysine yield in small scale cultures designed to assess product titer performance. Small scale cultures are conducted using media from industrial scale cultures. Product titer is optically measured at carbon exhaustion (i.e., representative of single batch yield) with a standard colorimetric assay. Briefly, a concentrated assay mixture is prepared and is added to fermentation samples such that final concentrations of reagents are 160 mM sodium phosphate buffer, 0.2 mM Amplex Red, 0.2 U/mL Horseradish Peroxidase and 0.005 U/mL of lysine oxidase. Reactions proceed to completion and optical density is measured using a Tecan M1000 plate spectrophotometer at a 560 nm wavelength.

As shown in Table 8, the yield of L-lysine is increased by over 14% (e.g., recombinant strain 7000152451) over the parent strain that does not contain a heterologous promoter functionally linked to an off-pathway target gene. Among those promoter replacements applied in at least three different strains in Table 9, the best performing modifications overall are pcg0007_39-cg0725 (average of 6.5% yield change in six strains), pcg0007_39-ncg11262 (average of 6.3% yield change in nine strains), and pcg0007_39-cg2766 (average of 5.1% yield change in 23 strains).

Notably, the production of L-lysine is not a simple dependence on incorporating the most active promoters. The pcg3121-mutm22522 modification involves a weak promoter but improved yield by an average of 5% in four strains.

TABLE 8

Recombinant strains of C. glutamicum having modified expression of non-L-lysine

Biosynthetic Genes and yield change from base of at least 3%, where the promoter-

target modification has been applied in at least five different strain backgrounds

% Yield

Mean

Change

Strain
promoter-target
N
(A₅₆₀)
Std Error
From Base

7000011650
pcg0007_39-dnak
16
0.939907
0.005637
14.40616

7000011837
pcg0007_39-cg0074
8
0.924085
0.005601
12.48029

7000012092
pcg3121-cg0074
8
0.927409
0.006967
12.88495

7000051494
pcg1860-rhle_609
14
1.060682
0.005131
8.870004

7000051495
pcg3121-cg1144
16
1.055172
0.0054
8.30446

7000071062
pcg1860-rhle_609
18
0.744484
0.007673
3.518385

7000101786
pcg0007_39-cg2899_2194
20
1.059718
0.00367
3.920982

7000101932
pcg0007_39-cg1486
6
1.057967
0.008648
3.749293

7000101946
pcg0007_39-cg2766
448
1.075533
0.003077
5.471888

7000102382
pcg0007_39-cmk
8
1.056225
0.007802
3.57847

7000132573
pcg0007_39-rpob_383
12
1.055606
0.008687
3.517745

7000132579
pcg0007_39-ddl
4
1.099107
0.012557
4.686924

7000132585
pcg0007_39-cg0027
5
1.078981
0.007576
5.008844

7000132587
pcg0007_39-ddl
5
1.061015
0.009978
3.260317

7000132589
pcg0007_39-rpob_383
5
1.082512
0.007857
5.352516

7000132596
pcg0007_39-rpob_383
5
1.107812
0.007265
6.123558

7000134570
pcg0007_39-cg0027
12
1.071955
0.010682
9.944488

7000138780
pcg1860-cg1144
16
1.068026
0.016669
3.061599

7000139655
pcg0007_39-cg0725
28
1.06408
0.005703
5.890614

7000144050
pcg0007_39-cg0027
8
1.113507
0.009172
5.81486

7000144052
pcg0007_39-cg1527
6
1.122574
0.009961
6.676453

7000144055
pcg0007_39-ddl
8
1.114668
0.003544
5.925188

7000144056
pcg0007_39-rpob_383
8
1.122532
0.004265
6.672479

7000148399
pcg0007_39-cg0725
39
1.069788
0.007402
3.231703

7000148414
pcg0007_39-cg0725
20
1.06931
0.008863
6.411116

7000148433
pcg0007_39-ddl
4
0.999022
0.013036
3.363822

7000148440
pcg0007_39-cg0725
17
1.080813
0.009706
11.82635

7000148442
pcg0007_39-cg2766
15
1.09271
0.011011
13.0573

7000148453
pcg0007_39-cg0725
19
0.967318
0.008709
3.173838

7000148476
pcg0007_39-hspr
2
0.95376
0.0072
4.883646

7000148498
pcg0007_39-cg3352
7
0.956069
0.01208
5.137602

7000148526
pcg0007_39-cg2899_2194
4
0.9628
0.022005
5.877807

7000148917
pcg0007_39-cg2766
17
0.966282
0.007737
6.260712

7000148950
pcg0007_39-cg2766
11
1.101872
0.00755
6.318818

7000148952
pcg0007_39-cg2965
11
1.068518
0.006968
3.100537

7000148963
pcg0007_39-rpob_383
26
0.885703
0.01053
4.88738

7000148966
pcg0007_39-cg2766
19
1.078181
0.00924
5.142825

7000149002
pcg0007_39-cg2766
17
1.105547
0.003775
3.957767

7000149072
pcg0007_39-cg2899_2194
7
0.938323
0.009912
3.186081

7000149133
pcg0007_39-cg0074
4
0.949821
0.002412
4.45053

7000149138
pcg3121-cg0074
6
0.93946
0.002513
3.311153

7000151562
pcg0007_39-cg2766
22
1.069264
0.007822
4.196667

7000151586
pcg3121-cg1144
8
0.948437
0.006684
4.298347

7000151646
pcg0007_39-cg2766
37
1.073209
0.00667
3.660275

7000151755
pcg0007_39-cg2766
6
1.07101
0.010452
12.61841

7000151756
pcg0007_39-cg2899
2
1.080952
0.00245
13.66387

7000151757
pcg0007_39-rho
7
1.055369
0.010359
10.97379

7000151842
pcg0007_39-cg2766
14
0.950916
0.007382
4.092383

7000151844
pcg0007_39-cg2766
13
0.980145
0.005638
7.291958

7000151863
pcg0007_39-cg0725
347
1.037644
0.003105
8.621402

7000151867
pcg1860-cg1144
17
0.997554
0.016818
4.424723

7000151881
pcg0007_39-cg2766
21
1.057606
0.003115
3.190432

7000151906
pcg0007_39-cg2766
52
1.085568
0.008262
5.547884

7000152431
pcg0007_39-urer
27
1.082933
0.008288
4.599504

7000152450
pcg0007_39-nusg
27
0.990823
0.013909
5.68089

7000152451
pcg3121-mutm2_2522
8
0.98005
0.00757
4.531918

7000152503
pcg0007_39-ddl
8
0.972555
0.00636
3.732443

7000152510
pcg1860-cg1144
6
0.973355
0.005158
3.817762

7000152585
pcg0007_39-cg2899
6
0.92449
0.028472
6.198442

7000152586
pcg0007_39-cg2965
15
0.952111
0.014247
9.371358

7000152587
pcg0007_39-ddl
6
0.97185
0.010086
11.63879

7000152595
pcg3121-mutm2_2522
7
1.000598
0.005055
14.94122

7000154599
pcg0007_39-cg2766
32
1.047813
0.006632
5.038279

7000154607
pcg0007_39-cg2766
64
1.094538
0.01056
4.462625

7000154623
pcg0007_39-cg2766
12
1.084043
0.010304
5.347278

7000155554
pcg0007_39-cg2766
76
1.061757
0.006753
3.655825

7000172142
pcg0007_39-cg2766
23
0.98211
0.006367
5.011511

7000172150
pcg0007_39-cg2766
20
1.100986
0.007079
5.411028

7000174400
pcg0007_39-tyra
42
1.082497
0.011274
3.702717

7000174421
pcg0007_39-cg1486
34
1.087003
0.01147
4.134408

7000178668
pcg0007_39-cg2899
18
1.091477
0.021824
3.721322

7000178693
pcg0007_39-cg0027
18
1.091807
0.011304
3.519654

7000179790
pcg0007_39-ncgl1511
13
1.077263
0.020883
3.484369

7000179967
pcg0007_39-ncgl1262
55
1.127037
0.009118
8.2657

7000182541
pcg0007_39-cg3419
11
1.109639
0.009557
5.221902

7000182553
pcg0007_39-cg1486
10
1.121579
0.012964
6.354126

7000182556
pcg0007_39-cg3210
8
1.093494
0.013361
3.690927

7000182560
pcg0007_39-cg1486
9
1.111948
0.011145
5.440906

7000182594
pcg0007_39-cg1486
8
1.030912
0.015987
3.199736

7000182604
pcg0007_39-cg1486
8
1.080288
0.013249
4.919604

7000182620
pcg0007_39-cg1486
71
1.121599
0.007064
3.116152

7000183003
pcg0007_39-ncgl0767
8
1.1098
0.015313
5.23717

7000183123
pcg0007_39-ncgl2481
7
1.098826
0.017557
4.1966

7000183674
pcg0007_39-tyra
11
1.065235
0.007951
3.264175

7000187919
pcg0007_39-cg1486
8
1.055633
0.007257
3.038697

7000187929
pcg0007_39-ncgl1511
52
1.074922
0.006759
5.748624

7000187963
pcg0007_39-ncgl0827
8
1.093548
0.004974
4.32642

7000190043
pcg0007_39-tyra
12
1.105391
0.006829
3.253128

7000190074
pcg0007_39-cg1486
10
1.119157
0.007057
3.146392

7000190089
pcg0007_39-ncgl1262
24
1.132882
0.010445
7.067927

7000190098
pcg0007_39-cg1486
12
1.096864
0.010931
3.142087

7000190123
pcg0007_39-ncgl1262
144
1.11294
0.002915
5.948846

7000191520
pcg0007_39-ncgl1262
117
1.099061
0.004345
5.441041

7000191588
pcg0007_39-ncgl0767
12
1.096351
0.004382
4.369537

7000196624
pcg0007_39-ncgl0304
18
1.090471
0.0102
3.809772

7000196641
pcg0007_39-ncgl1511
12
1.173593
0.084735
4.635733

7000196649
pcg0007_39-ncgl0767
18
1.128663
0.008043
4.022545

7000196650
pcg0007_39-ncgl1262
19
1.127885
0.005913
3.95088

7000196651
pcg0007_39-ncgl1511
7
1.119959
0.018691
3.220332

7000196657
pcg0007_39-ncgl0767
8
1.11792
0.008145
3.032404

7000196668
pcg0007_39-ncgl0767
18
1.114954
0.005466
3.439875

7000196677
pcg0007_39-ncgl1262
14
1.131585
0.004536
4.982825

7000196687
pcg0007_39-cg1486
20
1.129246
0.004393
4.76576

7000196703
pcg0007_39-ncgl1262
20
1.17434
0.003597
8.949436

7000197878
pcg0007_39-ncgl0304
16
1.106952
0.005627
3.246974

7000197883
pcg0007_39-ncgl1262
19
1.129494
0.00609
5.076771

7000197896
pcg0007_39-ncgl0767
22
1.199136
0.005992
5.506654

7000197934
pcg0007_39-ncgl1262
20
1.112516
0.005336
7.136187

As shown in Table 9, off-pathway target genes that exhibit a significant increase in lysine production when operably linked to a heterologous promoter exhibit an overrepresentation of certain GOSlim terms.

TABLE 9

Recombinant strains of C. glutamicum having modified expression of non-L-lysine Biosynthetic

Genes and yield change from base of at least 3%, and associated GOSlim terms

promoter-

Improvement

target
N
Over Parent
GOSlims

pcg1860-pfka
1
4.39028632
GO:0051186; GO:0005975; GO:0016301; GO:0043167; GO:0034641;

GO:0008150; GO:0044281; GO:0003674; GO:0006091; GO:0009056

pcg0007_265-
1
3.34890197
GO:0051186; GO:0005975; GO:0016301; GO:0043167; GO:0034641;

pfka

GO:0008150; GO:0044281; GO:0003674; GO:0006091; GO:0009056

pcg3381-pfka
1
11.3980559
GO:0051186; GO:0005975; GO:0016301; GO:0043167; GO:0034641;

GO:0008150; GO:0044281; GO:0003674; GO:0006091; GO:0009056

pcg0007_39-
6
14.4061592
GO:0008150; GO:0006457; GO:0003674; GO:0051082; GO:0043167

dnak

pcg0007-rho
1
13.4898058
GO:0009058; GO:0008150; GO:0003723; GO:0016887; GO:0003674;

GO:0043167; GO:0034641; GO:0004386

pcg0007-
1
12.5839916
GO:0006457; GO:0003674; GO:0008150; GO:0043167

groel

pcg1860-rho
3
14.1730443
GO:0009058; GO:0008150; GO:0003723; GO:0016887; GO:0003674;

GO:0043167; GO:0034641; GO:0004386

pcg1860-
2
13.5818163
GO:0004518; GO:0016829; GO:0006950; GO:0016798; GO:0034641;

mutm2_2522

GO:0008150; GO:0003677; GO:0006259; GO:0003674; GO:0043167

pcg0007_265-
1
13.2635149
GO:0043167; GO:0003674; GO:0004386

rhle_609

pcg3381-gpsi
1
13.1192293
GO:0016779; GO:0004518; GO:0043167; GO:0008150; GO:0034641;

GO:0003674; GO:0003723; GO:0034655; GO:0009056

pcg3121-rho
1
12.3280267
GO:0009058; GO:0008150; GO:0003723; GO:0016887; GO:0003674;

GO:0043167; GO:0034641; GO:0004386

pcg0007_39-
5
12.4802928
GO:0003674

cg0074

pcg0007_39-
2
13.8438588
GO:0008150; GO:0016779; GO:0003674; GO:0043167

glne

pcg0007-
1
12.1722755
GO:0003674

cg0074

pcg0007-glne
1
14.1530559
GO:0008150; GO:0016779; GO:0003674; GO:0043167

pcg0007_265-
1
12.5857278
GO:0003674

cg0074

pcg0007_265-
1
17.1431663
GO:0008150; GO:0016779; GO:0003674; GO:0043167

glne

pcg3381-
1
12.266976
GO:0003674

cg0074

pcg3121-
7
12.8849506
GO:0003674

cg0074

pcg3121-glne
2
14.2546395
GO:0008150; GO:0016779; GO:0003674; GO:0043167

pcg1860-
7
8.87000429
GO:0043167; GO:0003674; GO:0004386

rhle_609

pcg1860-
7
3.51838501
GO:0043167; GO:0003674; GO:0004386

rhle_609

pcg0007_39-
4
−4.5845813
GO:0009058; GO:0008150; GO:0044281; GO:0003674; GO:0016301;

prsa

GO:0034641; GO:0043167

pcg0007_39-
3
3.06020314
GO:0034641; GO:0008150; GO:0001071; GO:0003677; GO:0003674;

cg2942

GO:0009058

pcg0007_39-
61
−0.6395119
GO:0003674; GO:0003677

cg1527

pcg0007_39-
4
0.76168482
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg3239

GO:0009058

pcg1860-
1
−0.5888931
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg3239

GO:0009058

pcg0007_39-
3
−3.8426999
GO:0034641; GO:0008150; GO:0044281; GO:0003677; GO:0003674;

cg2831_2140

GO:0009058

pcg1860-
1
−7.8963623
GO:0034641; GO:0003674; GO:0003723; GO:0008150; GO:0016829;

cg1615

GO:0016853

pcg0007_39-
4
−3.1295926
GO:0044403; GO:0006950; GO:0008150

cg2151_1597

pcg1860-
1
−7.3331102
GO:0044403; GO:0006950; GO:0008150

cg2151_1597

pcg1860-
1
−3.2598098
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2783_2117

GO:0009058

pcg0007_39-
2
−6.1189202
GO:0003674; GO:0001071; GO:0003677; GO:0008150; GO:0043167

cg2784

pcg0007_39-
1
−5.1371168
GO:0009058; GO:0034641; GO:0003674; GO:0003677; GO:0008150;

rpob_384

GO:0016779

pcg1860-ptsi
1
−12.991654
GO:0016301; GO:0003674; GO:0008150; GO:0006810; GO:0043167

pcg0007_39-
3
−7.9173341
GO:0009058; GO:0003674; GO:0008150; GO:0016779

galu2

pcg0007_39-
9
3.92098179
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899_2194

GO:0009058

pcg0007_39-
1
4.35468317
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg0646_412

pcg1860-
1
3.73948719
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg0646_412

pcg0007_39-
3
3.82606325
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1410

pcg0007_39-
32
3.74929273
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
45
5.47188771
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg1860-
1
3.01149306
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg0537

GO:0009058

pcg0007_39-
3
3.09596969
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

hrca

pcg1860-
1
3.74501997
GO:0034641; GO:0008150; GO:0001071; GO:0003677; GO:0003674;

cg2965

GO:0009058

pcg0007_39-
1
3.71827755
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg0702_459

GO:0009058

pcg0007_39-
2
3.55921201
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg0702_460

GO:0009058

pcg0007_39-
2
−6.7755339
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg1324_966

GO:0009058

pcg0007_39-
5
3.57846953
GO:0034641; GO:0003674; GO:0044281; GO:0008150; GO:0016301;

cmk

GO:0043167

pcg1860-cmk
1
4.52929794
GO:0034641; GO:0003674; GO:0044281; GO:0008150; GO:0016301;

GO:0043167

pcg0007_39-
52
−2.3418417
GO:0009058; GO:0008150; GO:0003723; GO:0016887; GO:0003674;

rho

GO:0043167; GO:0034641; GO:0004386

pcg0007_39-
59
3.51774505
GO:0009058; GO:0034641; GO:0003674; GO:0003677; GO:0008150;

rpob_383

GO:0016779

pcg0007_39-
39
4.68692368
GO:0071554; GO:0009058; GO:0003674; GO:0008150; GO:0016874;

ddl

GO:0043167

pcg0007_39-
26
5.00884445
GO:0003674; GO:0008150; GO:0003677

cg0027

pcg0007_39-
39
3.26031674
GO:0071554; GO:0009058; GO:0003674; GO:0008150; GO:0016874;

ddl

GO:0043167

pcg0007_39-
59
5.35251645
GO:0009058; GO:0034641; GO:0003674; GO:0003677; GO:0008150;

rpob_383

GO:0016779

pcg0007_39-
59
6.12355761
GO:0009058; GO:0034641; GO:0003674; GO:0003677; GO:0008150;

rpob_383

GO:0016779

pcg0007_39-
26
9.94448777
GO:0003674; GO:0008150; GO:0003677

cg0027

pcg0007_39-
39
−2.1858149
GO:0071554; GO:0009058; GO:0003674; GO:0008150; GO:0016874;

ddl

GO:0043167

pcg0007_39-
41
5.89061417
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg0725

GO:0009058

pcg0007_39-
26
5.81485995
GO:0003674; GO:0008150; GO:0003677

cg0027

pcg0007_39-
61
6.67645276
GO:0003674; GO:0003677

cg1527

pcg0007_39-
39
5.92518842
GO:0071554; GO:0009058; GO:0003674; GO:0008150; GO:0016874;

ddl

GO:0043167

pcg0007_39-
59
6.67247895
GO:0009058; GO:0034641; GO:0003674; GO:0003677; GO:0008150;

rpob_383

GO:0016779

pcg0007_39-
41
3.23170311
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg0725

GO:0009058

pcg0007_39-
41
6.41111591
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg0725

GO:0009058

pcg0007_39-
39
3.36382174
GO:0071554; GO:0009058; GO:0003674; GO:0008150; GO:0016874;

ddl

GO:0043167

pcg0007_39-
41
11.8263514
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg0725

GO:0009058

pcg0007_39-
45
13.0573003
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
41
3.17383831
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg0725

GO:0009058

pcg0007_39-
2
3.80588976
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg3315

GO:0009058

pcg0007_39-
5
4.88364585
GO:0003674; GO:0008150; GO:0003677

hspr

pcg0007_39-
4
3.39617637
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg2910

pcg0007_39-
5
5.137602
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg3352

pcg0007_39-
2
5.60015218
GO:0003674

cg2177_1627

pcg0007_39-
4
4.89731116
GO:0008150; GO:0016746; GO:0003674

plsc_1822

pcg0007_39-
9
5.87780715
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899_2194

GO:0009058

pcg0007_39-
4
2.27062338
GO:0034641; GO:0008150; GO:0009058

nusg_374

pcg0007_39-
4
4.03184061
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg0646_413

pcg0007_39-
5
2.89398198
GO:0003674; GO:0008150; GO:0016887

para2_1175

pcg0007_39-
4
3.31061965
GO:0044403; GO:0006950; GO:0008150

cg2151_1597

pcg0007_39-
45
6.26071191
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
6.31881824
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
34
3.10053677
GO:0034641; GO:0008150; GO:0001071; GO:0003677; GO:0003674;

cg2965

GO:0009058

pcg0007_39-
59
4.88737996
GO:0009058; GO:0034641; GO:0003674; GO:0003677; GO:0008150;

rpob_383

GO:0016779

pcg0007_39-
45
5.14282462
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
3.95776653
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
9
3.18608122
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899_2194

GO:0009058

pcg0007_39-
52
1.78153221
GO:0009058; GO:0008150; GO:0003723; GO:0016887; GO:0003674;

rho

GO:0043167; GO:0034641; GO:0004386

pcg0007_39-
5
4.45053021
GO:0003674

cg0074

pcg3121-
7
3.31115349
GO:0003674

cg0074

pcg0007_39-
4
−1.0859037
GO:0004518; GO:0003674

cg2453

pcg3121-
35
−0.238969
GO:0004518; GO:0016829; GO:0006950; GO:0016798; GO:0034641;

mutm2_2522

GO:0008150; GO:0003677; GO:0006259; GO:0003674; GO:0043167

pcg0007_39-
45
4.19666701
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
0.57574968
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
52
0.87040444
GO:0009058; GO:0008150; GO:0003723; GO:0016887; GO:0003674;

rho

GO:0043167; GO:0034641; GO:0004386

pcg0007_39-
45
3.66027494
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
12.6184078
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
51
13.6638701
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899

GO:0009058

pcg0007_39-
52
10.9737942
GO:0009058; GO:0008150; GO:0003723; GO:0016887; GO:0003674;

rho

GO:0043167; GO:0034641; GO:0004386

pcg0007_39-
45
4.09238291
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
7.29195777
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
41
8.62140196
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg0725

GO:0009058

pcg0007_39-
45
3.19043193
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
1.62135831
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
5.54788425
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
61
−0.9105898
GO:0003674; GO:0003677

cg1527

pcg1860-xerd
32
−1.0306474
GO:0034641; GO:0008150; GO:0007049; GO:0003677; GO:0006259;

GO:0003674; GO:0007059; GO:0032196; GO:0051301

pcg0007_39-
44
4.59950374
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

urer

GO:0009058

pcg1860-xerd
32
0.51774094
GO:0034641; GO:0008150; GO:0007049; GO:0003677; GO:0006259;

GO:0003674; GO:0007059; GO:0032196; GO:0051301

pcg0007_39-
52
0.80226785
GO:0034641; GO:0008150; GO:0009058

nusg

pcg0007_39-
52
5.6808904
GO:0034641; GO:0008150; GO:0009058

nusg

pcg3121-
35
4.53191788
GO:0004518; GO:0016829; GO:0006950; GO:0016798; GO:0034641;

mutm2_2522

GO:0008150; GO:0003677; GO:0006259; GO:0003674; GO:0043167

pcg1860-xerd
32
0.45401159
GO:0034641; GO:0008150; GO:0007049; GO:0003677; GO:0006259;

GO:0003674; GO:0007059; GO:0032196; GO:0051301

pcg0007_39-
39
3.73244268
GO:0071554; GO:0009058; GO:0003674; GO:0008150; GO:0016874;

ddl

GO:0043167

pcg0007_39-
51
6.19844239
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899

GO:0009058

pcg0007_39-
34
9.37135808
GO:0034641; GO:0008150; GO:0001071; GO:0003677; GO:0003674;

cg2965

GO:0009058

pcg0007_39-
39
11.6387877
GO:0071554; GO:0009058; GO:0003674; GO:0008150; GO:0016874;

ddl

GO:0043167

pcg3121-
35
14.9412244
GO:0004518; GO:0016829; GO:0006950; GO:0016798; GO:0034641;

mutm2_2522

GO:0008150; GO:0003677; GO:0006259; GO:0003674; GO:0043167

pcg0007_39-
51
0.8074962
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899

GO:0009058

pcg0007_39-
34
−0.9759507
GO:0034641; GO:0008150; GO:0001071; GO:0003677; GO:0003674;

cg2965

GO:0009058

pcg0007_39-
39
−0.1918499
GO:0071554; GO:0009058; GO:0003674; GO:0008150; GO:0016874;

ddl

GO:0043167

pcg3121-
35
0.62085394
GO:0004518; GO:0016829; GO:0006950; GO:0016798; GO:0034641;

mutm2_2522

GO:0008150; GO:0003677; GO:0006259; GO:0003674; GO:0043167

pcg0007_39-
45
5.03827938
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
4.46262469
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
5.34727763
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
51
0.8195123
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899

GO:0009058

pcg0007_39-
61
0.7420368
GO:0003674; GO:0003677

cg1527

pcg0007_39-
34
−0.9362595
GO:0034641; GO:0008150; GO:0001071; GO:0003677; GO:0003674;

cg2965

GO:0009058

pcg0007_39-
45
3.65582546
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg3381-
10
0.10953454
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1410

pcg3121-
1
3.04229587
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2783_2117

GO:0009058

pcg0007_39-
45
2.64354688
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg1860-xerd
32
−2.7110091
GO:0034641; GO:0008150; GO:0007049; GO:0003677; GO:0006259;

GO:0003674; GO:0007059; GO:0032196; GO:0051301

pcg0007_39-
59
−0.290262
GO:0009058; GO:0034641; GO:0003674; GO:0003677; GO:0008150;

rpob_383

GO:0016779

pcg0007_39-
45
5.01151095
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
45
5.41102769
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
3
0.45383689
GO:0034641; GO:0003674; GO:0001071; GO:0008150; GO:0009058

cg3082_2321

pcg0007_39-
9
0.66363765
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899_2194

GO:0009058

pcg0007_39-
23
3.70271695
GO:0009058; GO:0008150; GO:0044281; GO:0003674; GO:0006520;

tyra

GO:0016491

pcg0007_39-
32
4.13440802
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
44
−1.2304485
GO:0008150; GO:0003674; GO:0003677

cg0800_539

pcg0007_39-
45
2.65554177
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2766

GO:0009058

pcg0007_39-
52
−2.294756
GO:0009058; GO:0008150; GO:0003723; GO:0016887; GO:0003674;

rho

GO:0043167; GO:0034641; GO:0004386

pcg0007_39-
44
0.72184473
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

urer

GO:0009058

pcg1860-xerd
32
0.06000601
GO:0034641; GO:0008150; GO:0007049; GO:0003677; GO:0006259;

GO:0003674; GO:0007059; GO:0032196; GO:0051301

pcg0007_39-
51
3.72132191
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899

GO:0009058

pcg0007_39-
26
3.51965405
GO:0003674; GO:0008150; GO:0003677

cg0027

pcg0007_39-
26
1.29180691
GO:0003674; GO:0008150; GO:0003677

cg0027

pcg0007_39-
1
−17.04716
GO:0005975; GO:0009058; GO:0008150; GO:0016757; GO:0003674

ncgl2535

pcg0007_39-
1
0.34018726
GO:0051186; GO:0009058; GO:0043167; GO:0034641; GO:0003674;

ncgl2446

GO:0044281; GO:0008150; GO:0016874

pcg0007_39-
1
−0.3317848
GO:0008150; GO:0009058; GO:0003674; GO:0006629

ncgl0600

pcg0007_39-
5
0.41840795
GO:0034641; GO:0008150; GO:0044281; GO:0003674; GO:0016810;

ncgl0827

GO:0009058

pcg0007_39-
17
0.56929624
GO:0016829; GO:0009058; GO:0034641; GO:0008150; GO:0044281;

ncgl0306

GO:0004871; GO:0003674; GO:0007165

pcg0007_39-
11
−0.388723
GO:0034641; GO:0003674; GO:0044281; GO:0008150; GO:0016301;

ncgl1948

GO:0009058; GO:0043167

pcg0007_39-
1
0.57425156
GO:0003674; GO:0051186; GO:0009058; GO:0043167; GO:0016765;

ncgl1961

GO:0034641; GO:0008150; GO:0044281; GO:0006790

pcg0007_39-
1
−2.6435068
GO:0034641; GO:0008150; GO:0044281; GO:0003674; GO:0016874;

ncgl2669

GO:0009058; GO:0043167

pcg0007_39-
1
−2.1326692
GO:0051186; GO:0009058; GO:0034641; GO:0003674; GO:0008150;

ncgl1508

GO:0016491

pcg0007_39-
1
−1.7218114
GO:0051186; GO:0009058; GO:0022607; GO:0006790; GO:0008150

ncgl1503

pcg0007_39-
15
3.48436935
GO:0051186; GO:0009058; GO:0016765; GO:0034641; GO:0008150;

ncgl1511

GO:0003674

pcg0007_39-
2
−1.3907926
GO:0016765; GO:0009058; GO:0008150; GO:0006629; GO:0003674;

ncgl0598

GO:0044281

pcg0007_39-
1
−0.0604086
GO:0009058; GO:0008150; GO:0006629; GO:0003674; GO:0016829;

ncgl2569

GO:0044281; GO:0043167

pcg0007_39-
1
0.97131028
GO:0034641; GO:0051186; GO:0008150; GO:0003674; GO:0019748;

ncgl1905

GO:0009058; GO:0043167

pcg0007_39-
1
−0.5809877
GO:0034641; GO:0003674; GO:0044281; GO:0008150; GO:0016301;

ncgl2287

GO:0009058; GO:0043167

pcg0007_39-
1
0.29165558
GO:0009058; GO:0051186; GO:0006790; GO:0008150; GO:0043167;

ncgl1457

GO:0003674; GO:0016874

pcg0007_39-
1
−3.5394984
GO:0043167; GO:0009058; GO:0008150; GO:0006629; GO:0003674;

ncgl0874

GO:0016301; GO:0044281

pcg0007_39-
1
−1.532018
GO:0042592; GO:0003674; GO:0008150; GO:0043167; GO:0016491

ncgl1928

pcg0007_39-
1
−3.372779
GO:0006950; GO:0043167; GO:0034641; GO:0003674; GO:0016887;

ncgl1880

GO:0003677; GO:0006259; GO:0008150

pcg0007_39-
2
−3.3625411
GO:0003674; GO:0008150; GO:0016491

ncgl0663

pcg0007_39-
1
−1.659528
GO:0004518; GO:0016829; GO:0006950; GO:0016798; GO:0034641;

ncgl0813

GO:0008150; GO:0003677; GO:0006259; GO:0003674; GO:0043167

pcg0007_39-
1
−2.0046229
GO:0003674; GO:0008150; GO:0016491

ncgl2286

pcg0007_39-
2
−2.6375612
GO:0004518; GO:0034641; GO:0008150; GO:0006259; GO:0003674;

ncgl0641

GO:0006950

pcg0007_39-
1
−1.1543868
GO:0006457; GO:0034641; GO:0051082; GO:0043167; GO:0009058;

ncgl2210

GO:0006950; GO:0008150; GO:0006259; GO:0003674

pcg0007_39-
7
−1.6400734
GO:0034641; GO:0008150; GO:0008168; GO:0006259; GO:0003674;

ncgl2901

GO:0006950

pcg0007_39-
7
−3.1981371
GO:0003674; GO:0016853; GO:0008150; GO:0016491; GO:0043167

ncgl0877

pcg0007_39-
1
−3.302181
GO:0006950; GO:0003674; GO:0008150; GO:0016491

ncgl2984

pcg0007_39-
1
−3.0186755
GO:0034641; GO:0009058; GO:0006950; GO:0008150; GO:0003677;

ncgl1855

GO:0006259; GO:0003674; GO:0008233

pcg0007_39-
6
−2.9642887
GO:0004518; GO:0006950; GO:0043167; GO:0034641; GO:0003674;

ncgl1322

GO:0016887; GO:0003677; GO:0006259; GO:0008150

pcg0007_39-
1
−1.8831379
GO:0022607; GO:0006461; GO:0065003; GO:0008150; GO:0003674

ncgl0427

pcg0007_39-
1
−2.4009195
GO:0006950; GO:0034641; GO:0043167; GO:0009058; GO:0008150;

ncgl1196

GO:0006259; GO:0003674; GO:0016874

pcg0007_39-
1
−2.286499
GO:0016779; GO:0034641; GO:0043167; GO:0006950; GO:0003674;

ncgl2576

GO:0007165; GO:0003677; GO:0006259; GO:0008150

pcg0007_39-
2
−0.4397465
GO:0042592; GO:0003674; GO:0016853; GO:0008150; GO:0016491

ncgl0424

pcg0007_39-
1
−0.6772859
GO:0034641; GO:0008150; GO:0006259; GO:0006950

ncgl2204

pcg0007_39-
1
−0.2836098
GO:0022607; GO:0006461; GO:0065003; GO:0008150

ncgl0425

pcg0007_39-
1
−9.9973443
GO:0006950; GO:0008150

ncgl2755

pcg0007_39-
1
−0.9815224
GO:0006950; GO:0008150; GO:0006259; GO:0003674; GO:0034641;

ncgl0142

GO:0016798

pcg0007_39-
2
−0.3108331
GO:0006950; GO:0043167; GO:0034641; GO:0008150; GO:0003677;

ncgl0241

GO:0006259; GO:0003674

pcg0007_39-
1
−2.484144
GO:0008150; GO:0003674; GO:0016810; GO:0009056; GO:0043167

ncgl2789

pcg0007_39-
1
−2.1347389
GO:0006520; GO:0034641; GO:0043167; GO:0009058; GO:0008150;

ncgl2929

GO:0044281; GO:0016757; GO:0003674

pcg0007_39-
1
−2.7583354
GO:0009058; GO:0008150; GO:0044281; GO:0003674; GO:0016829;

ncgl0408

GO:0006520

pcg0007_39-
7
−2.8766171
GO:0006520; GO:0003674; GO:0044281; GO:0008150; GO:0016874

ncgl1484

pcg0007_39-
2
−2.1343277
GO:0003674; GO:0016829; GO:0006520; GO:0009058; GO:0008150;

ncgl2473

GO:0044281; GO:0006790

pcg0007_39-
2
−2.7490574
GO:0003674; GO:0016829; GO:0006520; GO:0009058; GO:0008150;

ncgl2473

GO:0044281; GO:0006790

pcg0007_39-
1
−2.7534611
GO:0005975; GO:0016301; GO:0043167; GO:0008150; GO:0044281;

ncgl2790

GO:0003674; GO:0009056

pcg0007_39-
1
−0.9048132
GO:0006520; GO:0016301; GO:0043167; GO:0009058; GO:0008150;

ncgl2274

GO:0044281; GO:0003674; GO:0003723

pcg0007_39-
1
−1.1695153
GO:0009058; GO:0003674; GO:0044281; GO:0008150; GO:0006520;

ncgl0398

GO:0016491

pcg0007_39-
16
8.26570011
GO:0016829; GO:0006520; GO:0043167; GO:0009058; GO:0008150;

ncgl1262

GO:0044281; GO:0003674

pcg0007_39-
1
0.01729616
GO:0034641; GO:0008150; GO:0044281; GO:0003674; GO:0006520;

ncgl1550

GO:0009058; GO:0043167

pcg0007_39-
1
−0.1957523
GO:0008150; GO:0003674; GO:0005975

ncgl2505

pcg0007_39-
1
0.0396492
GO:0008150; GO:0003674; GO:0016301; GO:0005975; GO:0043167

ncgl2399

pcg0007_39-
1
0.92749467
GO:0008150; GO:0044281; GO:0003674; GO:0016829; GO:0006520;

ncgl1500

GO:0006790; GO:0043167

pcg0007_39-
1
0.5029595
GO:0009058; GO:0016301; GO:0008150; GO:0044281; GO:0003674;

ncgl1137

GO:0006520; GO:0043167

pcg0007_39-
1
−2.0644554
GO:0006520; GO:0006790; GO:0043167; GO:0009058; GO:0008150;

ncgl2048

GO:0044281; GO:0008168; GO:0003674

pcg0007_39-
1
1.24260236
GO:0016829; GO:0006520; GO:0034641; GO:0009058; GO:0008150;

ncgl2019

GO:0044281; GO:0003674

pcg0007_39-
1
−0.3679826
GO:0008150; GO:0044281; GO:0003674; GO:0009056; GO:0005975;

ncgl2559

GO:0016853

pcg0007_39-
1
−0.8689167
GO:0008150; GO:0044281; GO:0003674; GO:0006091; GO:0016746;

ncgl2247

GO:0005975; GO:0043167

pcg0007_39-
1
−1.5472722
GO:0016779; GO:0008150; GO:0044281; GO:0003674; GO:0005975;

ncgl2002

GO:0043167

pcg0007_39-
1
−0.5896195
GO:0008150; GO:0003674; GO:0016301; GO:0005975

ncgl2905

pcg0007_39-
1
−0.8260018
GO:0003674; GO:0008150; GO:0008233; GO:0016746; GO:0034641;

ncgl0916

GO:0009056; GO:0006790

pcg0007_39-
1
−1.3961923
GO:0009058; GO:0008150; GO:0044281; GO:0005975; GO:0016829;

ncgl1583

GO:0003674

pcg0007_39-
1
−0.6491076
GO:0008150; GO:0003674; GO:0005975; GO:0016798

ncgl0853

pcg0007_39-
1
−0.5659838
GO:0016853; GO:0016829; GO:0006520; GO:0034641; GO:0009058;

ncgl2930

GO:0008150; GO:0044281; GO:0003674

pcg0007_39-
44
−0.2183031
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

urer

GO:0009058

pcg0007_39-
44
2.15803348
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

urer

GO:0009058

pcg0007_39-
51
−0.3543192
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899

GO:0009058

pcg0007_39-
44
−3.0417686
GO:0008150; GO:0003674; GO:0003677

cg0800_539

pcg0007_39-
9
5.22190199
GO:0061024; GO:0006810; GO:0008150

cg3419

pcg0007_39-
32
6.35412587
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
25
3.69092746
GO:0008150

cg3210

pcg0007_39-
32
5.44090596
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
44
0.86106557
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

urer

GO:0009058

pcg0007_39-
32
3.19973635
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
25
0.28172305
GO:0008150

cg3210

pcg0007_39-
44
0.40999284
GO:0008150; GO:0003674; GO:0003677

cg0800_539

pcg0007_39-
32
4.91960389
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
44
−1.8148135
GO:0008150; GO:0003674; GO:0003677

cg0800_539

pcg0007_39-
32
3.11615157
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
25
−0.2470034
GO:0008150

cg3210

pcg0007_39-
9
5.23717007
GO:0034641; GO:0003674; GO:0003723; GO:0008150; GO:0008135;

ncgl0767

GO:0009058; GO:0006412

pcg0007_39-
7
0.32038287
GO:0006399; GO:0034641; GO:0008150; GO:0003674; GO:0016301

ncgl0564

pcg0007_39-
2
7.04961811
GO:0006520; GO:0009058; GO:0043167; GO:0006399; GO:0034641;

ncgl1607

GO:0008150; GO:0044281; GO:0003674; GO:0016874; GO:0006412

pcg0007_39-
7
4.19660001
GO:0043167; GO:0006399; GO:0034641; GO:0008150; GO:0003674;

ncgl2481

GO:0016491

pcg0007_39-
8
2.21004426
GO:0034641; GO:0008150; GO:0051276; GO:0003677; GO:0006259;

ncgl0304

GO:0003674; GO:0043167; GO:0016853

pcg0007_39-
10
2.20963174
GO:0034641; GO:0003674; GO:0003723; GO:0008150; GO:0016829;

cg1615

GO:0016853

pcg0007_39-
6
−2.1901613
GO:0008150; GO:0016829; GO:0003674

ncgl2491

pcg0007_39-
21
−1.225098
GO:0071554; GO:0009058; GO:0003674; GO:0051301; GO:0008150;

murc

GO:0016874; GO:0007049; GO:0043167

pcg0007_39-
25
0.75098055
GO:0008150

cg3210

pcg0007_39-
32
0.92038074
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
3
−1.8936532
GO:0034641; GO:0003674; GO:0001071; GO:0008150; GO:0009058

cg3082

pcg0007_39-
8
0.57521516
GO:0003674; GO:0016301; GO:0008150; GO:0003677

cg0012

pcg0007_39-
44
−1.4559363
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

urer

GO:0009058

pcg0007_39-
26
−1.256254
GO:0003674; GO:0008150; GO:0003677

cg0027

pcg0007_39-
59
0.7674954
GO:0009058; GO:0034641; GO:0003674; GO:0003677; GO:0008150;

rpob_383

GO:0016779

pcg0007_39-
51
1.89896771
GO:0034641; GO:0003674; GO:0001071; GO:0003677; GO:0008150;

cg2899

GO:0009058

pcg0007_39-
3
1.41331708
GO:0034641; GO:0003674; GO:0001071; GO:0008150; GO:0009058

cg3082

pcg0007_39-
23
3.26417536
GO:0009058; GO:0008150; GO:0044281; GO:0003674; GO:0006520;

tyra

GO:0016491

pcg0007_39-
21
−0.5881867
GO:0071554; GO:0009058; GO:0003674; GO:0051301; GO:0008150;

murc

GO:0016874; GO:0007049; GO:0043167

pcg0007_39-
44
−0.0852691
GO:0008150; GO:0003674; GO:0003677

cg0800_539

pcg0007_39-
44
−0.2629748
GO:0008150; GO:0003674; GO:0003677

cg0800_539

pcg0007_39-
1
4.77198741
GO:0016746; GO:0003674

ncgl1208

pcg0007_39-
1
3.0707362
GO:0003674; GO:0008150; GO:0016491

ncgl2449

pcg0007_39-
2
4.12705375
GO:0008150; GO:0008233; GO:0003674

ncgl2327

pcg0007_39-
2
4.57292995
GO:0003674

ncgl2250

pcg0007_39-
3
5.38326154
GO:0006259; GO:0003674; GO:0034641; GO:0008150; GO:0003677

ncgl1545

pcg0007_39-
32
3.03869679
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
15
5.74862357
GO:0051186; GO:0009058; GO:0016765; GO:0034641; GO:0008150;

ncgl1511

GO:0003674

pcg0007_39-
5
4.32642044
GO:0034641; GO:0008150; GO:0044281; GO:0003674; GO:0016810;

ncgl0827

GO:0009058

pcg0007_39-
11
−0.2038558
GO:0034641; GO:0003674; GO:0044281; GO:0008150; GO:0016301;

ncgl1948

GO:0009058; GO:0043167

pcg0007_39-
44
−0.7937019
GO:0008150; GO:0003674; GO:0003677

cg0800_539

pcg0007_39-
23
3.25312756
GO:0009058; GO:0008150; GO:0044281; GO:0003674; GO:0006520;

tyra

GO:0016491

pcg0007_39-
32
3.14639212
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
32
−3.5839145
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
32
−1.8163957
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
23
−3.5273781
GO:0009058; GO:0008150; GO:0044281; GO:0003674; GO:0006520;

tyra

GO:0016491

pcg0007_39-
15
−4.7165343
GO:0016829; GO:0006520; GO:0034641; GO:0009058; GO:0008150;

ncgl2931

GO:0044281; GO:0003674

pcg0007_39-
16
7.06792721
GO:0016829; GO:0006520; GO:0043167; GO:0009058; GO:0008150;

ncgl1262

GO:0044281; GO:0003674

pcg0007_39-
32
3.14208716
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
16
5.94884625
GO:0016829; GO:0006520; GO:0043167; GO:0009058; GO:0008150;

ncgl1262

GO:0044281; GO:0003674

pcg0007_39-
7
−1.7262637
GO:0006520; GO:0003674; GO:0044281; GO:0008150; GO:0016874

ncgl1484

pcg0007_39-
15
−1.3365563
GO:0051186; GO:0009058; GO:0016765; GO:0034641; GO:0008150;

ncgl1511

GO:0003674

pcg0007_39-
16
5.44104119
GO:0016829; GO:0006520; GO:0043167; GO:0009058; GO:0008150;

ncgl1262

GO:0044281; GO:0003674

pcg0007_39-
9
4.36953707
GO:0034641; GO:0003674; GO:0003723; GO:0008150; GO:0008135;

ncgl0767

GO:0009058; GO:0006412

pcg0007_39-
4
−0.3074247
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg2614_2011

pcg0007_39-
5
−3.8871473
GO:0003674; GO:0008150; GO:0003677

hspr

pcg0007_39-
4
1.05794426
GO:0003674; GO:0003677

cg1392_1013

pcg0007_39-
32
2.84208976
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
3
3.6771318
GO:0003674; GO:0008150; GO:0003677

cg0979_688

pcg0007_39-
4
1.74233214
GO:0003674

ptsx′

pcg0007_39-
1
−0.6808701
GO:0006810; GO:0008150; GO:0055085

ncgl0546

pcg0007_39-
2
−0.264575
GO:0009058; GO:0008150; GO:0044281; GO:0003674; GO:0006520;

ncgl0242

GO:0034641

pcg0007_39-
1
3.52470923
GO:0009058; GO:0043167; GO:0034641; GO:0008150; GO:0044281;

ncgl0578

GO:0003674; GO:0016491

pcg1860-xerd
32
0.29279276
GO:0034641; GO:0008150; GO:0007049; GO:0003677; GO:0006259;

GO:0003674; GO:0007059; GO:0032196; GO:0051301

pcg0007_39-
7
−0.8917066
GO:0006399; GO:0034641; GO:0008150; GO:0003674; GO:0016301

ncgl0564

pcg0007_39-
7
−1.1006911
GO:0034641; GO:0008150; GO:0008168; GO:0006259; GO:0003674;

ncgl2901

GO:0006950

pcg0007_39-
11
−0.3328713
GO:0034641; GO:0003674; GO:0044281; GO:0008150; GO:0016301;

ncgl1948

GO:0009058; GO:0043167

pcg0007_39-
7
−1.1884484
GO:0008150; GO:0009058; GO:0003674

ncgl1065

pcg0007_39-
8
3.8097718
GO:0034641; GO:0008150; GO:0051276; GO:0003677; GO:0006259;

ncgl0304

GO:0003674; GO:0043167; GO:0016853

pcg0007_39-
15
4.63573323
GO:0051186; GO:0009058; GO:0016765; GO:0034641; GO:0008150;

ncgl1511

GO:0003674

pcg0007_39-
9
4.02254461
GO:0034641; GO:0003674; GO:0003723; GO:0008150; GO:0008135;

ncgl0767

GO:0009058; GO:0006412

pcg0007_39-
16
3.95087992
GO:0016829; GO:0006520; GO:0043167; GO:0009058; GO:0008150;

ncgl1262

GO:0044281; GO:0003674

pcg0007_39-
15
3.22033208
GO:0051186; GO:0009058; GO:0016765; GO:0034641; GO:0008150;

ncgl1511

GO:0003674

pcg0007_39-
9
3.03240412
GO:0034641; GO:0003674; GO:0003723; GO:0008150; GO:0008135;

ncgl0767

GO:0009058; GO:0006412

pcg0007_39-
9
3.43987546
GO:0034641; GO:0003674; GO:0003723; GO:0008150; GO:0008135;

ncgl0767

GO:0009058; GO:0006412

pcg0007_39-
25
−0.2786013
GO:0008150

cg3210

pcg0007_39-
16
4.9828246
GO:0016829; GO:0006520; GO:0043167; GO:0009058; GO:0008150;

ncgl1262

GO:0044281; GO:0003674

pcg0007_39-
32
4.76576015
GO:0034641; GO:0008150; GO:0003677; GO:0003674; GO:0009058

cg1486

pcg0007_39-
16
8.94943618
GO:0016829; GO:0006520; GO:0043167; GO:0009058; GO:0008150;

ncgl1262

GO:0044281; GO:0003674

pcg0007_39-
8
3.24697423
GO:0034641; GO:0008150; GO:0051276; GO:0003677; GO:0006259;

ncgl0304

GO:0003674; GO:0043167; GO:0016853

pcg0007_39-
16
5.07677112
GO:0016829; GO:0006520; GO:0043167; GO:0009058; GO:0008150;

ncgl1262

GO:0044281; GO:0003674

pcg0007_39-
25
0.46752889
GO:0008150

cg3210

pcg0007_39-
7
0.00474009
GO:0008150; GO:0009058; GO:0003674

ncgl1065

pcg0007_39-
7
−1.1311402
GO:0006520; GO:0003674; GO:0044281; GO:0008150; GO:0016874

ncgl1484

pcg0007_39-
9
5.50665417
GO:0034641; GO:0003674; GO:0003723; GO:0008150; GO:0008135;

ncgl0767

GO:0009058; GO:0006412

pcg0007_39-
16
7.1361868
GO:0016829; GO:0006520; GO:0043167; GO:0009058; GO:0008150;

ncgl1262

GO:0044281; GO:0003674

pcg0007_39-
11
2.17493085
GO:0034641; GO:0003674; GO:0044281; GO:0008150; GO:0016301;

ncgl1948

GO:0009058; GO:0043167

pcg0007_39-
2
3.0247232
GO:0008150; GO:0008233; GO:0003674

ncgl2327

pcg0007_39-
3
5.33577683
GO:0006259; GO:0003674; GO:0034641; GO:0008150; GO:0003677

ncgl1545

Example 4: Assessing L-Lysine Biosynthesis Modulation by Genes Belonging to Different Shells

Genetic loci across the C. glutamicum genome were tested for potential impact on lysine production by generating strains in which the native promoter regulating the gene's expression was substituted with a promoter selected from SEQ ID NOs: 1-8. The impact of each locus was tested by individually substituting the native promoter with one or more promoters from the promoters defined by SEQ ID NOs: 1-8.

The strains were tested for lysine production in multiple experiments and the mean performance of each strain across all experiments was calculated and compared to a control strain lacking the promoter modification. All strain pairs which differ by a single genetic change were calculated and the difference in lysine production at 96 hours between the strain with the change and the strain without the change was calculated. A change is defined as a hit if this performance difference is significantly greater than 0 (at p=0.01) across all strain pairs that differ by this change.

Table 10 provides each genetic locus/promoter modification combination tested and the performance thereof. Each genetic locus is provided with a shell designation as defined herein. Table 10 provides the locus id of each modified genetic locus, the promoter used to modify expression, whether any strains containing the modification had a significant difference in strain performance, and the shell allocation of the gene associated with the particular locus. As shown in Table 10, by testing the same genetic locus using multiple different promoters, applicants were able to identify loci encoding genes impacting strain performance, including shell 4 genes for which no known relationship with strain performance for lysine production was previously identified (e.g., see rows 49-54).

TABLE 10

Systematic sampling of promoter/target gene combinations from

different target gene shells for biomolecule production

locus_id
promoter
any_above_threshold
Shell

ncgl0009
pcg0007_39
TRUE
3

ncgl0009
pcg3121
FALSE
3

ncgl0015
pcg0007_39
TRUE
3

ncgl0015
pcg3381
FALSE
3

ncgl0019
pcg0007_39
TRUE
3

ncgl0019
pcg0755
FALSE
3

ncgl0019
pcg3121
FALSE
3

ncgl0019
pcg3381
FALSE
3

ncgl0026
pcg0007_39
TRUE
3

ncgl0031
pcg0007_39
FALSE
3

ncgl0031
pcg1860
TRUE
3

ncgl0031
pcg3381
FALSE
3

ncgl0042
pcg0007_39
TRUE
3

ncgl0042
pcg1860
TRUE
3

ncgl0054
pcg0007_39
TRUE
4

ncgl0054
pcg3121
FALSE
4

ncgl0082
pcg0007_119
FALSE
3

ncgl0082
pcg0007_39
TRUE
3

ncgl0082
pcg1860
TRUE
3

ncgl0082
pcg3121
FALSE
3

ncgl0082
pcg3381
FALSE
3

ncgl0082
wt
FALSE
3

ncgl0111
pcg0007_39
TRUE
4

ncgl0167
pcg0007_39
TRUE
3

ncgl0167
pcg1860
TRUE
3

ncgl0167
pcg3121
FALSE
3

ncgl0182
pcg0007_39
FALSE
2

ncgl0182
pcg1860
FALSE
2

ncgl0182
pcg3121
TRUE
2

ncgl0211
pcg0007_39
TRUE
2

ncgl0211
pcg0007_39
TRUE
3

ncgl0211
pcg3121
FALSE
2

ncgl0211
pcg3121
FALSE
3

ncgl0211
pcg3381
FALSE
2

ncgl0211
pcg3381
FALSE
3

ncgl0223
pcg0007_39
TRUE
3

ncgl0223
pcg0755
FALSE
3

ncgl0223
pcg2613
FALSE
3

ncgl0223
pcg3381
FALSE
3

ncgl0253
pcg0007_39
TRUE
3

ncgl0253
pcg1860
TRUE
3

ncgl0253
pcg3121
FALSE
3

ncgl0253
pcg3381
FALSE
3

ncgl0254
pcg0007_39
TRUE
3

ncgl0280
pcg1860
TRUE
3

ncgl0281
pcg0007_39
TRUE
3

ncgl0281
pcg3381
FALSE
3

ncgl0304
pcg0007_119
FALSE
4

ncgl0304
pcg0007_39
TRUE
4

ncgl0304
pcg0755
FALSE
4

ncgl0304
pcg1860
FALSE
4

ncgl0304
pcg2613
FALSE
4

ncgl0304
wt
FALSE
4

ncgl0306
pcg0007_39
TRUE
4

ncgl0306
pcg0755
FALSE
4

ncgl0306
pcg2613
FALSE
4

ncgl0306
pcg3381
FALSE
4

ncgl0355
pcg0007_39
TRUE
2

ncgl0355
pcg0755
FALSE
2

ncgl0355
pcg1860
FALSE
2

ncgl0355
pcg2613
FALSE
2

ncgl0355
pcg3121
FALSE
2

ncgl0355
pcg3381
FALSE
2

ncgl0355
wt
FALSE
2

ncgl0359
pcg0007_39
FALSE
2

ncgl0359
pcg1860
TRUE
2

ncgl0378
pcg0007_39
TRUE
3

ncgl0378
pcg3121
FALSE
3

ncgl0378
pcg3381
FALSE
3

ncgl0380
pcg0007_39
TRUE
3

ncgl0386
pcg0007_39
TRUE
3

ncgl0386
pcg1860
TRUE
3

ncgl0411
pcg0007_39
TRUE
3

ncgl0411
pcg1860
TRUE
3

ncgl0411
pcg3121
FALSE
3

ncgl0419
pcg0007_39
TRUE
3

ncgl0419
pcg1860
TRUE
3

ncgl0419
pcg3381
FALSE
3

ncgl0439
pcg0007_39
FALSE
3

ncgl0439
pcg1860
TRUE
3

ncgl0439
pcg3121
FALSE
3

ncgl0439
pcg3381
FALSE
3

ncgl0445
pcg0007_39
TRUE
3

ncgl0445
pcg3381
FALSE
3

ncgl0453
pcg0007_39
TRUE
3

ncgl0453
pcg3121
FALSE
3

ncgl0453
pcg3381
FALSE
3

ncgl0456
pcg3381
TRUE
4

ncgl0458
pcg0007_39
TRUE
3

ncgl0458
pcg0007_39
TRUE
3

ncgl0458
pcg0007_39
FALSE
3

ncgl0458
pcg3381
FALSE
3

ncgl0458
pcg3381
FALSE
3

ncgl0465
pcg0007_39
TRUE
3

ncgl0465
pcg3381
FALSE
3

ncgl0471
pcg0007_39
FALSE
3

ncgl0471
pcg0007_39
TRUE
3

ncgl0471
pcg0007_39
FALSE
3

ncgl0471
pcg1860
FALSE
3

ncgl0471
pcg1860
FALSE
3

ncgl0471
pcg1860
FALSE
3

ncgl0471
wt
FALSE
3

ncgl0472
pcg0007_39
TRUE
3

ncgl0482
pcg0007_39
TRUE
3

ncgl0482
pcg3381
FALSE
3

ncgl0509
pcg0007_39
TRUE
3

ncgl0509
pcg1860
TRUE
3

ncgl0509
pcg3121
FALSE
3

ncgl0509
pcg3381
FALSE
3

ncgl0527
pcg0007_39
TRUE
3

ncgl0527
pcg1860
TRUE
3

ncgl0527
pcg3121
FALSE
3

ncgl0527
pcg3381
FALSE
3

ncgl0531
pcg0007_39
TRUE
3

ncgl0531
pcg0007_39
TRUE
3

ncgl0531
pcg1860
TRUE
3

ncgl0531
pcg3121
FALSE
3

ncgl0531
pcg3381
FALSE
3

ncgl0531
pcg3381
FALSE
3

ncgl0546
pcg0007_39
TRUE
4

ncgl0548
pcg0007_39
TRUE
3

ncgl0548
pcg1860
TRUE
3

ncgl0548
pcg3121
FALSE
3

ncgl0548
pcg3381
FALSE
3

ncgl0565
pcg0007_39
FALSE
3

ncgl0565
pcg1860
TRUE
3

ncgl0565
pcg3121
FALSE
3

ncgl0565
pcg3381
FALSE
3

ncgl0578
pcg0007_39
TRUE
4

ncgl0578
pcg3381
FALSE
4

ncgl0581
pcg0007_39
TRUE
3

ncgl0581
pcg0007_39
TRUE
3

ncgl0581
pcg3121
FALSE
3

ncgl0581
pcg3121
FALSE
3

ncgl0581
pcg3381
FALSE
3

ncgl0581
pcg3381
FALSE
3

ncgl0598
pcg0007_39
TRUE
4

ncgl0598
pcg3381
FALSE
4

ncgl0601
pcg0007_119
FALSE
3

ncgl0601
pcg0007_39
TRUE
3

ncgl0601
pcg3121
FALSE
3

ncgl0601
pcg3381
FALSE
3

ncgl0601
wt
FALSE
3

ncgl0631
pcg0007_39
TRUE
2

ncgl0631
pcg1860
FALSE
2

ncgl0631
pcg3121
FALSE
2

ncgl0634
pcg0007
TRUE
1

ncgl0634
pcg0007
TRUE
2

ncgl0634
pcg0007_265
FALSE
1

ncgl0634
pcg0007_265
FALSE
2

ncgl0634
pcg0007_39
FALSE
1

ncgl0634
pcg0007_39
FALSE
2

ncgl0634
pcg0755
FALSE
1

ncgl0634
pcg0755
FALSE
2

ncgl0634
pcg1860
TRUE
1

ncgl0634
pcg1860
TRUE
2

ncgl0634
pcg3121
FALSE
1

ncgl0634
pcg3121
FALSE
2

ncgl0634
pcg3381
FALSE
1

ncgl0634
pcg3381
FALSE
2

ncgl0634
wt
FALSE
1

ncgl0634
wt
FALSE
2

ncgl0634
wt
FALSE
1

ncgl0634
wt
FALSE
2

ncgl0634
wt
FALSE
1

ncgl0634
wt
FALSE
2

ncgl0634
wt
FALSE
1

ncgl0634
wt
FALSE
2

ncgl0634
wt
FALSE
1

ncgl0634
wt
FALSE
2

ncgl0636
pcg0007_39
TRUE
3

ncgl0636
pcg3121
FALSE
3

ncgl0636
pcg3381
FALSE
3

ncgl0637
pcg0007_39
TRUE
3

ncgl0637
pcg1860
TRUE
3

ncgl0637
pcg3121
FALSE
3

ncgl0637
pcg3381
FALSE
3

ncgl0638
pcg0007_39
TRUE
3

ncgl0638
pcg0755
FALSE
3

ncgl0640
pcg1860
TRUE
3

ncgl0640
pcg3121
FALSE
3

ncgl0640
pcg3381
FALSE
3

ncgl0645
pcg0007_39
TRUE
3

ncgl0645
pcg3121
FALSE
3

ncgl0645
pcg3381
FALSE
3

ncgl0646
pcg0007_39
TRUE
3

ncgl0646
pcg3121
FALSE
3

ncgl0646
pcg3381
FALSE
3

ncgl0655
pcg0007_39
TRUE
3

ncgl0655
pcg1860
TRUE
3

ncgl0655
pcg1860
TRUE
3

ncgl0655
pcg3121
FALSE
3

ncgl0655
pcg3121
FALSE
3

ncgl0655
pcg3381
FALSE
3

ncgl0655
pcg3381
FALSE
3

ncgl0668
pcg0007_39
TRUE
3

ncgl0668
pcg0755
FALSE
3

ncgl0668
pcg2613
FALSE
3

ncgl0668
pcg3121
FALSE
3

ncgl0668
pcg3381
FALSE
3

ncgl0679
pcg0007_39
TRUE
3

ncgl0679
pcg1860
FALSE
3

ncgl0679
pcg3121
FALSE
3

ncgl0679
pcg3381
FALSE
3

ncgl0689
pcg0007_39
FALSE
3

ncgl0689
pcg0007_39
TRUE
3

ncgl0689
pcg3381
FALSE
3

ncgl0694
pcg0007_39
FALSE
3

ncgl0694
pcg1860
TRUE
3

ncgl0694
pcg3121
FALSE
3

ncgl0694
pcg3381
FALSE
3

ncgl0697
pcg0007_39
FALSE
3

ncgl0697
pcg0007_39
TRUE
3

ncgl0697
pcg3121
FALSE
3

ncgl0697
pcg3381
FALSE
3

ncgl0698
pcg0007_119
FALSE
3

ncgl0698
pcg0007_39
TRUE
3

ncgl0698
pcg0007_39
TRUE
3

ncgl0698
pcg1860
FALSE
3

ncgl0698
pcg3121
FALSE
3

ncgl0698
pcg3121
FALSE
3

ncgl0708
pcg0007_39
TRUE
3

ncgl0743
pcg0007_39
FALSE
4

ncgl0743
pcg3381
TRUE
4

ncgl0767
pcg0007_39
TRUE
4

ncgl0767
pcg2613
FALSE
4

ncgl0768
pcg0007_39
FALSE
3

ncgl0768
pcg0007_39
TRUE
3

ncgl0768
pcg3121
FALSE
3

ncgl0768
pcg3381
FALSE
3

ncgl0780
pcg0007
TRUE
1

ncgl0780
pcg0007_265
TRUE
1

ncgl0780
pcg0755
TRUE
1

ncgl0780
pcg3121
TRUE
1

ncgl0780
pcg3381
TRUE
1

ncgl0817
pcg0007
TRUE
1

ncgl0817
pcg0007_119
FALSE
1

ncgl0817
pcg0007_119
TRUE
1

ncgl0817
pcg0007_265
TRUE
1

ncgl0817
pcg0007_39
TRUE
1

ncgl0817
pcg0755
FALSE
1

ncgl0817
pcg0755
TRUE
1

ncgl0817
pcg1860
TRUE
1

ncgl0817
pcg2613
FALSE
1

ncgl0817
pcg3121
TRUE
1

ncgl0817
pcg3121
TRUE
1

ncgl0817
pcg3381
TRUE
1

ncgl0817
pcg3381
TRUE
1

ncgl0823
pcg0007_39
FALSE
3

ncgl0823
pcg0007_39
TRUE
3

ncgl0823
pcg3381
FALSE
3

ncgl0827
pcg0007_39
TRUE
4

ncgl0827
pcg3381
FALSE
4

ncgl0847
pcg0007_39
TRUE
2

ncgl0847
pcg0755
FALSE
2

ncgl0847
pcg2613
FALSE
2

ncgl0847
pcg3381
FALSE
2

ncgl0860
pcg0007_39
TRUE
4

ncgl0861
pcg0007_39
TRUE
3

ncgl0861
pcg1860
TRUE
3

ncgl0861
pcg3381
FALSE
3

ncgl0865
pcg0007_39
FALSE
1

ncgl0865
pcg0755
FALSE
1

ncgl0865
pcg3121
TRUE
1

ncgl0865
pcg3381
FALSE
1

ncgl0877
pcg0007_119
FALSE
4

ncgl0877
pcg0007_39
TRUE
4

ncgl0877
pcg3381
FALSE
4

ncgl0877
wt
FALSE
4

ncgl0893
pcg0007_39
TRUE
3

ncgl0893
pcg3121
FALSE
3

ncgl0893
pcg3381
FALSE
3

ncgl0897
pcg0007_39
TRUE
3

ncgl0897
pcg3381
FALSE
3

ncgl0901
pcg0007_39
TRUE
3

ncgl0901
pcg1860
TRUE
3

ncgl0901
pcg3121
FALSE
3

ncgl0901
pcg3381
FALSE
3

ncgl0909
pcg0007_39
FALSE
3

ncgl0909
pcg3121
TRUE
3

ncgl0909
pcg3381
FALSE
3

ncgl0965
pcg1860
TRUE
3

ncgl0966
pcg1860
FALSE
4

ncgl0966
pcg3121
TRUE
4

ncgl0966
wt
FALSE
4

ncgl0976
pcg0007
FALSE
1

ncgl0976
pcg0007_119
FALSE
1

ncgl0976
pcg0007_39
TRUE
1

ncgl0976
pcg0755
FALSE
1

ncgl0976
pcg1860
FALSE
1

ncgl0976
pcg3121
FALSE
1

ncgl0976
pcg3381
FALSE
1

ncgl1016
pcg0007_39
TRUE
3

ncgl1016
pcg1860
TRUE
3

ncgl1016
pcg3121
FALSE
3

ncgl1016
pcg3381
FALSE
3

ncgl1025
pcg0007_39
TRUE
3

ncgl1025
pcg1860
TRUE
3

ncgl1025
pcg3121
FALSE
3

ncgl1044
pcg0007_39
TRUE
4

ncgl1049
pcg0007_39
TRUE
3

ncgl1049
pcg3381
FALSE
3

ncgl1062
pcg0007_39
TRUE
3

ncgl1062
pcg2613
FALSE
3

ncgl1064
pcg0007
FALSE
1

ncgl1064
pcg0007_119
FALSE
1

ncgl1064
pcg0007_39
FALSE
1

ncgl1064
pcg0755
TRUE
1

ncgl1064
pcg1860
FALSE
1

ncgl1064
pcg2613
FALSE
1

ncgl1064
pcg3381
FALSE
1

ncgl1064
wt
FALSE
1

ncgl1065
pcg0007_39
TRUE
4

ncgl1080
pcg0007_39
TRUE
3

ncgl1080
pcg3121
FALSE
3

ncgl1080
pcg3381
FALSE
3

ncgl1084
pcg0007_119
FALSE
2

ncgl1084
pcg0007_39
TRUE
2

ncgl1084
wt
FALSE
2

ncgl1129
pcg0007_39
TRUE
3

ncgl1129
pcg3121
FALSE
3

ncgl1129
pcg3381
FALSE
3

ncgl1133
pcg0007
TRUE
1

ncgl1133
pcg0007_119
FALSE
1

ncgl1133
pcg0007_265
TRUE
1

ncgl1133
pcg0007_39
TRUE
1

ncgl1133
pcg0755
FALSE
1

ncgl1133
pcg3121
FALSE
1

ncgl1133
pcg3381
TRUE
1

ncgl1136
pcg0007
FALSE
1

ncgl1136
pcg0007_119
FALSE
1

ncgl1136
pcg0007_39
TRUE
1

ncgl1136
pcg0755
TRUE
1

ncgl1136
pcg1860
TRUE
1

ncgl1136
pcg2613
FALSE
1

ncgl1136
pcg3121
TRUE
1

ncgl1136
pcg3381
TRUE
1

ncgl1136
wt
FALSE
1

ncgl1140
pcg0007_39
TRUE
2

ncgl1140
pcg0755
FALSE
2

ncgl1140
pcg2613
FALSE
2

ncgl1140
pcg3121
FALSE
2

ncgl1140
wt
FALSE
2

ncgl1143
pcg0007_39
TRUE
2

ncgl1143
pcg0007_39
TRUE
3

ncgl1143
pcg3121
FALSE
2

ncgl1143
pcg3121
FALSE
3

ncgl1143
pcg3381
FALSE
2

ncgl1143
pcg3381
FALSE
3

ncgl1147
pcg0007_39
TRUE
3

ncgl1147
pcg3121
FALSE
3

ncgl1147
pcg3381
FALSE
3

ncgl1152
pcg0007_39
TRUE
2

ncgl1152
pcg0007_39
TRUE
3

ncgl1152
pcg0755
FALSE
2

ncgl1152
pcg0755
FALSE
3

ncgl1152
pcg1860
FALSE
2

ncgl1152
pcg1860
FALSE
3

ncgl1152
pcg3381
FALSE
2

ncgl1152
pcg3381
FALSE
3

ncgl1152
wt
FALSE
2

ncgl1152
wt
FALSE
3

ncgl1175
pcg0007_39
TRUE
3

ncgl1175
pcg1860
TRUE
3

ncgl1175
pcg3381
FALSE
3

ncgl1179
pcg0007_39
TRUE
3

ncgl1179
pcg0755
FALSE
3

ncgl1179
pcg1860
FALSE
3

ncgl1179
pcg2613
FALSE
3

ncgl1179
pcg3121
FALSE
3

ncgl1179
pcg3381
FALSE
3

ncgl1179
wt
FALSE
3

ncgl1181
pcg0007_39
TRUE
3

ncgl1181
pcg3121
FALSE
3

ncgl1181
pcg3381
FALSE
3

ncgl1202
pcg0007
FALSE
4

ncgl1202
pcg0007_265
FALSE
4

ncgl1202
pcg0007_39
FALSE
4

ncgl1202
pcg1860
FALSE
4

ncgl1202
pcg3381
TRUE
4

ncgl1203
pcg0007_39
TRUE
3

ncgl1203
pcg3381
FALSE
3

ncgl1208
pcg0007_39
TRUE
4

ncgl1209
pcg0007_39
TRUE
3

ncgl1209
pcg1860
TRUE
3

ncgl1209
pcg3121
FALSE
3

ncgl1209
pcg3121
FALSE
3

ncgl1209
pcg3381
FALSE
3

ncgl1209
pcg3381
FALSE
3

ncgl1214
pcg0007
TRUE
1

ncgl1214
pcg0007_119
TRUE
1

ncgl1214
pcg0007_265
FALSE
1

ncgl1214
pcg0007_39
TRUE
1

ncgl1214
pcg0755
FALSE
1

ncgl1214
pcg1860
FALSE
1

ncgl1214
pcg3121
FALSE
1

ncgl1214
pcg3381
FALSE
1

ncgl1224
pcg0007_39
TRUE
4

ncgl1241
pcg0007
TRUE
1

ncgl1241
pcg0007_119
FALSE
1

ncgl1241
pcg0007_39
TRUE
1

ncgl1241
pcg0755
FALSE
1

ncgl1241
pcg1860
FALSE
1

ncgl1241
pcg3121
FALSE
1

ncgl1241
pcg3381
FALSE
1

ncgl1261
pcg0007_119
FALSE
3

ncgl1261
pcg0007_39
TRUE
3

ncgl1261
pcg0755
FALSE
3

ncgl1261
pcg2613
FALSE
3

ncgl1261
pcg3381
FALSE
3

ncgl1261
wt
FALSE
3

ncgl1262
pcg0007_39
TRUE
4

ncgl1262
pcg0755
TRUE
4

ncgl1262
pcg1860
TRUE
4

ncgl1262
pcg2613
TRUE
4

ncgl1262
pcg3121
FALSE
4

ncgl1262
pcg3381
FALSE
4

ncgl1262
wt
FALSE
4

ncgl1263
pcg0007_39
TRUE
4

ncgl1263
pcg3381
FALSE
4

ncgl1267
pcg0007_39
TRUE
3

ncgl1267
pcg0755
FALSE
3

ncgl1267
pcg1860
FALSE
3

ncgl1267
pcg2613
FALSE
3

ncgl1267
pcg3381
FALSE
3

ncgl1267
wt
FALSE
3

ncgl1267
wt
FALSE
3

ncgl1267
wt
FALSE
3

ncgl1267
wt
FALSE
3

ncgl1267
wt
FALSE
3

ncgl1267
wt
FALSE
3

ncgl1267
wt
FALSE
3

ncgl1277
pcg0007_39
TRUE
3

ncgl1277
pcg0755
FALSE
3

ncgl1277
pcg2613
FALSE
3

ncgl1277
pcg3121
FALSE
3

ncgl1277
pcg3381
FALSE
3

ncgl1277
wt
FALSE
3

ncgl1301
pcg0007_119
FALSE
3

ncgl1301
pcg0007_39
TRUE
3

ncgl1301
pcg1860
FALSE
3

ncgl1301
pcg2613
FALSE
3

ncgl1301
pcg3121
FALSE
3

ncgl1301
pcg3381
FALSE
3

ncgl1305
pcg0007
FALSE
1

ncgl1305
pcg0007
FALSE
3

ncgl1305
pcg0007
FALSE
1

ncgl1305
pcg0007
FALSE
3

ncgl1305
pcg0007_265
TRUE
1

ncgl1305
pcg0007_265
TRUE
3

ncgl1305
pcg0007_39
FALSE
1

ncgl1305
pcg0007_39
FALSE
3

ncgl1305
pcg0007_39
TRUE
1

ncgl1305
pcg0007_39
TRUE
3

ncgl1305
pcg0755
TRUE
1

ncgl1305
pcg0755
TRUE
3

ncgl1305
pcg0755
FALSE
1

ncgl1305
pcg0755
FALSE
3

ncgl1305
pcg1860
TRUE
1

ncgl1305
pcg1860
TRUE
3

ncgl1305
pcg1860
TRUE
1

ncgl1305
pcg1860
TRUE
3

ncgl1305
pcg3121
FALSE
1

ncgl1305
pcg3121
FALSE
3

ncgl1305
pcg3121
TRUE
1

ncgl1305
pcg3121
TRUE
3

ncgl1305
pcg3381
FALSE
1

ncgl1305
pcg3381
FALSE
3

ncgl1305
pcg3381
FALSE
1

ncgl1305
pcg3381
FALSE
3

ncgl1322
pcg0007_39
TRUE
4

ncgl1322
pcg0755
FALSE
4

ncgl1322
pcg1860
FALSE
4

ncgl1322
pcg2613
FALSE
4

ncgl1322
pcg3121
FALSE
4

ncgl1322
pcg3381
FALSE
4

ncgl1322
wt
FALSE
4

ncgl1330
pcg0007_39
TRUE
3

ncgl1330
pcg3381
FALSE
3

ncgl1332
pcg0007_119
FALSE
3

ncgl1332
pcg0007_39
TRUE
3

ncgl1332
pcg0755
FALSE
3

ncgl1332
pcg1860
TRUE
3

ncgl1332
pcg3121
FALSE
3

ncgl1332
pcg3381
FALSE
3

ncgl1332
wt
FALSE
3

ncgl1344
pcg0007_39
TRUE
4

ncgl1344
pcg3381
FALSE
4

ncgl1347
pcg0007_39
TRUE
4

ncgl1362
pcg0007_39
FALSE
3

ncgl1362
pcg1860
TRUE
3

ncgl1362
pcg3121
FALSE
3

ncgl1362
pcg3381
FALSE
3

ncgl1363
pcg0007_39
TRUE
3

ncgl1363
pcg3121
FALSE
3

ncgl1364
pcg0007_119
FALSE
3

ncgl1364
pcg0007_39
TRUE
3

ncgl1364
pcg0755
FALSE
3

ncgl1364
pcg1860
TRUE
3

ncgl1364
pcg2613
FALSE
3

ncgl1364
pcg3121
FALSE
3

ncgl1364
pcg3381
FALSE
3

ncgl1364
wt
FALSE
3

ncgl1366
pcg0007_39
FALSE
3

ncgl1366
pcg0007_39
TRUE
3

ncgl1366
pcg0007_39
TRUE
3

ncgl1366
pcg1860
TRUE
3

ncgl1366
pcg3121
FALSE
3

ncgl1366
pcg3121
FALSE
3

ncgl1366
pcg3381
FALSE
3

ncgl1366
pcg3381
FALSE
3

ncgl1367
pcg0007_39
TRUE
3

ncgl1367
pcg3121
FALSE
3

ncgl1367
pcg3381
FALSE
3

ncgl1370
pcg0007_39
FALSE
3

ncgl1370
pcg1860
TRUE
3

ncgl1370
pcg3121
FALSE
3

ncgl1370
pcg3381
FALSE
3

ncgl1371
pcg0007_119
FALSE
3

ncgl1371
pcg0007_39
TRUE
3

ncgl1371
pcg0755
FALSE
3

ncgl1371
pcg1860
FALSE
3

ncgl1371
pcg2613
FALSE
3

ncgl1371
wt
FALSE
3

ncgl1372
pcg0007_39
TRUE
3

ncgl1372
pcg1860
TRUE
3

ncgl1372
pcg3121
FALSE
3

ncgl1372
pcg3381
FALSE
3

ncgl1378
pcg0007_39
TRUE
3

ncgl1378
pcg3121
FALSE
3

ncgl1399
pcg0007_39
TRUE
3

ncgl1399
pcg3121
FALSE
3

ncgl1399
pcg3381
FALSE
3

ncgl1402
pcg0007_39
FALSE
3

ncgl1402
pcg1860
TRUE
3

ncgl1454
pcg0007_39
TRUE
3

ncgl1454
pcg3121
FALSE
3

ncgl1454
pcg3381
FALSE
3

ncgl1455
pcg0007_39
TRUE
3

ncgl1455
pcg3121
FALSE
3

ncgl1455
pcg3381
FALSE
3

ncgl1484
pcg0007_119
FALSE
4

ncgl1484
pcg0007_39
TRUE
4

ncgl1484
pcg0755
FALSE
4

ncgl1484
pcg2613
FALSE
4

ncgl1484
pcg3121
FALSE
4

ncgl1484
wt
FALSE
4

ncgl1506
pcg0007_39
TRUE
3

ncgl1506
pcg1860
TRUE
3

ncgl1506
pcg3121
FALSE
3

ncgl1506
pcg3381
FALSE
3

ncgl1507
pcg1860
TRUE
3

ncgl1507
pcg3121
FALSE
3

ncgl1507
pcg3381
FALSE
3

ncgl1511
pcg0007_119
FALSE
4

ncgl1511
pcg0007_39
TRUE
4

ncgl1511
pcg0755
FALSE
4

ncgl1511
pcg2613
FALSE
4

ncgl1511
pcg3121
FALSE
4

ncgl1511
pcg3381
FALSE
4

ncgl1511
wt
FALSE
4

ncgl1512
pcg0007
FALSE
1

ncgl1512
pcg0007_39
FALSE
1

ncgl1512
pcg0007_39
TRUE
1

ncgl1512
pcg0755
FALSE
1

ncgl1512
pcg0755
FALSE
1

ncgl1512
pcg1860
FALSE
1

ncgl1512
pcg1860
TRUE
1

ncgl1512
pcg3381
FALSE
1

ncgl1512
pcg3381
TRUE
1

ncgl1514
pcg0007
TRUE
1

ncgl1514
pcg0007_119
TRUE
1

ncgl1514
pcg0007_265
FALSE
1

ncgl1514
pcg0007_39
TRUE
1

ncgl1514
pcg0755
FALSE
1

ncgl1514
pcg1860
FALSE
1

ncgl1514
pcg3121
FALSE
1

ncgl1514
pcg3381
TRUE
1

ncgl1521
pcg0007_39
TRUE
2

ncgl1521
pcg0007_39
TRUE
3

ncgl1521
pcg1860
FALSE
2

ncgl1521
pcg1860
FALSE
3

ncgl1521
pcg3121
FALSE
2

ncgl1521
pcg3121
FALSE
3

ncgl1523
pcg0007
FALSE
1

ncgl1523
pcg0007_119
FALSE
1

ncgl1523
pcg0007_39
TRUE
1

ncgl1523
pcg0755
TRUE
1

ncgl1523
pcg1860
FALSE
1

ncgl1523
pcg3121
TRUE
1

ncgl1525
pcg0007_39
FALSE
4

ncgl1525
pcg3381
TRUE
4

ncgl1545
pcg0007_39
TRUE
4

ncgl1545
wt
FALSE
4

ncgl1590
pcg3381
FALSE
4

ncgl1590
wt
FALSE
4

ncgl1590
wt
FALSE
4

ncgl1590
wt
FALSE
4

ncgl1590
wt
TRUE
4

ncgl1592
pcg0007_39
TRUE
3

ncgl1592
pcg3121
FALSE
3

ncgl1592
pcg3381
FALSE
3

ncgl1607
pcg0007_39
TRUE
4

ncgl1607
pcg0755
FALSE
4

ncgl1607
pcg2613
FALSE
4

ncgl1607
wt
FALSE
4

ncgl1817
pcg0007_39
TRUE
3

ncgl1817
pcg1860
TRUE
3

ncgl1817
pcg3121
FALSE
3

ncgl1817
pcg3381
FALSE
3

ncgl1844
pcg0007_39
TRUE
3

ncgl1844
pcg1860
TRUE
3

ncgl1868
pcg0007
TRUE
1

ncgl1868
pcg0007_119
TRUE
1

ncgl1868
pcg0007_39
TRUE
1

ncgl1868
pcg0755
TRUE
1

ncgl1868
pcg1860
TRUE
1

ncgl1868
pcg3121
FALSE
1

ncgl1868
pcg3381
FALSE
1

ncgl1875
pcg0007_39
TRUE
3

ncgl1875
pcg1860
TRUE
3

ncgl1875
pcg3121
FALSE
3

ncgl1875
pcg3381
FALSE
3

ncgl1875
pcg3381
FALSE
3

ncgl1877
pcg0007_39
TRUE
3

ncgl1877
pcg3121
FALSE
3

ncgl1885
pcg0007_39
TRUE
3

ncgl1885
pcg1860
TRUE
3

ncgl1896
pcg0007
FALSE
1

ncgl1896
pcg0007
FALSE
1

ncgl1896
pcg0007_265
FALSE
1

ncgl1896
pcg0007_265
FALSE
1

ncgl1896
pcg0007_39
TRUE
1

ncgl1896
pcg0007_39
FALSE
1

ncgl1896
pcg0755
FALSE
1

ncgl1896
pcg1860
FALSE
1

ncgl1896
pcg3121
FALSE
1

ncgl1896
pcg3381
FALSE
1

ncgl1896
pcg3381
FALSE
1

ncgl1896
wt
FALSE
1

ncgl1898
pcg0007_265
TRUE
1

ncgl1898
pcg0755
FALSE
1

ncgl1898
pcg1860
FALSE
1

ncgl1899
pcg0007_39
TRUE
3

ncgl1899
pcg3381
FALSE
3

ncgl1911
pcg0007_119
FALSE
3

ncgl1911
pcg0007_39
TRUE
3

ncgl1911
pcg0755
FALSE
3

ncgl1911
pcg1860
TRUE
3

ncgl1911
pcg2613
FALSE
3

ncgl1911
pcg3121
FALSE
3

ncgl1911
pcg3121
FALSE
3

ncgl1911
pcg3381
FALSE
3

ncgl1911
pcg3381
FALSE
3

ncgl1912
pcg0007_39
TRUE
3

ncgl1913
pcg0007_39
TRUE
3

ncgl1913
pcg3121
FALSE
3

ncgl1913
pcg3381
FALSE
3

ncgl1915
pcg0007_39
FALSE
3

ncgl1915
pcg0007_39
TRUE
3

ncgl1915
pcg3121
FALSE
3

ncgl1917
pcg0007_39
TRUE
3

ncgl1917
pcg3381
FALSE
3

ncgl1918
pcg1860
TRUE
3

ncgl1918
pcg3121
FALSE
3

ncgl1926
pcg0007_39
FALSE
2

ncgl1926
pcg1860
FALSE
2

ncgl1926
pcg3121
FALSE
2

ncgl1926
pcg3381
TRUE
2

ncgl1948
pcg0007_39
TRUE
4

ncgl1948
wt
FALSE
4

ncgl1978
pcg0007_39
TRUE
3

ncgl1978
pcg3121
FALSE
3

ncgl1997
pcg0007_119
FALSE
3

ncgl1997
pcg0007_39
TRUE
3

ncgl1997
pcg0755
FALSE
3

ncgl1997
pcg1860
FALSE
3

ncgl1997
pcg2613
TRUE
3

ncgl1997
pcg3121
FALSE
3

ncgl1997
pcg3381
FALSE
3

ncgl1997
wt
FALSE
3

ncgl2017
pcg0007_39
TRUE
3

ncgl2017
pcg3381
FALSE
3

ncgl2025
pcg0007_39
TRUE
3

ncgl2025
pcg1860
TRUE
3

ncgl2031
pcg0007_39
TRUE
3

ncgl2031
pcg3121
FALSE
3

ncgl2031
pcg3381
FALSE
3

ncgl2032
pcg0007_39
TRUE
3

ncgl2032
pcg3121
FALSE
3

ncgl2032
pcg3381
FALSE
3

ncgl2060
pcg0007_39
TRUE
3

ncgl2060
pcg1860
TRUE
3

ncgl2060
pcg3121
FALSE
3

ncgl2066
pcg0007_39
TRUE
3

ncgl2066
pcg1860
TRUE
3

ncgl2066
pcg3121
FALSE
3

ncgl2066
pcg3381
FALSE
3

ncgl2081
pcg0007_39
TRUE
3

ncgl2082
pcg0007_39
TRUE
3

ncgl2082
pcg3381
FALSE
3

ncgl2083
pcg0007_39
TRUE
3

ncgl2083
pcg0755
FALSE
3

ncgl2083
wt
FALSE
3

ncgl2084
pcg0007_39
TRUE
3

ncgl2084
pcg3381
FALSE
3

ncgl2091
pcg0007_39
TRUE
4

ncgl2091
pcg3381
FALSE
4

ncgl2104
pcg0007_39
TRUE
3

ncgl2104
pcg0007_39
TRUE
3

ncgl2104
pcg3121
FALSE
3

ncgl2104
pcg3121
FALSE
3

ncgl2104
pcg3381
FALSE
3

ncgl2104
wt
FALSE
3

ncgl2126
pcg0007
FALSE
2

ncgl2126
pcg0007_39
FALSE
2

ncgl2126
pcg1860
TRUE
2

ncgl2126
pcg3121
FALSE
2

ncgl2133
pcg0007_39
TRUE
2

ncgl2133
pcg0007_39
TRUE
3

ncgl2133
pcg1860
FALSE
2

ncgl2133
pcg1860
FALSE
3

ncgl2133
wt
FALSE
2

ncgl2133
wt
FALSE
3

ncgl2163
pcg0007_39
TRUE
3

ncgl2163
pcg1860
TRUE
3

ncgl2167
pcg0007_39
FALSE
2

ncgl2167
pcg0007_39
FALSE
2

ncgl2167
pcg0755
FALSE
2

ncgl2167
pcg1860
FALSE
2

ncgl2167
pcg1860
TRUE
2

ncgl2167
pcg2613
FALSE
2

ncgl2167
pcg3121
FALSE
2

ncgl2167
pcg3381
FALSE
2

ncgl2167
pcg3381
TRUE
2

ncgl2168
pcg0007_39
TRUE
3

ncgl2169
pcg0007_39
TRUE
3

ncgl2169
pcg0755_promoter
FALSE
3

ncgl2169
pcg1860
TRUE
3

ncgl2169
pcg3121
FALSE
3

ncgl2169
pcg3381
FALSE
3

ncgl2211
pcg0007_39
TRUE
3

ncgl2211
pcg3381
FALSE
3

ncgl2230
pcg0007_39
FALSE
3

ncgl2230
pcg0007_39
TRUE
3

ncgl2230
pcg0007_39
TRUE
3

ncgl2230
pcg3121
FALSE
3

ncgl2230
pcg3381
FALSE
3

ncgl2239
pcg0007_39
TRUE
3

ncgl2239
pcg3121
FALSE
3

ncgl2239
pcg3381
FALSE
3

ncgl2240
pcg0007_119
FALSE
3

ncgl2240
pcg0007_39
TRUE
3

ncgl2240
pcg0755
FALSE
3

ncgl2240
pcg1860
TRUE
3

ncgl2240
pcg2613
FALSE
3

ncgl2240
pcg3121
FALSE
3

ncgl2240
pcg3381
FALSE
3

ncgl2240
wt
FALSE
3

ncgl2241
pcg0007_39
FALSE
3

ncgl2241
pcg1860
TRUE
3

ncgl2241
pcg3121
FALSE
3

ncgl2241
pcg3381
FALSE
3

ncgl2245
pcg0007_39
TRUE
3

ncgl2245
pcg3121
FALSE
3

ncgl2245
pcg3381
FALSE
3

ncgl2250
pcg0007_119
FALSE
4

ncgl2250
pcg0007_39
TRUE
4

ncgl2250
pcg0755
FALSE
4

ncgl2250
pcg2613
FALSE
4

ncgl2250
pcg3121
FALSE
4

ncgl2250
pcg3381
FALSE
4

ncgl2257
pcg0007_39
TRUE
3

ncgl2257
pcg3121
FALSE
3

ncgl2257
pcg3381
FALSE
3

ncgl2298
pcg0007_39
TRUE
3

ncgl2298
pcg0007_39
TRUE
3

ncgl2298
pcg1860
FALSE
3

ncgl2298
pcg3381
FALSE
3

ncgl2327
pcg0007_39
TRUE
4

ncgl2327
pcg2613
FALSE
4

ncgl2327
pcg3381
FALSE
4

ncgl2327
wt
FALSE
4

ncgl2350
pcg0007_39
TRUE
3

ncgl2350
pcg3121
FALSE
3

ncgl2350
pcg3381
FALSE
3

ncgl2373
pcg0007_39
TRUE
3

ncgl2374
pcg0007_39
TRUE
3

ncgl2377
pcg0007_39
TRUE
3

ncgl2377
pcg3121
FALSE
3

ncgl2385
pcg0007_39
TRUE
3

ncgl2385
pcg3381
FALSE
3

ncgl2405
pcg0007_39
FALSE
3

ncgl2405
pcg1860
TRUE
3

ncgl2406
pcg0007_39
TRUE
3

ncgl2406
pcg3121
FALSE
3

ncgl2406
pcg3381
FALSE
3

ncgl2425
pcg0007_119
FALSE
3

ncgl2425
pcg0007_39
TRUE
3

ncgl2425
pcg3121
FALSE
3

ncgl2425
pcg3381
FALSE
3

ncgl2425
wt
FALSE
3

ncgl2440
pcg0007_39
FALSE
3

ncgl2440
pcg1860
FALSE
3

ncgl2440
pcg3121
TRUE
3

ncgl2440
pcg3381
FALSE
3

ncgl2449
pcg0007_39
TRUE
4

ncgl2449
wt
FALSE
4

ncgl2465
pcg0007_39
FALSE
3

ncgl2465
pcg0007_39
TRUE
3

ncgl2465
pcg3381
FALSE
3

ncgl2481
pcg0007_39
TRUE
4

ncgl2481
wt
FALSE
4

ncgl2482
pcg0007_39
TRUE
3

ncgl2482
pcg0007_39
FALSE
3

ncgl2482
pcg0755
FALSE
3

ncgl2482
pcg2613
FALSE
3

ncgl2482
pcg3121
FALSE
3

ncgl2482
pcg3381
TRUE
3

ncgl2482
pcg3381
FALSE
3

ncgl2482
wt
FALSE
3

ncgl2483
pcg0007_39
TRUE
3

ncgl2483
pcg1860
TRUE
3

ncgl2483
pcg3381
FALSE
3

ncgl2485
pcg0007_39
TRUE
3

ncgl2485
pcg3381
FALSE
3

ncgl2491
pcg0007_119
FALSE
4

ncgl2491
pcg0007_39
TRUE
4

ncgl2491
pcg2613
FALSE
4

ncgl2491
pcg3381
FALSE
4

ncgl2491
wt
FALSE
4

ncgl2499
pcg0007_39
TRUE
4

ncgl2500
pcg0007_39
TRUE
4

ncgl2500
pcg3381
FALSE
4

ncgl2509
pcg0007_39
TRUE
4

ncgl2509
pcg2613
FALSE
4

ncgl2509
pcg3121
FALSE
4

ncgl2509
pcg3381
FALSE
4

ncgl2509
wt
FALSE
4

ncgl2514
pcg0007_39
TRUE
3

ncgl2514
pcg3121
FALSE
3

ncgl2514
pcg3381
FALSE
3

ncgl2516
pcg0007_39
TRUE
3

ncgl2516
pcg3381
FALSE
3

ncgl2527
pcg0007_39
TRUE
3

ncgl2527
pcg0007_39
TRUE
3

ncgl2527
pcg2613
FALSE
3

ncgl2527
pcg3381
FALSE
3

ncgl2527
pcg3381
FALSE
3

ncgl2527
wt
FALSE
3

ncgl2541
pcg0007_119
FALSE
3

ncgl2541
pcg0007_39
TRUE
3

ncgl2541
pcg1860
FALSE
3

ncgl2541
pcg2613
FALSE
3

ncgl2541
pcg3121
FALSE
3

ncgl2541
pcg3381
FALSE
3

ncgl2541
wt
FALSE
3

ncgl2553
pcg0007_39
FALSE
3

ncgl2553
pcg0007_39
TRUE
3

ncgl2553
pcg3121
FALSE
3

ncgl2572
pcg1860
TRUE
3

ncgl2572
pcg3121
FALSE
3

ncgl2587
pcg0007_119
FALSE
3

ncgl2587
pcg0007_39
TRUE
3

ncgl2587
pcg0755
FALSE
3

ncgl2587
pcg1860
TRUE
3

ncgl2587
pcg2613
FALSE
3

ncgl2587
pcg3121
FALSE
3

ncgl2587
pcg3381
FALSE
3

ncgl2587
wt
FALSE
3

ncgl2599
pcg0007_39
FALSE
4

ncgl2599
pcg3381
TRUE
4

ncgl2614
pcg0007_39
TRUE
3

ncgl2614
pcg3121
FALSE
3

ncgl2614
pcg3381
FALSE
3

ncgl2650
pcg0007_39
TRUE
3

ncgl2650
pcg3121
FALSE
3

ncgl2650
pcg3381
FALSE
3

ncgl2684
pcg0007_39
FALSE
3

ncgl2684
pcg0007_39
TRUE
3

ncgl2684
pcg1860
FALSE
3

ncgl2684
pcg3121
FALSE
3

ncgl2684
pcg3381
FALSE
3

ncgl2699
pcg0007_39
TRUE
3

ncgl2699
pcg1860
FALSE
3

ncgl2699
pcg3121
FALSE
3

ncgl2699
pcg3381
FALSE
3

ncgl2713
pcg0007_39
TRUE
3

ncgl2713
pcg3381
FALSE
3

ncgl2717
pcg0007_39
TRUE
4

ncgl2724
pcg0007_39
TRUE
3

ncgl2724
pcg3121
FALSE
3

ncgl2724
pcg3381
FALSE
3

ncgl2725
pcg0007_39
TRUE
3

ncgl2725
pcg3381
FALSE
3

ncgl2726
pcg0007_39
TRUE
3

ncgl2733
pcg0007_39
TRUE
3

ncgl2733
pcg1860
TRUE
3

ncgl2765
pcg0007
FALSE
1

ncgl2765
pcg0007
FALSE
2

ncgl2765
pcg0007_119
FALSE
1

ncgl2765
pcg0007_119
FALSE
2

ncgl2765
pcg0007_39
FALSE
1

ncgl2765
pcg0007_39
FALSE
2

ncgl2765
pcg0755
TRUE
1

ncgl2765
pcg0755
TRUE
2

ncgl2765
pcg1860
FALSE
1

ncgl2765
pcg1860
FALSE
2

ncgl2765
pcg3121
FALSE
1

ncgl2765
pcg3121
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2765
wt
FALSE
1

ncgl2765
wt
FALSE
2

ncgl2772
pcg0007_119
FALSE
3

ncgl2772
pcg0007_39
TRUE
3

ncgl2772
pcg0755
FALSE
3

ncgl2772
pcg2613
FALSE
3

ncgl2772
pcg3121
TRUE
3

ncgl2772
pcg3381
FALSE
3

ncgl2774
pcg0007_39
FALSE
3

ncgl2774
pcg0007_39
TRUE
3

ncgl2792
pcg0007_39
TRUE
3

ncgl2792
pcg3121
FALSE
3

ncgl2792
pcg3381
FALSE
3

ncgl2802
pcg0007_39
TRUE
3

ncgl2802
pcg0007_39
TRUE
3

ncgl2802
pcg0755
FALSE
3

ncgl2802
pcg1860
TRUE
3

ncgl2802
pcg3381
FALSE
3

ncgl2802
pcg3381
FALSE
3

ncgl2802
wt
FALSE
3

ncgl2816
pcg0007_39
TRUE
1

ncgl2816
pcg0007_39
TRUE
3

ncgl2828
pcg0007_39
TRUE
3

ncgl2828
pcg3121
FALSE
3

ncgl2828
pcg3381
FALSE
3

ncgl2846
pcg1860
TRUE
3

ncgl2846
pcg3121
FALSE
3

ncgl2871
pcg0007_119
FALSE
3

ncgl2871
pcg0007_39
TRUE
3

ncgl2871
pcg0755
FALSE
3

ncgl2871
pcg2613
FALSE
3

ncgl2871
pcg3121
FALSE
3

ncgl2871
pcg3381
FALSE
3

ncgl2877
pcg0007_39
TRUE
3

ncgl2877
pcg3121
FALSE
3

ncgl2877
pcg3381
FALSE
3

ncgl2884
pcg0007_39
TRUE
3

ncgl2884
pcg3121
FALSE
3

ncgl2884
pcg3381
FALSE
3

ncgl2892
pcg0007_39
TRUE
3

ncgl2892
pcg3381
FALSE
3

ncgl2898
pcg0007_39
FALSE
2

ncgl2898
pcg0007_39
FALSE
2

ncgl2898
pcg1860
FALSE
2

ncgl2898
pcg3121
TRUE
2

ncgl2898
pcg3381
FALSE
2

ncgl2901
pcg0007_119
FALSE
4

ncgl2901
pcg0007_39
TRUE
4

ncgl2901
pcg0755
FALSE
4

ncgl2901
pcg1860
FALSE
4

ncgl2901
pcg3121
FALSE
4

ncgl2901
pcg3381
FALSE
4

ncgl2901
wt
FALSE
4

ncgl2908
pcg0007_39
TRUE
2

ncgl2908
pcg1860
FALSE
2

ncgl2908
pcg3121
FALSE
2

ncgl2921
pcg0007_119
FALSE
3

ncgl2921
pcg0007_39
TRUE
3

ncgl2921
pcg3121
FALSE
3

ncgl2921
pcg3381
FALSE
3

ncgl2921
wt
FALSE
3

ncgl2931
pcg0007_119
FALSE
4

ncgl2931
pcg0007_39
TRUE
4

ncgl2931
pcg1860
FALSE
4

ncgl2931
pcg2613
FALSE
4

ncgl2931
pcg3121
FALSE
4

ncgl2931
pcg3381
FALSE
4

ncgl2931
wt
FALSE
4

ncgl2941
pcg0007_39
FALSE
3

ncgl2941
pcg1860
TRUE
3

ncgl2941
pcg3121
FALSE
3

ncgl2941
pcg3381
FALSE
3

ncgl2950
pcg0007_39
TRUE
3

ncgl2950
pcg1860
FALSE
3

ncgl2950
pcg3121
FALSE
3

ncgl2953
pcg0007_39
TRUE
3

ncgl2953
pcg3381
FALSE
3

ncgl2961
pcg0007_39
TRUE
3

ncgl2961
pcg0755
FALSE
3

ncgl2961
pcg3121
FALSE
3

ncgl2961
pcg3121
FALSE
3

ncgl2961
pcg3381
FALSE
3

ncgl2961
pcg3381
FALSE
3

ncgl2961
pcg3381
FALSE
3

ncgl2977
pcg0007_39
TRUE
3

ncgl2977
pcg3121
FALSE
3

ncgl2977
pcg3381
FALSE
3

ncgl2982
pcg0007_39
TRUE
3

ncgl2986
pcg0007_39
TRUE
3

ncgl2986
pcg1860
TRUE
3

ncgl2986
pcg3381
FALSE
3

ncgl2989
pcg0007_39
TRUE
3

ncgl2989
pcg3121
FALSE
3

ncgl2989
pcg3381
FALSE
3

Example 5: Allocation of Genes in the C. glutamicum Genome into Various Shells for Systematic Genome-Wide Perturbation
Identification of Genes:

Identified genes were separated into four shells (1-4) based on the relevance of their impact to lysine production.

For shells 1 and 2, the genes in the genome of C. glutamicum were annotated by homology to the sequence of type strain ATCC 13032. The function of each gene in shells 1 and 2 was determined by using the KEGG pathway database. For shell 3, the genes in the genome of C. glutamicum were annotated using the RAST server. The function of each gene in shell 3 was determined using natural language search terms in the annotated description of each gene. These search terms were strings taken from the name of the metabolic area of interest.

Allocation into Each Shell:

The identified genes were allocated into shell 1 if they were involved in the conversion of direct metabolic intermediates between the substrate glucose and the product lysine. This included the transport of glucose into the cell, the transport of lysine out of the cell, and the enzymes involved in the conversion of carbon originally contained in glucose into each intermediate that ultimately was converted to lysine.

The identified genes were allocated into shell 2 if they were identified as being part of nitrogen metabolism, the TCA cycle, or the RNA degradasome KEGG pathway map. These areas of metabolism were chosen based on their relatedness to lysine production: Lysine contains significant nitrogen as compared to biomass, the TCA cycle generates energy for synthesis of lysine and biomass, and the RNA degradasome controls protein expression which is important to maximize for sufficient production during industrial fermentation.

The identified genes were allocated into shell 3 if they were identified as being part of cellular membrane transport, transcription, peptidoglycan biosynthesis, fatty acid biosynthesis, and biotin metabolism. These areas of metabolism are related to the production of lysine in industrial fermentation, but less so than the areas identified in shell 2. Transport is important to increase the productivity of each cell; altering genes related to transcription allows for the systematic modification of genes throughout the cell; peptidoglycan and fatty acid synthesis are involved in cell wall biosynthesis, which is the end point of one of the intermediates of lysine; biotin is an important cofactor for enzymes that are in the lysine metabolic pathway.

The identified genes were allocated into shell 4 if they did not fall into any of shells 1-3.

All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety to the extent not inconsistent with the present description.

From the foregoing it will be appreciated that, although specific embodiments described herein have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope described herein. Accordingly, the disclosure is not limited except as by the appended claims.

PROMOTERS FROM CORYNEBACTERIUM GLUTAMICUM AND USES THEREOF IN REGULATING ANCILLARY GENE EXPRESSION

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