Patent Application
20220127311
The content of the Sequence Listing submitted electronically herewith (name: 113322-0003_Sequence_Listing_27122021.txt); size 1,100,758 bytes; and date of creation: Dec. 27, 2021) is hereby incorporated by reference in its entirety.
The invention relates to a genetically modified prokaryotic cell capable of improved iron-sulfur cluster delivery, characterized by a modified gene encoding a mutant Iron Sulfur Cluster Regulator (IscR) as well as one or more transgenes or upregulated endogenous genes encoding iron-sulfur (Fe—S) cluster polypeptides that catalyze complex radical-mediated molecular rearrangements, electron transfer, radical or non-redox reactions, sulfur donation or perform regulatory functions. One example are radical SAM enzymes, which are involved in the biosynthesis of vitamins, cofactors, antibiotics and natural products, metalloprotein cluster formation, enzyme activation as well as amino acid, nucleic acid and sugar post-transcriptional and post-translational modification like methylation. Protein production and metabolic pathways for the synthesis of a wide range of biological compounds are dependent on their Fe—S clusters. Prokaryotic cells of the invention are characterized by enhanced activity of these iron-sulfur (Fe—S) cluster polypeptides, thereby enhancing their respective functional capacity, as well as facilitating enhanced yields of diverse compounds, including heme, δ-aminolevulinic acid, amino acids (glutamate and branched-chain amino acids), vitamins (B3, B5 and B12 derivatives), biofuels, isoprenoids, pyrroloquinoline quinone, ammonia, indigo or their precursors, as well as proteins containing said compounds in protein-bound form (tetrapyrrole proteins, hemoproteins, quinoproteins and quinohemoproteins); and whose biosynthesis depends on their activity. The invention further relates to a method for producing each of said compounds, or their precursors using the genetically modified prokaryotic cell of the invention; as well as the use of the genetically modified prokaryotic cell.
The production of a wide range of compounds (such as vitamins, pharmaceuticals, food supplements, flavors, fragrances, biofuels, fertilizers, and dyes) currently relies on chemical synthesis, which is costly. Biosynthetic methods for their manufacture would provide an alternative, more cost-effective means for meeting current and future needs. Biosynthetic pathways suitable for synthesis of many such compounds are dependent on one or more iron-sulfur (Fe—S) cluster proteins. Development of cell factories tailor-made for the production of those compounds whose biosynthesis depends on such pathways include: Biotin (also known as vitamin B7 or vitamin H), vitamin B3 (NAD, NR, and NMN), cobalamin (also known as vitamin B12) and pantothenate (vitamin B5), and vitamin K are essential dietary vitamins for humans, because in common with other metazoans, they cannot produce biotin, nicotinamide-derived vitamins or cobalamin.
Heme is a member of the tetrapyrrole family, encompassing several important molecules of diverse metabolic functions (e.g. vitamin B12, chlorophyll, heme, siroheme, chlorophyll, and cofactor F430). Heme plays an essential role in both prokaryotes and eukaryotes for heme-containing proteins. Hemoproteins are involved in enzymatic reactions (e.g. cytochrome P450, cytochrome c oxidase, peroxidase, ligninase, catalase, tryptophan 2,3-dioxygenase, nitric oxide synthase), oxygen transport (e.g. myoglobin, hemoglobin, neuroglobin, cytoglobin and leghemoglobin) and electron transport (e.g. cytochromes in respiratory chain). Heme and hemoproteins find various applications such as in iron supplies for treating anemia, iron supplements for artificial meat, and use in biocatalysts to produce a wide range of compounds in the food, feed, pharmaceutical, chemical and cosmetic industries.
Isoprenoids and terpenoids are large families of natural compounds with many applications in the food, feed, pharmaceutical, flavor, fragrance, chemical and cosmetics industries.
Pyrroloquinoline quinone (PQQ), also called methoxatin, is a redox cofactor found in several enzymes. It has been shown to have potent antioxidant properties, as well as being a growth promoting agent, with applications as a food or pharmaceutical ingredient. Quinoproteins (e.g. methanol dehydrogenase, glucose dehydrogenase) can be also used in biosensing and bioconversion of useful compounds for medical applications as well as in bioremediation.
Additional dietary compounds that might advantageously be produced in a cell factory include the amino acids L-valine, L-leucine, L-isoleucine and L-glutamate. A biosynthetic route for their production, unlike chemical synthesis, ensures that the product is exclusively L-isomers. Since L-glutamate is the precursor of monosodium glutamate, this finds use as a food flavor. Biofuel production (such as butanol and isobutanol) may also be achieved using such a cell factory, since a biosynthetic route for production of such biofuels shares key catalytic steps in common with the branched-chain amino acids. Glutamate is a precursor of δ-aminolevulinic acid, which is the building block of pharmaceuticals, plastics and many different chemicals. δ-aminolevulinic acid is also the precursor of tetrapyrroles (porphyrins), which are compounds that can be produced in free- or protein-bound form with many applications in the medical, pharmaceutical, electronics and chemical industries.
Ammonium, obtained by means of biological Nitrogen Fixation (BNF) in a cell factory, would provide a source of natural fertilizers for plants, and replace current polluting nitrogen fertilizers used by agricultural industries. BNF also takes place in plants that form symbiotic associations with diazotrophs, where the efficiency of nitrogen deficiency determines the need for nitrogen fertilizers. Indigo is a blue dye in high demand in the global textile Industry whose industrial production currently relies on a chemical and polluting process.
The use of prokaryote-based cell factories is a potential route for the biosynthetic production of the above nutrient supplements. The advantages of recombinant E. coli as a cell factory for production of bio-products are widely recognized due to the fact that: (i) it has unparalleled fast growth kinetics; with a doubling time of about 20 minutes when cultivated in glucose-salts media and under optimal environmental conditions, (ii) it easily achieves a high cell density; where the theoretical density limit of an E. coli liquid culture is estimated to be about 200 g dry cell weight/L or roughly 1×1013 viable bacteria/mL. Additionally, there are many molecular tools and protocols at hand for genetic modification of E. coli; as well as being an organism that is amenable to the expression of heterologous proteins; both of which may be essential for obtaining high-level production of desired bio-products.
In general, there exists a need to identify the bottlenecks in those complex biosynthetic pathways that are dependent on Fe—S clusters and often required to facilitate the production of diverse compounds in prokaryote-based cell factories (e.g. E. coli), and to provide tailor-made cell factories that overcome the diversity of factors that may limit their use and productivity.
According to a first embodiment the invention provides a genetically modified prokaryotic cell comprising:
In a further embodiment, the amino acid sequence of said mutant IscR polypeptide comprises at least one amino acid modification and exits only as an apoprotein.
In a further embodiment, the genetically unmodified iscR gene is endogenous or heterologous to the cell.
In a further embodiment, the at least one Fe—S cluster polypeptide is heterologous or endogenous to the cell.
In a further embodiment, said at least one Fe—S cluster polypeptide is selected from the group consisting of: a HemN polypeptide having oxygen-independent coproporphyrinogen III oxidase synthase (EC: 1.3.98.3) activity; a HemZ polypeptide having oxygen-independent, coproporphyrinogen III oxidase synthase (EC: 1.3.98.3) activity; a P450 polypeptide having monooxygenase activity (EC: 1.14.14.1); a NadA polypeptide having quinolate synthase (EC: 2.5.1.72) activity; a CobG polypeptide having precorrin-3B synthase (EC: 1.3.98.3); an IlvD polypeptide having dihydroxy-acid dehydratase activity (EC: 4.2.1.9); an IspG polypeptide having 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC: 1.17.7.3); an IspH polypeptide having 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity (EC: 1.17.7.4); a large chain GltB polypeptide and a small chain GltD polypeptide having glutamate synthase [NADPH] activity (EC: 1.4.1.13); a PqqE polypeptide having PqqA peptide cyclase activity (EC: 1.21.98.4); a NifB polypeptide having nitrogenase FeMo cofactor biosynthesis activity; a NdoB and NdoC polypeptide having naphthalene 1,2-dioxygenase activity (EC: 1.14.12.12) together with a NdoA and NdoR polypeptide having ferredoxin-NAD(P)+ reductase activity (EC: 1.18.1.7); and a hydA polypeptide having hydrogenase activity (EC: 1.18.99.1)
In a further embodiment, the invention provides a cell culture, comprising the genetically modified prokaryotic cell, and a growth medium.
According to a second embodiment, the invention provides a method for producing a compound resulting from catalytic activity of said Fe—S cluster polypeptide, comprising the steps of:
introducing a genetically modified prokaryote of the invention, into a growth medium to produce a culture;
cultivating the culture; and
recovering said compound produced by said culture, and optionally purifying the recovered compound.
According to a third embodiment, the invention for a use of a genetically modified gene encoding a mutant iscR polypeptide to increase production of at least one compound resulting from the catalytic activity of at least one Fe—S cluster polypeptide in a genetically modified prokaryotic cell as compared to a prokaryotic cell comprising the genetically unmodified iscR gene, wherein said Fe—S cluster polypeptides not any one of biotin synthase (EC: 2.8.1.6), lipoic acid synthase (EC: 2.8.1.8), HMP-P synthase (EC: 4.1.99.17), and tyrosine lyase (EC: 4.1.99.19), wherein the mutant IscR polypeptide as compared to a non-mutant IsR polypeptide has an increased apoprotein:holoprotein ratio in the cell.
In a further aspect the invention provides a cell culture comprising the cell of the invention.
In a further aspect the invention provides a fermentation liquid comprising the cell culture of the invention, and its contents of a compound resulting from catalytic activity of the Fe—S cluster protein.
In a further aspect the invention provides a composition comprising the fermentation liquid of the invention and one or more agents, additives and/or excipients.
Amino acid sequence identity: The term “sequence identity” as used herein, indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length. The two sequences to be compared must be aligned to give a best possible fit, by means of the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as ((Nref−Ndif)100)/(Nref), wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Sequence identity calculations are preferably automated using the BLAST program e.g. the BLASTP program (Pearson W. R and D. J. Lipman (1988)) (www.ncbi.nlm.nih.gov/cgi-bin/BLAST). Multiple sequence alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al 1994, available at http://www2.ebi.ac.uk/clustalw/.
Preferably, the numbers of substitutions, insertions, additions or deletions of one or more amino acid residues in the polypeptide as compared to its comparator polypeptide is limited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deletions. Preferably the substitutions are conservative amino acid substitutions: limited to exchanges within members of group 1: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); group 2: Serine (S), Cysteine (C), Selenocysteine (U), Threonine (T), Methionine (M); group 3: proline (P); group 4: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Group 5: Aspartate (D), Glutamate (E), Asparagine (N), Glutamine (Q) and Group 6: Arginine (R), histidine (H) and lysine (K).
Endogenous gene: is a gene in a prokaryotic cell genome that is homologous in origin to a host prokaryotic cell (i.e. a native gene of the host prokaryotic cell). The endogenous gene may be genetically modified using tools known in the art whereby the genetically modified endogenous gene encodes a mutant polypeptide whose amino acid sequence differs at one or more position from the polypeptide encoded by the parent endogenous gene from which it was derived.
Genome: is the genetic material present in a prokaryotic cell; said genome comprising all of the information needed to build and maintain that cell or organism; and includes the genetic material in both chromosome(s) and any episomal genetic element(s) (including plasmid(s)) present within the cell or organism.
Genetically modified regulatory sequence: is a regulatory sequence that is operably linked to a gene comprising a protein coding or non-coding sequence; wherein said regulatory sequence is capable of enhancing transcription of said operably linked gene as compared to the native regulatory sequence of said gene; wherein said regulatory sequence is selected from 1) a promoter region sequence and any enhancer element sequence therein; and 2) is-regulatory elements (for example ribosomal binding site) that provide binding sites of transcription factors that are capable of enhancing transcription of said gene.
GFP: Green Fluorescent Protein.
gi number: (genInfo identifier) is a unique integer which identifies a particular sequence, independent of the database source, which is assigned by NCBI to all sequences processed into Entrez, including nucleotide sequences from DDBJ/EMBL/GenBank, protein sequences from SWISS-PROT, PIR and many others.
Heterologous gene: encodes a polypeptide derived from an organism that is different from the organism into which the heterologous gene is expressed.
Isc pathway: iron sulfur cluster pathway; encoded by the isc operon including the iscR gene.
Multiskan: filter-based microplate photometer; for measuring absorbance from 96 or 384-well plate formats in the wavelength range of 340 to 850 nm, including 600-620 nm. Plates are incubated in the photometer at the selected temperature, of up to 50° C. The photometer is supplied by Thermo Scientific.
Native gene: endogenous coding or non-coding gene in a bacterial cell genome, homologous to host bacterium.
Non-native promoter: in the context of a genetically modified prokaryotic cell of the present invention, is a promoter that is operably-linked to a gene or transgene in said cell, where said promoter would not be found operably-linked to said gene or transgene in a prokaryotic cell found in nature.
OD: Optical Density
Promoter activity: is the measured strength in arbitory units i.e. measured relative activity of a promoter to drive expression of a reporter gene encoding Fluorescent Protein in E. coli, as described by Mutalik et al. 2013. A promoter that is capable of enhancing expression of an operably linked endogenous gene as compared to the native regulatory sequence of said gene is defined herein as a promoter having a measured strength of 370 AU, which is the coincidentally the value for promoter apFAB306.
Transgene: is an exogenous gene that has been introduced into the genome of a prokaryotic cell by means of genetic engineering. In the context of the present invention, said exogenous gene may have a nucleotide sequence identical to a native or non-native gene of said prokaryotic cell and may encode a protein that is native or non-native to said prokaryotic cell; wherein said genome includes both chromosomal and episomal genetic elements.
Upregulated endogenous gene: is an endogenous gene operably linked to a genetically modified regulatory sequence capable of enhancing expression of said endogenous gene as compared to the native regulatory sequence of said gene.
A common feature of many biological compounds when produced by fermentation in prokaryotes is that their biosynthesis employs a step catalyzed by, or dependent on, the activity of one or more iron-sulfur (Fe—S) cluster protein. Optimal product titers by microbial fermentation however are hampered by the fact that overexpression of such pathways leads to growth inhibition. For example, to produce biotin the overexpression of the biotin operon, or even a mutant biotin operon insusceptible to feedback regulation by the BirA repressor, led to a strong inhibition of growth (Ifuku, O. et al., 1995). In the absence of any evidence-based explanation for the observed growth inhibition; alternative approaches were needed to identify the cellular factors that may account for the toxicity of elevated synthesis of Fe—S cluster proteins.
The solution to this problem, provided by the present invention, is shown to be equally applicable for enhancing the synthesis of any Fe—S cluster protein in a prokaryotic cell factory (for example E. coli). The approach pursued to solve this problem was to generate libraries of E. coli cells having evolved genomic diversity due to the accumulation of background mutations generated by imperfect error-correcting polymerases. Cells of such libraries were transformed with a plasmid comprising an IPTG-inducible bioB gene expression cassette, encoding a polypeptide of the Fe—S cluster protein, biotin synthase. Candidate mutants were those cells in a library that were capable of growth in the presence of IPTG at a concentration sufficient to induce BioB expression toxicity in the parent E. coli strain from which the mutant cells were derived.
The genetic basis for the growth of the selected BioB-expressing mutant strains was established by whole genome sequencing. Surprisingly three of the strains were found to have mutations in the native Iron Sulfur Cluster Regulator gene (iscR); which encodes a pleiotropic transcription factor (IscR) [SEQ ID No.: 2]. Fe—S clusters are co-factors of many proteins and essential enzymes, endowing them with diverse biochemical abilities that are not solely required for the synthesis of S-containing compounds, but also as regulatory proteins in the form of sensors for redox- or iron-related stress conditions, as well as for mediating electron transfer, radical or non-redox reactions, and sulfur donation.
IscR exists in two states, either as an Fe—S cluster holo-protein, or as the apo-protein without the Fe—S cluster. Assembly of the Fe—S cluster of IscR is catalyzed by the Isc pathway encoded by the isc operon. The isc operon encodes firstly the regulator (IscR), followed by a cysteine desulfurase (IscS), a scaffold (IscU), an A-type protein (IscA), a DnaJ-like co-chaperone (HscB), a DnaK-like chaperone (HscA) and a ferredoxin (Fdx). In addition to being essential for the assembly of the IscR holoenzyme, the Isc pathway is the primary pathway for Fe—S cluster biogenesis in E. coli(
The ratio between the two forms of IscR is determined by the cellular level of [2Fe-2S] clusters, which in turn is influenced by several factors including iron- and oxygen levels (Py, B. & Barras, 2010). Under iron-rich conditions, IscR exists mainly as the holoenzyme, which then acts as a transcriptional repressor of the isc operon. However, under iron-low conditions (low level of [2Fe-2S] clusters), IscR returns to its apo-protein state, allowing transcription of the isc operon. In its apo-protein state, IscR serves as an activator of the sufABCDSE operon, which catalyzes Fe—S cluster biogenesis under oxidative stress.
In addition to regulating expression of the two Fe—S-cluster assembly systems in E. coli, IscR regulates >40 genes involved in diverse mechanisms of action such as improved assembly of Fe—S clusters (e.g., suf operon), oxidative stress mechanisms (e.g. sodA), specific and global regulators (e.g. yqjl and soxS), amino acid biosynthesis (e.g argE) and a range of genes with unknown functions. The role of IscR is further complicated by the fact that the IscR regulatory landscape changes between aerobic and anaerobic conditions (Giel et al., 2006).
In view of the homeostatic role of IscR and its role in global gene regulation; the consequences of any modification of its regulatory properties are unpredictable and probably profound for cellular metabolism. Furthermore, cellular conditions where Fe—S cluster biogenesis is increased, due to elevated expression of both the sulfur formation (suf) and isc pathways creates the risk that the accumulated Fe—S clusters generate peroxide radicals by Fenton reactions.
In this light, it was highly unexpected that IscR should be found so important for the activity and toxicity of a cellular Fe—S cluster protein, such as BioB, as demonstrated by the three isolated individual mutants. Furthermore, the impact of the mutant IscR protein on end-product (e.g. biotin) biosynthesis was unexpected, since over-expression of the isc operon or suf operons giving an increased capacity to synthesize and assemble Fe—S clusters was not found to enhance biotin production in cells over-expressing bioB (see example 1,
The three different mutations in the IscR protein that eliminated the toxicity of BioB expression in the mutant cells, were single amino acid substitutions of the amino acids L15 [SEQ ID No.: 16], C92 [SEQ ID No.: 18] and H107 [SEQ ID No.: 20] (
While not wishing to be bound by theory, this suggests that homeostatic control of Fe—S cluster biogenesis and global gene regulation required for cell growth are uniquely preserved in cells expressing a mutant iscR gene of the invention, while facilitating the assembly of Fe—S cluster containing enzymes, even during their over-expression.
In summary, the inventors have identified a mutant iscR gene encoding a mutant IscR polypeptide, characterized by the lack of one or more of amino acid residues required for ligation of Fe—S clusters, such that the expressed mutant IscR protein exists solely in the apo-protein form. Synthesis of iron-sulfur cluster containing polypeptides is shown to constitute a significant bottleneck in efforts to enhance production of the biochemical products of pathways requiring such Fe—S proteins in prokaryotes. The solution to this problem, as provided by the present invention, is facilitated by over-expression of these Fe—S polypeptides in a cell factory comprising a gene encoding a mutant IscR polypeptide that exists only in the apo-protein state.
The various embodiments of the invention are described in more detail below.
According to a first embodiment, the invention provides a genetically modified prokaryotic cell comprises a genetically modified iscR gene encoding a mutant IscR, as well as either at least one transgene, or at least one endogenous gene operably linked to a genetically modified regulatory sequence capable of enhancing expression of said endogenous gene (herein called an upregulated endogenous gene); wherein the transgene or endogenous gene encodes any Fe—S cluster polypeptide excluding biotin synthase (EC: 2.8.1.6), lipoic acid synthase (EC: 2.8.1.8), HMP-P synthase (EC: 4.1.99.17), and tyrosine lyase (EC: 4.1.99.19).
The mutant IscR polypeptide is derived from a wild-type member of a family of IscR polypeptides, whereby the mutant IscR is characterized by an amino acid sequence comprising at least one amino acid substitution when compared to its wild-type parent IscR polypeptide; and as a consequence of said at least one substitution the mutants IscR polypeptide, when expressed in a genetically modified prokaryotic cell of the invention, exits only as an apoprotein (i.e. it is unable to exist in a Fe—S cluster holoprotein state). The prokaryotic cell may comprise a genetically modified endogenous iscR gene; or alternatively a transgene encoding a mutant IscR polypeptide but where the native IscR gene is inactive. When a Fe—S cluster polypeptide is over-expressed in a genetically modified prokaryotic cell of the invention, the cells exhibit enhanced growth, and the activity of the respective Fe—S cluster polypeptide is enhanced, as compared to over-expression in a prokaryotic cell having gene encoding a wild-type IscR polypeptide.
The Fe—S cluster polypeptide, encoded by a gene expressed in a prokaryotic cell of the invention, has one of a wide diversity of biochemical abilities, and may be one selected from the group set out in Table A:
A genetically modified regulatory sequence capable of enhancing expression of said endogenous gene encoding an Fe—S cluster polypeptide as compared to the native regulatory sequence of said endogenous gene includes both a promoter sequence (for example having a measured promoter strength of at least 370 AU (see definitions); or a RBS conferring increased transcription (see example 7).
According to a further embodiment, the present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of heme and hemoproteins. The prokaryotic cell is genetically modified to express a mutant IscR in substitution for a wild type IscR, as well as comprising a transgene or up-regulated endogenous hemN gene (i.e an endogenous hemN gene operably linked to a genetically modified regulatory sequence capable of enhancing expression of said endogenous gene, as described in section I) encoding a HemN polypeptide having oxygen-independent coproporphyrinogen III oxidase (EC: 1.3.98.3) activity. Optionally, the genetically modified prokaryotic cell may further comprise one or more additional transgenes or upregulated endogenous genes encoding polypeptides that catalyze additional steps in the heme pathway (
The mutant IscR polypeptide is derived from a wild-type member of a family of IscR polypeptides, whereby the mutant IscR as compared to a corresponding non-mutant IscR polypeptide has an increased apoprotein:holoprotein ratio in the cell. In a special embodiment the apoprotein:holoprotein ratio in the cell of the invention is increased by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 80%, such as at least 100%, such as at least 150%, such as at least 200% compared to a corresponding cell with non-mutant IscR.
In one embodiment, mutant IscR polypeptide has an amino acid sequence comprising at least one amino acid modification (by substitution, addition or deletion), when compared to its wild-type parent IscR polypeptide and can only exit as an apoprotein. In one embodiment the amino acid sequence of a wild-type member of a family of IscR polypeptides has at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 2, 4, 6, 8, 10, 12 and 14, 15-26. The amino acid sequence of the mutant IscR polypeptide according to the invention differs from the amino acid sequence of the corresponding wild-type IscR polypeptide from which it was derived by at least one amino acid substitution; wherein said substitution is selected from L15X, C92X, C98X, C104X, and H107X; wherein X, the substituting amino acid, is any amino acid other than the amino acid found at the corresponding position in the wild type IscR from which the mutant was derived.
In alternative embodiments, the amino acid substitution in the mutant IscR is selected from either L15X, wherein X is any amino acid other than L, more preferably X is selected from phenylalanine (F), tyrosine (Y), methionine (M) and tryptophan (W); C92X, wherein X is any amino acid other than C, more preferably X is selected from tyrosine (Y), alanine (A), methionine (M), phenylalanine (F) and tryptophan (W); C98X, wherein X is any amino acid other than C, more preferably X is selected from alanine (A), valine (V), isoleucine (I), leucine (L), phenylalanine (F) and tryptophan (W); Cys104X, wherein X is any amino acid other than C, more preferably X is selected from alanine (A), valine (V), isoleucine (I), leucine (L), phenylalanine (F), and tryptophan (W); and His107X, wherein X is any amino acid other than H, more preferably X is selected from alanine (A), tyrosine (Y), valine (V), isoleucine (I), and leucine (L). For example, the amino acid substitution in the mutant IscR may be selected from among L15F, C92Y, C92A, C98A, Cys104A, H107Y, and H107A.
The mutant IscR expressed by the genetically modified prokaryotic cell of the invention (instead of a wild type IscR), is encoded by a genetically modified gene, located in the genome of the cell, either on the chromosome or on a self-replicating plasmid. The genetically modified iscR gene in the chromosome can be located in the genome at the same position of the wild-type iscR gene in the native genome. The genome of the genetically modified prokaryotic cell of the invention lacks a native wild type iscR gene, since the native wild type iscR gene is either deleted or directly substituted by the genetically modified iscR gene. The promoter driving expression of the genetically modified iscR gene may be the native promoter of the wild type iscR gene from which the genetically modified iscR gene was derived or replaced by. Alternatively, the promoter may be a heterologous constitutive or inducible promoter. When the promoter is a heterologous constitutive promoter, then a suitable promoter includes: apFab family [SEQ ID Nos.:230-232] while a suitable inducible promoter includes: pBad (arabinose-inducible) [SEQ ID No.:38] and Lacl [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 41].
A polypeptide having oxygen-independent coproporphyrinogen III oxidase synthase (EC: 1.3.98.3) activity, according to the invention, catalyzes the conversion of coproporphyrin III into protoporphyrinogen IX, consuming two molecules of the co-factor S-adenosyl-methionine (SAM) (see
The polypeptides that are encoded by additional transgenes or additional upregulated endogenous genes (as defined herein) in the genetically modified prokaryotic cell, and whose activity serves to enhance the synthesis of both intermediates and products of the heme pathway, are as follows:
The genetically modified prokaryotic cell of the invention can also make use of heme as a cofactor for the production of heme-containing proteins or molecules whose production is dependent on hemoproteins. Such hemoproteins include the enzymes: cytochrome P450 monooxygenase (EC: 1.14.-.-); polypeptides having Fe—S clusters (EC: 1.14.15.-), peroxidase (EC: 1.11.1.-), perooxygenase (EC: 1.11.2.-), catechol oxidase (EC: 1.10.3.-), hydroperoxide dehydratase (EC: 4.2.1.-), tryptophan 2,3-dioxygenase (EC: 1.13.11.-), and cytochrome c oxidase (EC: 1.9.3.-). Additionally, the hemoproteins myoglobin, hemoglobin, neuroglobin, cytoglobin, leghemoglobin can be produced using the genetically modified prokaryotic cell of the invention.
Accordingly, the prokaryotic cell may comprise transgenes or upregulated endogenous genes (as defined herein) encoding a HemN polypeptide, and preferably also a HemB polypeptide, as well as comprising one or more additional transgenes or upregulated endogenous genes (as defined herein) encoding such hemopolypeptides. For example, cytochrome P450s catalyze the stereoselective insertion of two hydroxy groups into indole in two consecutive enzymatic steps into cis-indole-2,3-dihydrodiol, which spontaneously oxidizes to indigo. Accordingly, in one embodiment the cytochrome P450 comprises a monooxygenase, e.g. BM3 having monooxygenase activity (EC: 1.14.14.1), which is a self-sufficient enzyme composed of a single polypeptide with a heme domain and a reductase domain having NAD(P)H reductase activity (EC: 1.6.2.4).
A P450-BM3 polypeptide having monooxygenase activity (EC: 1.14.14.1) and NAD(P)H reductase activity (EC: 1.6.2.4), is characterized by an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to: SEQ ID No.: 115 (origin: Bacillus megaterium). The amino acid sequence of a mutant derivative of said P450-BM3 (called P450-BM3*) has the following amino acid mutations: A74G, F87V, L188Q and V445A; which enable P450 BM3* to oxidise indole into indigo.
When the gene(s) encoding HemN together with any additional polypeptides that catalyze the above listed additional steps in the heme pathway, as well as any cytochrome P450 polypeptide, are transgenes, they are located in the genome of the genetically modified prokaryotic cell, either integrated into the cell chromosome or on a self-replicating plasmid. The transgenes encoding said heme pathway enzymes (HemALBCDEGH) and/or cytochrome P450 polypeptide may be present in the genome within one or more operon.
The promoter driving expression of the transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When expression driven by the promoter is constitutive, then a suitable promoter includes apFab family [SEQ ID Nos.:93] while a suitable inducible promoter includes: pBad (arabinose-inducible) [SEQ ID No.:38] and lac promoter lac p, which is regulated by repressor lacl [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 41]. The selected promoter and terminator may be operably linked to the coding sequence for HemN; and to the coding sequences of the one or more coding sequences for the HemA, L, C, B, C, D, E, G, and H polypeptides and cytochrome P450 polypeptide or may be operably linked to the one or more operon encoding the selected Hem polypeptides.
Heme can be produced and exported using genetically modified prokaryotic cells of the invention (e.g. genetically modified E. coli cells) by introducing the cells into a culture medium suitable for supporting growth as well as comprising a carbon source suitable for the biosynthesis of heme; and finally recovering the heme produced by the culture, as illustrated in Example 3,
The genetically modified prokaryotic cells of the invention comprising a transgene encoding a HemN polypeptide, and preferably a transgene encoding HemB, will produce enhanced levels of heme when the supplied carbon source includes aminolaevulinic acid (ALA). When the genetically modified prokaryotic cells of the invention additionally comprise transgenes encoding each of HemA, L, C, B, C, D, E, G, and H polypeptides, they will produce heme when the supplied carbon source is selected from among glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose (example 3,
A method for producing and quantifying heme and porphyrin produced by a genetically modified prokaryotic cell of the invention is described in example 3.01. Production of heme under anaerobic conditions, employing the HemN catalyzed pathway has the additional advantage of (i) reducing equipment cost (air compressor, gas processing); (ii) reducing electricity cost (air management, reduced stirring needs, reducing cooling); and (iii) reducing contamination risks due to unfavorable conditions for opportunistic organisms.
According to a further embodiment, the present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of B3 vitamins and/or the intermediate quinolate. The prokaryotic cell is genetically modified to express a mutant IscR, according to the invention (see Section I and II), in substitution for a wild type IscR, as well as comprising a transgene or up-regulated nadA endogenous gene (i.e an endogenous nadA gene operably linked to a genetically modified regulatory sequence capable of enhancing expression of said endogenous gene, as described in section I) encoding a NadA polypeptide having quinolate synthase activity (EC: 2.5.1.72). NadA is an [4Fe-4S] cluster-dependent enzyme that catalyzes the condensation and cyclisation of 2-iminosuccinate with dihydroxyacetone to synthesize quinolate (
An increase in the levels of those polypeptides that catalyze steps in the NR pathway enhances the synthesis of both intermediates and end products of the NR pathway in the prokaryotic cell.
Polypeptides having quinolate synthase activity (EC: 2.5.1.72) are encoded by genes found in a wide range of bacteria and plants belonging to a wide range of genera. The amino acid sequence of the NadA polypeptide having quinolate synthase activity has at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 117 (origin: Escherichia coli); SEQ ID No.: 118 (origin: Thermotoga maritima); SEQ ID No.: 119 (origin: Acidobacterium capsulatum); SEQ ID No.: 120 (origin: Aquifex aeolicus); SEQ ID No.: 121 (origin: Bacillus subtilis); SEQ ID No.: 122 (origin: Corynebacterium glutamicum) SEQ ID No.: 123 (origin: Pseudomonas putida); SEQ ID No.: 124 (origin: Sulfolobus solfataricus); SEQ ID No.: 125 (origin: Synechococcus elongatus); SEQ ID No.: 126 (origin: Thermus thermophilus); and SEQ ID No.: 127 (origin: Arabidopsis thaliana).
The polypeptides that are encoded by the additional transgenes or upregulated endogenous genes in the genetically modified prokaryotic cell, and whose activity serves to enhance the synthesis of both intermediates and products of the NR pathway, are as follows:
The promoter driving expression of the transgene encoding NadB and one or more additional transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When expression driven by the promoter is constitutive, then a suitable promoter includes apFab family [SEQ ID Nos.:97] while a suitable inducible promoter includes: pBad (arabinose-inducible) [SEQ ID No.:38] and lac promoter lac p, which is regulated by repressor lacl [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 41]. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
B3 vitamins and quinolate can be produced using genetically modified prokaryotic cells of the invention (e.g. genetically modified E. coli cells) by introducing the cells into a suitable culture medium; and finally recovering the said products of the cells, as illustrated in the example 4.
The genetically modified prokaryotic cells of the invention comprising a transgene encoding a quinolate synthase (NadB) will produce quinolate when supplied with a suitable carbon source for example a source selected from among glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose.
A method for quantifying cellular quinolate and NR produced by a genetically modified prokaryotic cell of the invention is described in example 4.
According to a further embodiment, the present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of cobalamin. The prokaryotic cell is genetically modified to express a mutant IscR, according to the invention (see section I and II), in substitution for a wild type IscR, as well as comprising a transgene or up-regulated endogenous cobG gene (i.e an endogenous cobG gene operably linked to a genetically modified regulatory sequence capable of enhancing expression of said endogenous gene, as described in section 1) encoding a CobG polypeptide having precorrin-38 synthase (EC: 1.3.98.3).
CobG is an [4Fe-4S] cluster-dependent enzyme that catalyzes the conversion of precorrin-3-A to Precorrin-3-B, using dioxygen (O2) and nicotinamide adenine dinucleotide (NADH) as co-factors (
CobG polypeptides having precorrin-38 synthase (EC: 1.14.13.83) are encoded by genes found in a wide range of microorganisms belonging to a wide range of genera. The amino acid sequence of the polypeptide having precorrin-3B synthase activity has at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 135 (origin: Pseudomonas denitrificans); SEQ ID No.: 136 (origin: Corynebacterium glutamicum); SEQ ID No.: 137 (origin: Frankia canadensis); SEQ ID No.: 138 (origin: Nostoc sp. CENA543); SEQ ID No.: 139 (origin: Rhizobium leguminosarum); SEQ ID No.: 140 (origin: Mycoplana dimorpha) SEQ ID No.: 141 (origin: Rhodobacter sphaeroides); SEQ ID No.: 142 (origin: Granulicella tundricola); SEQ ID No.: 143 (origin: Sinorhizobium meliloti); SEQ ID No.: 144 (origin: Streptomyces cattleya); and SEQ ID No.: 145 (origin: Pannonibacter phragmitetus).
The polypeptides that are encoded by the additional transgenes or upregulated endogenous genes (as defined herein) in the genetically modified prokaryotic cell, and whose activity serves to enhance the synthesis of both intermediates of the Cob pathway, are as follows:
When the gene(s) encoding the CobG polypeptide, precorrin-3B synthase, together with one or more additional polypeptides that catalyze additional steps in the Cob pathway are transgenes, they are located in the genome of the genetically modified prokaryotic cell, either integrated into the prokaryotic cell chromosome or on a self-replicating plasmid. The transgene encoding CobG and one or more of the transgenes Cob pathway enzymes may be present in the genome within one or more operon.
The promoter driving expression of the transgene encoding CobG and one or more additional transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When expression driven by the promoter is constitutive, then a suitable promoter includes apFab family [SEQ ID Nos.:97] while a suitable inducible promoter includes: pBad (arabinose-inducible) [SEQ ID No.:38] and lac promoter lac p, which is regulated by repressor lacl [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 98]. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
Cobalamin can be produced using genetically modified prokaryotic cells of the invention (e.g. genetically modified E. coli cells) by introducing the cells into a suitable culture medium; and finally recovering the cobalamin, as illustrated in the Example 5. A suitable culture medium includes a carbon source selected from among glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose.
A method for quantifying cobalamin produced by a genetically modified prokaryotic cell of the invention is described in example 5; and may include the use of High Pressure Liquid Chromatography, relative to a cobalamin standard.
According to a further embodiment, the present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of the branched chain amino acids L-valine, L-leucine and L-isoleucine and vitamin B5 (pantothenate). The prokaryotic cell is genetically modified to express a mutant IscR, according to the invention (see section I and II), in substitution for a wild type IscR, as well as comprising a transgene or up-regulated endogenous ilvD gene (i.e an endogenous ilvD gene operably linked to a genetically modified regulatory sequence capable of enhancing expression of said endogenous gene, as described in section I) encoding a IlvD polypeptide having dihydroxy-acid dehydratase activity (EC: 4.2.1.9).
IlvD is an [4Fe-4S] cluster-dependent enzyme that catalyzes the dehydration of 2,3-dihydroxy-3-methylbutanoate into 3-methyl-2-oxobutanoate (
Additionally, the genetically modified cells of the invention may further comprise transgenes or upregulated endogenous genes (as defined herein) encoding the L-valine exporter ygaZH and global regulator leucine responsive protein (Lrp), to enhance net L-valine export.
Polypeptides having dihydroxy-acid dehydratase activity (EC: 4.2.1.9) are encoded by genes found in a wide range of microorganisms belonging to a wide range of genera. The amino acid sequence of the IlvD polypeptide having dihydroxy-acid dehydratase activity has at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 172 (origin: E. coli); SEQ ID No.: 173 (origin: Acidobacterium capsulatum); SEQ ID No.: 174 (origin: Saccharomyces cerevisiae); SEQ ID No.: 175 (origin: Aquifex aeolicus); SEQ ID No.: 176 (origin: Bacillus subtilis); SEQ ID No.: 177 (origin: Corynebacterium glutamicum) SEQ ID No.: 178 (origin: Deinococcus radiodurans); SEQ ID No.: 179 (origin: Methanococcus maripaludis); SEQ ID No.: 180 (origin: Pseudomonas putida); SEQ ID No.: 181 (origin: Synechococcus elongatus); and SEQ ID No.: 182 (origin: Thermotoga maritima).
The polypeptides that are encoded by the additional transgenes in the genetically modified prokaryotic cell, and whose activity serves to enhance the synthesis of both intermediates and products of the ilv pathway, are as follows:
When the gene(s) encoding the IlvD polypeptide, dihydroxy-acid dehydratase activity, together with one or more additional polypeptides that catalyze additional steps in the ilv pathway (encoding IlvB, IlvN or IlvNbis, IlvC, and optionally IlvE) as well as polypeptides conferring net valine export (YgaZ, YgaH and LrP) are transgenes, they are located in the genome of the genetically modified prokaryotic cell, either integrated into the prokaryotic cell chromosome or on a self-replicating plasmid. The transgene encoding IlvD and one or more of the transgenes encoding liv pathway enzymes may be present in the genome within one or more operon.
The promoter driving expression of the transgene encoding IlvD and one or more additional transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When the promoter is a heterologous constitutive promoter, then a suitable promoter includes the apFab family [SEQ ID Nos.:97], while a suitable inducible promoter includes: pBad (arabinose inducible [SEQ ID No.:38] and Lac [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 98]. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
Branched chain amino acids (valine, leucine and isoleucine) as well as pantothenoate can be produced using genetically modified prokaryotic cells of the invention (e.g. genetically modified E. coli cells) by introducing the cells into a suitable culture medium; and finally recovering the synthesized products, as illustrated in the Example 6. A suitable culture medium includes a carbon source selected from among glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose.
A method for quantifying branched chain amino acids and pantothenate produced by a genetically modified prokaryotic cell of the invention is described in example 6.
The present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of the branched chain amino acids L-valine, L-leucine and L-isoleucine and vitamin B5 (pantothenate).
According to a further embodiment, the present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of isoprenoids and the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the MEP pathway (
The prokaryotic cell is genetically modified to express a mutant IscR, according to the invention (see section I and II), in substitution for a wild type IscR, as well as comprising transgenes or up-regulated endogenous genes (i.e an endogenous genes operably linked to a genetically modified regulatory sequence capable of enhancing expression of said endogenous gene, as described in section 1) encoding an IspG polypeptide having 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC: 1.17.7.3) and an IspH polypeptide having 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity (EC: 1.17.7.4).
Both IspG and IspH are [4Fe-4S] cluster-dependent enzymes that catalyze the final two steps in the MEP pathway. IspG catalyzes the synthesis of HMBPP from MEcPP; while IspH catalyzes the synthesis of IPP and DMAPP in a 3:1 molar ratio from the substrate HMBPP.
Genetically modified prokaryotic cells of the invention that further comprise one or more additional transgenes or upregulated endogenous genes (as defined herein) encoding polypeptides are able to synthesize enhanced levels of both IPP and DMAPP.
IspG polypeptides having 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC: 1.17.7.3) are encoded by genes found in a wide range of microorganisms and plants belonging to a wide range of genera. The amino acid sequence of the polypeptide having 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase activity has at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 192 (origin: E. coli); SEQ ID No.: 193 (origin: Acidobacterium capsulatum); SEQ ID No.: 194 (origin: Aquifex aeolicus); SEQ ID No.: 195 (origin: Arabidopsis thaliana); SEQ ID No.: 196 (origin: Bacillus subtilis); SEQ ID No.: 197 (origin: Clostridium acetobutylicum) SEQ ID No.: 198 (origin: Corynebacterium glutamicum); SEQ ID No.: 199 (origin: Deinococcus radiodurans); SEQ ID No.: 200 (origin: Pseudomonas putida); and SEQ ID No.: 201 (origin: Synechococcus elongatus).
Correspondingly, IspH polypeptides having 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity (EC: 1.17.7.4) are encoded by genes found in a wide range of microorganisms and plants. The amino acid sequence of the polypeptide having 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity has at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 203 (origin: E. coli); SEQ ID No.: 204 (origin: Acidobacterium capsulatum); SEQ ID No.: 205 (origin: Aquifex aeolicus); SEQ ID No.: 206 (origin: Arabidopsis thaliana); SEQ ID No.: 207 (origin: Bacillus subtilis); SEQ ID No.: 208 (origin: Clostridium acetobutylicum) SEQ ID No.: 209 (origin: Corynebacterium glutamicum); SEQ ID No.: 210 (origin: Deinococcus radiodurans); SEQ ID No.: 211 (origin: Pseudomonas putida); SEQ ID No.: 212 (origin: Synechococcus elongatus) and SEQ ID No.: 213 (origin: Thermotoga maritima).
The genetically modified prokaryotic cell may be further modified in order to overexpress one or more polypeptides whose activity serves to enhance the flux through the isoprenoid biosynthesis pathway and thereby enhance synthesis of both intermediates and products of the pathway, as follows:
Optionally the genetically modified prokaryotic cell may be further modified in order to delete the ytjC gene encoding a phosphoglycerate mutase enzyme (EC: 5.4.2.-) in order to increase metabolic flux through the isoprenoid biosynthesis pathway.
When the genes encoding the IspG and IspH polypeptides, together with one or more additional polypeptides that catalyze additional steps that enhance synthesis of isoprenoids and their precursors IPP and DMAPP are transgenes, they are located in the genome of the genetically modified prokaryotic cell, either integrated into the prokaryotic cell chromosome or on a self-replicating plasmid. The transgene encoding IspG and IspH and one or more of the transgenes encoding other MEP pathway enzymes may be present in the genome within one or more operon.
The promoter driving expression of the transgene encoding IspG and IspH and one or more additional transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When expression driven by the promoter is constitutive, then a suitable promoter includes apFab family [SEQ ID Nos.:97] while a suitable inducible promoter includes: pBad (arabinose-inducible) [SEQ ID No.:38] and lac promoter lac p, which is regulated by repressor lacl [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 98]. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
XI A Method for Producing Isoprenoid Precursors and their Derivatives Using a Genetically Modified Bacterium of the Invention
The isoprenoid precursors IPP and DMAPP and their derivatives can be produced using genetically modified prokaryotic cell s of the invention (e.g. genetically modified E. coli cells) by introducing the cells into a suitable culture medium; and finally recovering the isoprenoid precursors or their derivatives, as illustrated in the Example 7. A suitable culture medium includes a carbon source selected from among glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose.
A method for quantifying IPP and DMAPP produced by a genetically modified prokaryotic cell of the invention is described in example 7.
The present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of the IPP and DMAPP as well as their isoprenoid derivatives.
Micro-organisms can produce L-glutamic acid by means of the glutamine synthase-glutamate synthase (glutamine:2-oxoglutarate aminotransferase (GS-GOGAT) pathway, particularly at low ammonia concentrations. The present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of the L-glutamic acid via the GS-GOGAT pathway as well as δ-aminolevulinic acid (ALA) (
The small and large chain polypeptides, GltB and GltD, together form the [4Fe-4S] cluster-dependent enzyme, GOGAT that catalyze GS-GOGAT reaction.
A GltB polypeptide comprising the glutamate synthase [NADPH] large chain of GOGAT, (EC: 1.4.1.13) has an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 219 (origin: E. coli) or SEQ ID No.: 220 (origin: Pseudomonas putida); SEQ ID No.: 221 (origin: Deinococcus swuensis); SEQ ID No.: 222 (origin: Methanoculleus chikugoensis); SEQ ID No.: 223 (origin: Acidobacterium sp.); SEQ ID No.: 224 (origin: Corynebacterium glutamicum); SEQ ID No.: 225 (origin: Bacillus subtilis); SEQ ID No.: 226 (origin: Aquifex aeolicus); SEQ ID No.: 227 (origin: Synechocystis sp); and SEQ ID No.: 228 (origin: Petrotoga miotherma).
A GltD polypeptide comprising the glutamate synthase [NADPH] small chain of GOGAT, (EC: 1.4.1.13) has an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 230 (origin: E. coli); SEQ ID No.: 231 (origin: Pseudomonas putida). SEQ ID No.: 232 (origin: Deinococcus swuensis); SEQ ID No.: 233 (origin: Methanoculleus chikugoensis); SEQ ID No.: 234 (origin: Acidobacterium sp.); SEQ ID No.: 235 (origin: Corynebacterium glutamicum); SEQ ID No.: 236 (origin: Bacillus subtilis); SEQ ID No.: 237 (origin: Aquifex aeolicus); SEQ ID No.: 238 (origin: Synechocystis sp); and SEQ ID No.: 239 (origin: Petrotoga miotherma).
A cell factory relying on increased GltDB catalysis can also be used to produce considerable amount of ALA, which can be used as a molecule itself or as precursors for other molecules of interest. Accordingly, a genetically modified prokaryotic cell of the invention, comprising transgenes or upregulated endogenous genes encoding GltB and GltD, may additionally comprise transgenes or upregulated endogenous genes encoding GltX, HemA and HemL.
A GltX polypeptide having glutamyl-tRNA synthetase activity, (EC: 6.1.1.17) has an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 240 (origin: E. coli).
A HemA polypeptide having glutamyl-tRNA reductase activity, (EC: 1.2.1.70) has an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 107 (origin: E. coli).
A HemL polypeptide having Glutamate-1-semialdehyde 2,1-aminomutase activity, (EC: 5.4.3.8) has an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 108 (origin: E. coli).
When the genes encoding the GltB and GltD polypeptides and any additional polypeptides, optionally within an operon, are transgenes, they are located in the genome of the genetically modified prokaryotic cell, either integrated into the prokaryotic cell chromosome or on a self-replicating plasmid.
The promoter driving expression of each of the transgenes encoding GltB, GltD and any additional polypeptides is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When the promoter is a heterologous constitutive promoter, then a suitable promoter includes the apFab family [SEQ ID Nos.:97], while a suitable inducible promoter includes: pBad (arabinose inducible [SEQ ID No.:38] and Lacl [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 98]. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
L-glutamic acid and δ-aminolevulinic acid can be produced using genetically modified prokaryotic cells of the invention (e.g. genetically modified E. coli cells) by introducing the cells into a suitable culture medium; and finally recovering the L-glutamic acid, as illustrated in the Example 8. A suitable culture medium includes a carbon source selected from among glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose.
A method for quantifying L-glutamic acid and δ-aminolevulinic acid produced by a genetically modified prokaryotic cell of the invention is described in example 8. The present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of L-glutamic acid.
PQQ is synthesized in nature from a precursor which is small peptide PqqA, containing the sequence motif -E-X-X-X-Y-, where an L-glutamate and an L-tyrosine are separated by three amino acid residues. The enzyme PqqA peptide cyclase (PqqE) catalyzes PQQ synthesis by means of a radical driven C—C bond formation linking the glutamate and tyrosine residues at atoms C9 and C9a of PQQ. All carbon and nitrogen atoms of PQQ are derived from the tyrosine and glutamate residues of the PqqA peptide. The PqqE enzyme features a tunnel through the whole protein and a cave at one end, which harbors the active site with an iron-sulfur-cluster and bound SAM. PqqA is suggested to move through the tunnel to the iron-sulfur cluster where the Glutamate and Tyrosine side chains are then connected. PqqE is the first catalytic step in PQQ synthesis from its precursor PqqA, the subsequent steps requiring genes expressing PqqB, PqqC, PqqD, PqqF as well as a gene encoding the precursor.
According to a further embodiment, the present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of PQQ via the PQQ biosynthetic pathway (
The prokaryotic cell is genetically modified to express a mutant IscR, according to the invention (see section I and II), in substitution for a wild type IscR, as well as comprising a transgene or up-regulated endogenous gene (i.e an endogenous gene operably linked to a genetically modified regulatory sequence capable of enhancing expression of said endogenous gene, as described in section I) encoding an PqqE polypeptide having PqqA peptide cyclase activity (EC: 1.21.98.4).
Genetically modified prokaryotic cells of the invention may further comprise one or more additional transgenes ot upregulated endogenous genes (as defined herein) encoding polypeptides catalyzing additional steps of the PQQ pathway, in particular encoding one or more of PqqA, PqqB, PqqC, PqqD, or PqqF such as to further enhance levels of both PQQ produced.
A PqqE polypeptide having PqqA peptide cyclase activity (EC: 1.21.98.4) is characterized by an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to: SEQ ID No.: 242 (origin: Klebsiella pneumoniae); SEQ ID No.: 243 (origin: Planctomycetaceae bacterium); SEQ ID No.: 244 (origin: Chroococcidiopsis cubana); SEQ ID No.: 245 (origin: Azotobacter vinelandii); and SEQ ID No.: 246 (origin: Klebsiella pneumoniae).
A PqqA polypeptide contains the sequence motif -E-X-X-X-Y-, where an L-glutamate and an L-tyrosine are separated by three amino acid residues, and is characterized by an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to: SEQ ID No.: 247 (origin: Klebsiella pneumoniae).
A PqqB polypeptide having a putative PQQ carrier function is characterized by an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to: SEQ ID No.: 248 (origin: Klebsiella pneumoniae).
A PqqC polypeptide having Pyrroloquinoline-quinone synthase activity (EC: 1.3.3.11) is characterized by an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to: SEQ ID No.: 249 (origin: Klebsiella pneumoniae).
A PqqD polypeptide having PqqA binding protein is characterized by an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to: SEQ ID No.: 250 (origin: Klebsiella pneumoniae).
A PqqF polypeptide having metalloendopeptidase (EC: 3.4.24.-), involved in processing of the tyrosine and glutamate of PqqA at R1-R3, is characterized by an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to: SEQ ID No.: 251 (origin: Klebsiella pneumoniae).
When the genes encoding PqqE, together with one or more additional PqqABCD and F polypeptides that play a role in the PQQ pathway and enhance its synthesis are transgenes, they are located in the genome of the genetically modified prokaryotic cell, either integrated into the prokaryotic cell chromosome or on a self-replicating plasmid. The transgene encoding PqqE and one or more of the transgenes encoding PqqABCD and F polypeptides may be present in the genome within one or more operon.
The promoter driving expression of the transgene encoding PqqE and one or more additional transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When expression driven by the promoter is constitutive, then a suitable promoter includes apFab family [SEQ ID Nos.:97] while a suitable inducible promoter includes: pBad (arabinose-inducible) [SEQ ID No.:38] and lac promoter lac p, which is regulated by repressor lacl [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 98]. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
The pyrroloquinoline quinone (PQQ), its precursors, and quinoproteins can be produced using genetically modified prokaryotic cells of the invention (e.g. genetically modified E. coli cells) by introducing the cells into a suitable culture medium; and finally recovering the isoprenoid precursors or their derivatives, as illustrated in the Example 9. A suitable culture medium includes a carbon source selected from among glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose.
A method for quantifying IPP and DMAPP produced by a genetically modified prokaryotic cell of the invention is described in example 9.
The present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of PQQ as well as its precursors.
According to a further embodiment, the present invention provides a genetically modified prokaryotic cell capable of enhanced assembly of nitrogenase that converts atmospheric di-nitrogen (N2) into ammonium that is then used in diverse metabolic pathway such as protein synthesis, as well in biological nitrogen fixation in diazotrophs. The assembly of the nitrogenase is dependent on the FeMo cofactor biosynthesis protein enzyme, NifB, which functions as a radical S-Adenosyl-Methionine (SAM) [4Fe-4S] enzyme (
The prokaryotic cell is genetically modified to express a mutant IscR, according to the invention (see section I and II), in substitution for a wild type IscR, as well as comprising a transgene or up-regulated endogenous gene (i.e an endogenous gene operably linked to a genetically modified regulatory sequence capable of enhancing expression of said endogenous gene, as described in section I and II) encoding a NifB polypeptide having nitrogenase iron-molybdenum cofactor biosynthesis protein activity.
The growth of cells over-expressing NifB enzyme is dependent on an increased supply of [4Fe-4S] clusters provided in cells expressing the mutant IscR (Example 10,
Polypeptides functioning as nifB, FeMo cofactor biosynthesis proteins are encoded by genes found in a wide range of microorganisms belonging to a wide range of genera. The amino acid sequence of the polypeptide having dihydroxy-acid dehydratase activity has at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to a sequence selected from any one of: SEQ ID No.: 253 (origin: Methanobacterium thermoautotrophicum); SEQ ID No.: 254 (origin: Paenibacillus polymyxa); SEQ ID No.: 255 (origin: Methanococcus infernus); SEQ ID No.: 256 (origin: Methanococcus acetivorans); SEQ ID No.: 257 (origin: Azotobacter vinelandii); SEQ ID No.: 258 (origin: Rhizobium leguminosarum) SEQ ID No.: 259 (origin: Thermocrinis albus); SEQ ID No.: 260 (origin: Frankia alni); SEQ ID No.: 261 (origin: Leptospirilum ferrodiazotrophum); and SEQ ID No.: 262 (origin: Cupriavidus taiwanensis).
The polypeptides that are encoded by the additional transgenes or upregulated endogenous gene (as defined herein) in the genetically modified prokaryotic cell, and whose activity serves to enhance the synthesis of both intermediates and products of the ilv pathway, are as follows:
The promoter driving expression of the transgene encoding NifB and one or more additional transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When the promoter is a heterologous constitutive promoter, then a suitable promoter includes the apFab family [SEQ ID Nos.:97], while a suitable inducible promoter includes: pBad (arabinose inducible [SEQ ID No.:38] and Lacl [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 98]. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
The assembly of the nitrogenase complex and nitrogen fixation in genetically modified prokaryotic cells of the invention (e.g. genetically modified E. coli cells) can be detected by introducing the cells into a suitable culture medium; and finally recovering the synthesized products, as illustrated in the Example 10. A suitable culture medium includes a carbon source selected from among glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose.
A method for the catalytic activity of the nitrogenase complex in a genetically modified prokaryotic cell of the invention is described in example 10.
The present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of the nitrogenase complex due to increased NifB mediated assembly, by measuring acetylene reduction.
According to a further embodiment, the present invention provides a genetically modified prokaryotic cell capable of indigo production via the indigo biosynthetic pathway (
A NdoB and NdoC polypeptides having naphthalene 1,2-dioxygenase activity (EC: 1.14.12.12), are characterized by an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to: SEQ ID No.: 272 and 274 respectively (origin: Pseudomonas pudita). A NdoR and NdoA polypeptides having ferredoxin-NAD(P)+ reductase (naphthalene dioxygenase ferredoxin-specific) activity (EC: 1.18.1.7), are characterized by an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence identity to: SEQ ID No.: 276 and 278 respectively (origin: Pseudomonas pudita).
When the genes encoding the RDO (e.g. NdoBCAR) Ndo, that mediate the indigo biosynthetic pathway are transgenes, they are each individually located in the genome of the genetically modified prokaryotic cell, either integrated into the prokaryotic cell chromosome or on a self-replicating plasmid. The transgenes RDO (e.g. NdoBCAR) may be present in the genome within one or more operon.
The promoter driving expression of the transgenes encoding RDO (e.g. NdoBCAR) is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When expression driven by the promoter is constitutive, then a suitable promoter includes apFab family [SEQ ID Nos.:97] while a suitable inducible promoter includes: pBad (arabinose-inducible) [SEQ ID No.:38] and lac promoter lac p, which is regulated by repressor lacl [SEQ ID No.:40]. Suitable terminators include members of the apFAB terminator family including [SEQ ID No.: 98]. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
The indigo can be produced using genetically modified prokaryotic cells of the invention (e.g. genetically modified E. coli cells) by introducing the cells into a suitable culture medium; and finally recovering the isoprenoid precursors or their derivatives, as illustrated in the Example 9. A suitable culture medium includes a carbon source selected from among glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose.
A method for quantifying IPP and DMAPP produced by a genetically modified prokaryotic cell of the invention is described in example 9.
The present invention provides a genetically modified prokaryotic cell capable of producing enhanced levels of PQQ as well as its precursors.
Fe_S cluster enzymes containing an oxidized [4Fe-4S]2+ cluster, e.g oxygen-independent coproporphyrinogen III oxidase synthase (EC: 1.3.98.3); NifH nitrogenase iron protein (EC: 1.18.6.1); IspG polypeptides having 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC: 1.17.7.3); HemN and IspH polypeptides having 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity (EC: 1.17.7.4), need electron transfer for reduction to a [4Fe-4S]+ cluster. Only the reduced [4Fe-4S]+ cluster is able generate the SAM-radical needed for catalysis. The electron transfer from the electron donor NADPH to the [4Fe-4S]2+ cluster can be mediated by a flavodoxin/ferredoxin reductase (Fpr) and flavodoxin (FldA) reduction system or by a Pyruvate-flavodoxin/ferredoxin oxidoreductase system.
In a further embodiment, the genetically modified prokaryotic cell according to the present invention, further comprises one or more genes selected from the group: a gene encoding a flavodoxin/ferredoxin-NADP reductase (EC: 1.18.1.2 and EC 1.19.1.1); a gene encoding a pyruvate-flavodoxin/ferredoxin oxidoreductase (EC: 1.2.7); a gene encoding a flavodoxin; a gene encoding a ferredoxin; a gene encoding a flavodoxin and a ferredoxin-NADP reductase. Promoter(s) or RBS sequences, operably-linked to each of said one or more genes are capable of enhancing expression of said one or more genes in said cell; wherein each said one or more genes may be a endogenous native gene or a transgene. Preferably, the operably-linked promoter or RBS, enhances expression of said one or more genes in said cell to a level greater than in the parent cell from which the genetically-modified bacterium of the invention was derived. Preferably, the genetically modified prokaryotic cell according to the present invention comprises a gene encoding a flavodoxin/ferredoxin-NADP reductase (EC: 1.18.1.2 and EC 1.19.1.1) and a gene encoding a flavodoxin; or a single gene comprising coding sequences for both a flavodoxin and a ferredoxin-NADP reductase. Additionally said genetically modified prokaryotic cell may further comprise a gene encoding a ferredoxin.
Overexpression of genes expressing components of the electron transfer pathway in genetically modified prokaryotic cells of the present invention, enhances the cellular activity of their SAM-radical iron-sulfur cluster enzymes (as illustrated in Example 2 for biotin-producing cells of the invention). Preferably, when the polypeptide encoded by a native gene or transgene in the genetically modified prokaryotic cell of the invention has flavodoxin/ferredoxin reductase activity (EC: 1.18.1.2 and EC 1.19.1.1), it has an amino acid sequence having 80, 85, 90, 95 or 100% sequence identity to a sequence selected from any one of: SEQ ID No.: 43 (origin: fpr gene from E. coli); SEQ ID No.:45 (origin: yum C gene from Bacillus subtilis 168); SEQ ID No.:47 (origin: fpr-I gene from Pseudomonas putida KT2440); SEQ ID No.:48 (origin: SVEN_0113 gene from Streptomyces venezuelae ATCC 10712-); SEQ ID No.:51 (origin: Cgl2384 gene from Corynebacterium glutamicum ATTCC 13032), and SEQ ID No.:53 (origin: SJN15614.1 gene from Sphingobacterium sp. JB170.
Preferably, when the polypeptide encoded by an endogenous native gene or transgene in the genetically modified prokaryotic cell of the invention has pyruvate-flavodoxin/ferredoxin oxidoreductase activity (EC: 1.2.7), it has an amino acid sequence having 80, 85, 90, 95 or 100% sequence identity to a sequence selected from any one of: SEQ ID No.: 55 (origin: YdbK gene from E. coli K12 MG1655); SEQ ID No.: 57 (origin: por gene from Geobacter sulfurreducens AM-1); SEQ ID No.: 59 (origin: Sfla_2592 gene from Streptomyces pratensis ATCC 33331; SEQ ID No.: 61 (origin: RM25_0186 gene from Propionibacterium freudenreichii DSM 20271); SEQ ID No.: 63 (origin: nifJ gene from Synechocystis sp. PCC 6803)
Preferably, when the polypeptide encoded by a endogenous native gene or transgene in the genetically modified prokaryotic cell of the invention Is a flavodoxin, it has an amino acid sequence having 80, 85, 90, 95 or 100% sequence Identity to a sequence selected from any one of: SEQ ID No.: 65 (origin: fldA gene from Escherichia coli K12 MG1655); SEQ ID No.: 67 (origin: fldB gene from Escherichia coli K12 MG1655); SEQ ID No.: 69 (origin: ykuN gene from Bacillus subtilis 168); SEQ ID No.: 71 (origin: isiB gene from Synechocystis sp. PCC 6803; SEQ ID No.: 73 (origin: wrbA gene from Streptomyces venezuelae ATCC 10712); SEQ ID No.: 75 (origin: PRK06242 gene from Methanococcus aeolicus Nankai-3).
Preferably, when the polypeptide encoded by a endogenous native gene or transgene in the genetically modified prokaryotic cell of the invention is a ferredoxin, it has an amino acid sequence having 80, 85, 90, 95 or 100% sequence identity to a sequence selected from any one of: SEQ ID No.: 77 (origin: fdx gene from E. coli); SEQ ID No.: 79 (origin: fer gene from Bacillus subtilis 168); SEQ ID No.: 81 (origin: fdxB gene from Corynebacterium glutamicum ATTCC 13032); SEQ ID No.: 83 (origin: fdx gene from Synechocystis sp. PCC 6803); SEQ ID No.: 85 (origin: SVEN_7039 gene from Streptomyces venezuelae ATCC 10712); SEQ ID No.: 87 (origin: fdx gene from Methanococcus aeolicus Nankai-3).
When a promoter is employed to enhance gene expression of an operably-linked endogenous native gene or to a transgene encoding a polypeptide of the electron transport pathway in said cell, it is preferably a non-native promoter. Said promoter may be a member of the constitutive apFAB309 promoter family [SEQ ID Nos.:93]. Preferably said non-native promoter, when operably-linked to said native gene or transgene enhances expression of said encoded polypeptide(s) in said genetically modified bacterium to a level greater than the parent bacterium from which it was derived. Suitable terminators that may be operably-linked to said endogenous native gene or transgene includes the apFAB terminator family [SEQ ID No.: 98].
Prokaryotic cells are genetically engineered by the introduction into the cells of transgenes or by the upregulation of expression of endogenous genes as illustrated in the Examples.
Genetic modification of endogenous genes in a prokaryotic cell of the invention can be performed by deletion (knockout) of the endogenous gene and insertion/substitution with a transgene encoding a mutant polypeptide as defined in section I and II, by applying standard recombineering methods to a suitable parent prokaryotic cell (Datsenko K A, et al.; 2000). Genetic modification of an endogenous gene sequence and/or regulatory sequence that are operatively linked to said endogenous gene can be performed by using a range of techniques known in the art, including recombineering (e.g. MAGE with single-strand DNA) and CRISPR-Cas gene editing.
The genetically modified prokaryotic cell according to the invention, may be a bacterium, a non-exhaustive list of suitable bacteria is given as follows: a species belonging to the genus selected from the group consisting of: Escherichia, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Pseudomonas, Serratia, Lactobacillus, Lactococcus, Acetobacter, Acinetobacter, Pseudomonas, etc.
Preferred bacterial species of the invention are Escherichia coli, Pseudomonas putida, Serratia marcescens and Corynebacterium glutamicum.
In the case of nitrogen fixation, a preferred genetically modified bacterial species of the invention belongs to the genus Rhizobium, associated with leguminous plants (e.g., various members of the pea family); Frankia, associated with certain dicotyledonous species (actinorhizal plants); and Azospirillum, associated with cereal grasses.
Fe_S cluster enzymes containing an oxidized [4Fe-4S]2+ duster, e.g oxygen-independent coproporphyrinogen III oxidase synthase (EC: 1.3.98.3); NifH nitrogenase iron protein (EC: 1.18.6.1); IspG polypeptides having 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC: 1.17.7.3); HemN and IspH polypeptides having 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity (EC: 1.17.7.4), need electron transfer for reduction to a [4Fe-4S]+ cluster. Only the reduced [4Fe-4S]+ cluster is able generate the SAM-radical needed for catalysis. The electron transfer from the electron donor NADPH to the [4Fe-4S]2+ cluster can be mediated by a flavodoxin/ferredoxin reductase (Fpr) and flavodoxin (FldA) reduction system or by a Pyruvate-flavodoxin/ferredoxin oxidoreductase system.
In a further embodiment, the genetically modified prokaryotic cell according to the present invention, further comprises one or more genes selected from the group: a gene encoding a flavodoxin/ferredoxin-NADP reductase (EC: 1.18.1.2 and EC 1.19.1.1); a gene encoding a pyruvate-flavodoxin/ferredoxin oxidoreductase (EC: 1.2.7); a gene encoding a flavodoxin; a gene encoding a ferredoxin; a gene encoding a flavodoxin and a ferredoxin-NADP reductase. Promoter(s) or RBS sequences, operably-linked to each of said one or more genes are capable of enhancing expression of said one or more genes in said cell; wherein each said one or more genes may be a endogenous native gene or a transgene. Preferably, the operably-linked promoter or RBS, enhances expression of said one or more genes in said cell to a level greater than in the parent cell from which the genetically-modified bacterium of the invention was derived. Preferably, the genetically modified prokaryotic cell according to the present invention comprises a gene encoding a flavodoxin/ferredoxin-NADP reductase (EC: 1.18.1.2 and EC 1.19.1.1) and a gene encoding a flavodoxin; or a single gene comprising coding sequences for both a flavodoxin and a ferredoxin-NADP reductase. Additionally said genetically modified prokaryotic cell may further comprise a gene encoding a ferredoxin.
Overexpression of genes expressing components of the electron transfer pathway in genetically modified prokaryotic cells of the present invention, enhances the cellular activity of their SAM-radical iron-sulfur cluster enzymes (as illustrated in Example 2 for biotin-producing cells of the invention).
Preferably, when the polypeptide encoded by a native gene or transgene in the genetically modified prokaryotic cell of the invention has flavodoxin/ferredoxin reductase activity (EC: 1.18.1.2 and EC 1.19.1.1), it has an amino acid sequence having 80, 85, 90, 95 or 100% sequence identity to a sequence selected from any one of: SEQ ID No.: 241 (origin: fpr gene from E. coli); SEQ ID No.:243 (origin: yumC gene from Bacillus subtilis 168); SEQ ID No.:245 (origin: fpr-I gene from Pseudomonas putida KT2440); SEQ ID No.:247 (origin: SVEN_0113 gene from Streptomyces venezuelae ATCC 10712-); SEQ ID No.:249 (origin: Cgl2384 gene from Corynebacterium glutamicum ATTCC 13032), and SEQ ID No.:251 (origin: SJN15614.1 gene from Sphingobacterium sp. JB170.
Preferably, when the polypeptide encoded by an endogenous native gene or transgene in the genetically modified prokaryotic cell of the invention has pyruvate-flavodoxin/ferredoxin oxidoreductase activity (EC: 1.2.7), it has an amino acid sequence having 80, 85, 90, 95 or 100% sequence identity to a sequence selected from any one of: SEQ ID No.: 253 (origin: YdbK gene from E. coli K12 MG1655); SEQ ID No.: 255 (origin: par gene from Geobacter sulfurreducens AM-1); SEQ ID No.: 257 (origin: Sfla_2592 gene from Streptomyces pratensis ATCC 33331; SEQ ID No.: 259 (origin: RM25_0186 gene from Propionibacterium freudenreichii DSM 20271); SEQ ID No.: 261 (origin: nifJ gene from Synechocystis sp. PCC 6803)
Preferably, when the polypeptide encoded by a endogenous native gene or transgene in the genetically modified prokaryotic cell of the invention is a flavodoxin, it has an amino acid sequence having 80, 85, 90, 95 or 100% sequence identity to a sequence selected from any one of: SEQ ID No.: 263 (origin: fldA gene from Escherichia coli K12 MG1655); SEQ ID No.: 265 (origin: fldB gene from Escherichia coli K12 MG1655); SEQ ID No.: 267 (origin: ykuN gene from Bacillus subtilis 168); SEQ ID No.: 269 (origin: IsiB gene from Synechocystis sp. PCC 6803; SEQ ID No.: 271 (origin: wrbA gene from Streptomyces venezuelae ATCC 10712); SEQ ID No.: 273 (origin: PRK06242 gene from Methanococcus aeolicus Nankai-3).
Preferably, when the polypeptide encoded by a endogenous native gene or transgene in the genetically modified prokaryotic cell of the Invention is a ferredoxin, it has an amino add sequence having 80, 85, 90, 95 or 100% sequence identity to a sequence selected from any one of: SEQ ID No.: 275 (origin: fdx gene from E. coli); SEQ ID No.: 277 (origin: fer gene from Bacillus subtilis 168); SEQ ID No.: 279 (origin: fdxB gene from Corynebacterium glutamicum ATTCC 13032); SEQ ID No.: 281 (origin: fdx gene from Synechocystis sp. PCC 6803); SEQ ID No.: 283 (origin: SVEN_7039 gene from Streptomyces venezuelae ATCC 10712); SEQ ID No.: 285 (origin: fdx gene from Methanococcus aeolicus Nankai-3).
When a promoter is employed to enhance gene expression of an operably-linked endogenous native gene or to a transgene encoding a polypeptide of the electron transport pathway in said cell, it is preferably a non-native promoter. Said promoter may be a member of the constitutive apFAB309 promoter family [SEQ ID Nos.:93]. Preferably said non-native promoter, when operably-linked to said native gene or transgene enhances expression of said encoded polypeptide(s) in said genetically modified bacterium to a level greater than the parent bacterium from which it was derived. Suitable terminators that may be operably-linked to said endogenous native gene or transgene includes the apFAB378 terminator family [SEQ ID No.: 41].
1.01: The Following Strains of Escherichia coli Used in the Examples are Listed Below.
E. coli K-12 BW25113 parent strain having genotype:
1Nucleotide sequence of ΔbioB gene prior to deletion was SEQ ID No. 33
E. coli isc operon (iscSUA-hscBA-fdx) from an IPTG inducible T5 promoter
E. coli suf operon (sufABCDSE) from an IPTG inducible T5 promoter cloned
1Nucleotide sequence of bioB frameshift gene has SEQ ID No. 35
The growth media (mMOPS) used in each example had the following composition: 1.32 mM K2HPO4; 2 g/L D-glucose; 0.0476 mg/I calcium pantothenate; 0.0138 mg/L p-aminobenzoic acid; 0.0138 mg/L p-hydroxybenzoic acid; 0.0154 mg/L 2,3-dihydroxybenzoic acid, and 1× modified MOPS buffer. 10× modified MOPS comprises 0.4 M MOPS (3-(N-morpholino) propane sulfonic acid); 0.04 M Tricine; 0.1 mM FeSO4•7H2O; 95 mM NH4Cl; 2.76 mM K2SO4; 5 μM CaCl2•2H2O; 5.25 mM MgCl2; 0.5 M NaCl; and 5000× dilution of micronutrient stock solution.
The following antibiotic stocks were employed: ampicillin (amp, 100 mg/mL), kanamycin (kan, 50 mg/mL), zeocin (zeo, 40 mg/mL); that were added to growth media as indicated to obtain a 1000× dilution.
1.04 Establishing of E. coli Strain Libraries:
E. coli libraries having evolved genomic diversity were derived from cells of E. coli strain BS1011 comprising plasmid pBS412 by subjecting the cells to stationary overnight culture in mMOPS medium supplemented with kan (MOPS-kan), preparing a 100× dilution of resulting culture in mMOPS-kan and repeating the consecutive steps of overnight culture and dilution 5 times. This procedure creates genetic diversity by allowing the accumulation of background mutation generated by imperfect error-correcting polymerases. After each round of culture and dilution a sample of the cell culture was plated on mMOPS plates with IPTG (see below), to detect the evolution of cells adapted to tolerate enhanced BioB expression. Cells of each library were then transformed with the BioB over-expression plasmid, pBS412.
A selection assay was developed by plating respectively 104, 105, 106 and 107 cells, derived from an o/n culture in mMOPS-kan of BS1011 comprising pBS412, on a series of 1.5% agar plates comprising mMOPS (Ø=9 cm) comprising IPTG concentrations of either 0, 0.0001, 0.001, 0.01, 0.1 and 1 mM. The plates were then incubated at 37° C. for up to 36 hours and cell growth was evaluated at intervals. Under these conditions, induction of BioB expression from pBS412 with 0.1 mM IPTG was found to prevent growth of up to 105 cells, while induction with 1 mM IPTG prevented growth of at least 10′ cells, when plated on a single petri dish. A selection pressure comprising induction with 1 mM IPTG for a cell population of 105 cells was found optimal for identifying strains with higher robustness towards BioB expression; and accordingly was implemented as follows:
Pre-cultures were each prepared from a selected single cell colony in 400 μL mMOPS-kan in a 96 deep-well plate, incubated at 37° C. with shake at 275 rpm for 16-18 hours. Production cultures were produced by inoculating 400 μL mMOPS-kan, supplemented with 0.1 g/L desthiobiotin (DTB), and optionally comprising IPTG at a final concentration of up to 1 mM, in a 96 deep-well plate, with 4 μL of the pre-culture enough to provide an initial OD600 of ˜0.03. Cultures were then grown at 37° C. with 275 rpm shake for 24 hours. Cells in the 96 deep-well plate were pelleted by centrifuging at 4000 G for 8 minutes after measuring OD600 of the cultures. The supernatant from each culture supernatant was diluted to a concentration range of 0.05 nM to 0.50 nM biotin in ultrapure (Milli-Q) water. In parallel, >5 biotin standards in the concentration range of 0.1 nM (0.024 μg biotin/L) to 1 nM (0.24 μg biotin/L), were prepared in Milli-Q water. 15 μL of each diluted supernatant and each of the biotin standards was then added to a well of a microtiter plate; wherein each well comprised 135 μL of a biotin-starved overnight culture of BS1011 comprising plasmid pBS451; and where the overnight culture was diluted to an initial OD620 of 0.01 in mMOPS supplemented with zeocin. The plate was sealed with a breathable seal and incubated at 37° C. with 275 rpm shaking for 20 hours before OD620nm was measured. A biotin bioassay calibration curve obtained with this bioassay, using a range of biotin standards, is shown in
For all Next-Generation Sequencing (NGS) data, CLC genomic workbench version 9.5.3 (supplied by Qiagen) was used to identify mutations in the genome of cells of selected strains as compared to the parent strain genome (single or a few substitutions, deletions or insertions by Variant Detection and bigger insertions/deletions by InDels and Structural Variants). A cut-off of 85% were used to define “significant mutations” meaning that a mutation should be present in more than 85% of the population of DNA molecules (genomes) isolated from cells of a given bacterial strain, in order to distinguish genome mutations from erroneous nucleotides introduced by the sequencing procedure.
The genome accession number CP009273 from NCBI was used as the reference sequence, while taking account of the Keio ΔbioB scar mutation whose sequence was confirmed by sequencing.
1.08 Characterizing Proteomics Landscape of iscR Mutant
Protein content of BS1013+pBS430, 851011+pBS412 and BS1353+pBS412 at 0.025 mM IPTG induction levels as well as BS1353+pBS412 at 1 mM IPTG induction were determined by a recently developed approach combining LC-MS and efficient protein extraction (Schmidt et al, 2015). 3 peptides were chosen as minimum number of identified peptides for analysis along with a peptide threshold of 2.0% FDR. Significant changes in protein expression are reported with a 0.5% confidence interval based on Analysis of Variance (ANOVA) with Benjamini-Hochberg correction for multiple testing using a Scaffold Viewer 4.7.5.
Strains were grown in mMOPS with IPTG induction for approximately 10 generations, until OD600 of 0.5 were reached (exponential phase). 108 cells were harvested by centrifugation at 4° C. at maximum speed; washed once in ice-cold PBS buffer; re-pelleted by centrifugation at 4° C. at maximum speed and snap-frozen in liquid nitrogen, after removing PBS buffer.
E. coli retaining its native biotin synthase gene (bioB) but expressing an IPTG-inducible frameshifted E. coli bioB gene (encoding non-functional biotin synthase due to a premature stop codon) from a low-copy plasmid (Sc101 origin of replication) is able to grow aerobically in mMOPS-kan medium with or without IPTG. This is illustrated in
1.10 Isolation of iscR Mutant Strains Having Enhanced Biotin Production Titers
E. coli libraries having evolved genomic diversity (see sections 1.4 and 1.5) were screened for strains with improved tolerance for bioB gene expression and increased biotin production. Whole genome sequencing of the selected strains led to the identification of three unique mutants each comprising an Iron-sulfur cluster regulator (iscR) gene encoding an iscR polypeptide having one of the amino acid substitutions: L15F, C92Y and H107Y, and where the amino acid sequence of the encoded regulators is SEQ ID No.: 16, 18 and 20 respectively. Biotin production levels were measured using a bioassay, as described in section 1.6 (and
The growth profile and biotin production titer of the iscR (H107Y) mutant strain was characterized in 50 mL mMOPS supplemented with 0.1 g/L DTB in a 250 mL shake-flask experiment at two different IPTG induction levels (0.01 mM in
An enhanced biotin tolerance phenotype was clearly demonstrated for all three of the identified IscR mutant strains, as seen in
1.13 Overexpression of the Isc-Operon or Suf Operon in E. coli Strains Alone is not Sufficient to Enhance Biotin Production
In order to determine the direct effect of overexpressing the isc-operon (iscSUA-hscBA-fdx, corresponding to the native E. coli operon structure minus the iscR gene) or the suf-operon (sufABCDSE corresponding to the native E. coli operon structure) on biotin production in E. coli, each operon was cloned into a medium copy number plasmid (p15A ori) placed under the control of a strong RBS and an IPTG inducible T5 promoter. A plasmid, comprising a gene encoding a super folder Green Fluorescent Protein (sfGFP) in substitution for the isc- or suf-operon, was employed as a control. The respective plasmids were transformed into cells of an E. coli strain comprising an IPTG-inducible bioB expression plasmid. Biological triplicate colonies comprising one of: 1) IPTG-inducible isc-operon, 2) IPTG-inducible suf-operon or 3) IPTG-inducible GFP (control) in addition to the IPTG-inducible bioB expression plasmid were assayed for biotin production (as described in section 1.5) following cultivation in 400 μL mMOPS with 100 μg/mL ampicillin and 50 μg/mL spectinomycin under low (0.01 mM IPTG) and high (0.1 mM IPTG) induction.
Although biotin production was IPTG-inducible in all strains (
1.14 BioB Protein Contents Correlates with Biotin Production
To investigate the molecular effects of BioB overexpression in wild type and mutant background strains, proteomics measurements were carried out for a wild type background strain: BS1013 holding pBS430; a wild type iscR strain with a bioB production plasmid: BS1011 holding pBS412; and a mutant iscR strain with a bioB production plasmid: BS1353 holding pBS412. All strains were grown in mMOPS with 0.1 g/L DTB and 0.025 mM IPTG. The latter strain was additionally grown at 1 mM IPTG induction. Cells were harvested for proteomics analysis in exponential phase, while the remaining cell culture were kept incubating for 24 hours in total, before biotin production were measured using the bioassay described elsewhere.
From the graph (
1.15 Biotin Production is not Enhanced in iscR Knockout Mutants
A translational knockout of the iscR gene was introduced into a BW25113 ΔbioB strain by MAGE, by converting the codon encoding glutamic acid on position 22 in iscR (E, GAA) into a stopcodon (*, TGA). Successful conversion of the codon was verified by PCR amplification of the region followed by Sanger sequencing. Strains with genes encoding wild type iscR, iscR knockout (E22*), and mutant iscR (C92Y) were transformed with IPTG-inducible bioB plasmid pBS412, and tested for biotin production in biological replicates (n=3) grown in mMOPS supplemented with 0.1 g/L DTB and 50 μg/L kanamycin at three different IPTG induction levels (0, 0.01, and 0.1 mM) as described above.
No significant differences in biotin production were observed between the iscR knockout (iscR KO) and the wild type iscR (iscR WT) strains when inducing bioB expression by IPTG induction. This provides evidence that knocking out iscR does not improve biotin production. Significant improvement in biotin production was again observed for the mutant iscR encoding IscR C92Y substitution as compared to both iscR WT and iscR KO strains.
1.16 De-Novo Biotin Production is Enhanced in iscR Mutant Strains of the Invention
A BW25113 E. coli strain from which both the bioA gene and the entire biotin-operon (ΔbioB-bioD) were deleted and comprising either iscR WT, iscR H107Y mutant or iscR C92Y mutant genes, were transformed with a tetracycline resistant plasmid, constitutively overexpressing the native E. coli bioA and biotin-operon, with a single point mutation in the bioO operator site (Type 9 mutation, Ifuku et al., 1993). Biotin production was evaluated for the three different strains in biological replicates (n=4) in mMOPS (2 g glucose/L) with 10 μg/ml tetracycline with and without the addition of 0.1 g/L DTB as described above (
A significant increase in biotin titers were observed for all three strains when the substrate, DTB, was added to the growth medium, indicating that the bioB enzyme reaction itself, converting DTB to biotin, is no longer a bottleneck for biotin production in these strains (
2.01: The Following Strains of Escherichia coli Used in the Examples are Listed Below.
The following plasmids used in the example are listed below.
An IPTG-inducible transgene encoding BioB was cloned on plasmid pBS679; a constitutively-regulated transgene encoding GFP was cloned on plasmid pBS1054; and a constitutively-regulated transgene comprising a synthetic operon encoding FldA-Fpr was cloned on plasmid pBS1112. pBS679 was introduced into an E. coli host strain (BS1615) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution, as described in Example 1, and further comprising a knock-out of the bioAFCD genes resulting in the strain BS1937. The strain BS1937 was then further transformed with either plasmid pBS1054 or pBS1112 resulting in the strains BS2707 (control strain) and BS2185, respectively.
The strains were cultured in mMOPS medium (as described in example 1.3) with appropriate antibiotic(s), 0.1 g/L DTB as substrate for BioB-mediated catalysis, and supplemented with either 0, 0.01, 0.025, 0.05, 0.075 or 0.1 mM IPTG for inducing expression of the BioB gene. The cells were incubated for 24 hours at 37° C. in individual wells of a deep well culture plate. End OD600nm values were estimated, supernatants were harvested by centrifugation and biotin quantified from the supernatants by a biotin bioassay carried out as described in example 1.6.
As shown in
3.01 Heme Production by an IscR H107Y Mutant Strain Overexpressing hemN and hemB Genes.
The following strains of Escherichia coli used in the example are listed below.
The following plasmids used in the example are listed below.
IPTG-inducible genes encoding HemN and HemB were cloned to give plasmid pBS1610; and an empty plasmid pB51259 having a p15A backbone was used as a control. pBS1610 or pBS1259 were introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strains BS3129 and BS2630, respectively. Similarly, the plasmids p651610 and pBS1259 were introduced into E. coli strain 651011 carrying a wild-type version of the iscR gene, resulting in strains 653128 and BS2629, respectively.
In addition, IPTG-inducible genes encoding HemZ and HemB were cloned to give plasmid pBS1611; and an empty plasmid pBS1259 having a p15A backbone was used as a control. pBS1611 or pBS1259 were introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strains BS3131 and BS2630, respectively. Similarly, the plasmids pBS1611 and pBS1259 were introduced into E. coli strain BS1011 carrying a wild-type version of the iscR gene, resulting in strains BS3130 and BS2629, respectively.
Furthermore, IPTG-inducible genes encoding HemF and HemB were cloned to give plasmid pBS1612. pBS1611 was introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strains BS3133. Similarly, the plasmids pBS1612 was introduced into E. coli strain 651011 carrying a wild-type version of the iscR gene, resulting in strain BS3132, respectively. The non iron-sulfur cluster hemF strains where used as controls.
Cells of each strain (Table 6) are cultivated in mMOPS medium (as described in Example 1.03) supplemented with 1 nmol/L biotin and 100 μg/mL ampicillin at 37° C. until a cell density (OD600nm) of 0.6 is reached. IPTG is then added to the growth medium (to a final concentration of 0.1 mM) to induce HemN and HemB synthesis. Porphyrins including heme production were measured after 24 h by fluorescence spectroscopy in a microriter plate. Fluorescence values (ex. 240; em 620) are a quantitative measure of the production of porphyrin and heme production potential of the strains.
The free porphyrin and heme content of the cultured cells is determined according to Fyrestam and Oestman, 2017. Cells in each culture are centrifuged (17000 g, 5 min), the supernatant is discarded, and each cell pellet is re-suspended in Tris-EDTA pH 7.2 solution and the cells are sonicated in 5 sec bursts of 20-50 KHz. Each sonicated sample is then centrifuged (17000 g, 5 min) and the respective supernatant collected, followed by addition of 3 volumes of 100 w % acetonitrile. Each sample is vortexed for 5 min and centrifuged (2500 g, 5 min). Finally, the supernatant of each sample is collected for HPLC analysis using a C18 reverse-phase column; using a mobile phase consisting of water, acetonitrile and 0.1 M formic acid (pH 5.1-5.2). Free porphyrins and free heme titer of each collected sample is also quantified using a Quantichrom heme assay kit from BioAssay systems (coloration of porphyrins and reading at 400 nm).
The tests demonstrate that over-expression of a hemN gene encoding a hemN Fe—S polypeptide in combination with a HemB gene in an E. coli strain comprising a gene encoding a mutant form of the IscR protein (IscR protein having an C92Y or H107Y substitution) leads to both a more stable production and increased in the heme and prophyrin titer as compared to over-expression of the HemN and HemB-encoding gene in a parent E. coli strain comprising a gene encoding the native, wild-type form of the IscR protein. Furthermore, the test demonstrate that over-expression of a hemZ gene encoding a HemZ Fe—S polypeptide in combination with a HemB gene in an E. coli strain comprising a gene encoding a mutant form of the IscR protein (IscR protein having a H107Y substitution) leads to both a more stable production and increase in the heme and prophyrin titer as compared to over-expression of the HemN and HemB-encoding gene in a parent E. coli strain comprising a gene encoding the native, wild-type form of the IscR protein.
3.02 Heme Production in an IscR H107Y Mutant Strain Overexpressing ‘hemN or hemF’ Gene in Combination with hemALBCDEGH Genes
The parent E. coli used in the example listed below, is modified to knock-out the yfeX gene encoding a putative heme degradation enzyme; as well as to knock-out the pta and IdhA genes encoding phosphate acetyl transferase and lactate dehydrogenase, respectively, in order to enhance metabolic flux from glucose to L-glutamate. A derivative of the E. coli ΔyfeX-LdhA-Pta strain further comprises an iscR gene encoding an H107Y mutation in the iscR protein.
The heme pathway genes, hemALBCDEGH and either hemN or hemF, cloned into 3 plasmid (see table BA), are transformed into a derivative of an E. coli parent strain comprising either wild-type or H107Y mutant IscR protein. Additionally, strains are constructed wherein the hemA gene is mutated to express either of the heme feedback-insensitive hemA proteins: hemA(kk) comprising (L2K; L3K) substitutions or hemA comprising an (C107A) substitution.
Cells of each strain (Table BA) are cultivated in biotin-supplemented mMOPS medium supplemented with 100 μg/mL ampicillin, 50 μg/mL spectinomycin and 50 μg/mL kanamycin at 37° C., including IPTG-induction of the heme pathway genes, and the heme and prophyrin titer of the cultured cells is determined as described in Example 3.01. Strains carrying the hemF gene are cultured under aerobic conditions. Hemne and prophyrin production by an E. coli strain over-expressing the genes of the heme pathway (i.e. hemALBCDEGH and hemN), and lacking yfeX-LdhA-Pta genes, is enhanced when the host strain expresses a gene encoding a mutant form of the IscR protein (IscR protein having an H107Y substitution) as compared to a strain expressing a gene encoding a native, wild-type IscR protein. This enhancement does not occur in strains expressing Hem F instead of HemN.
3.03 Hemeprotein Production in an IscR H107Y Mutant Strain Overexpressing ‘hemN or hemF’ in Combination with hemALBCDEGH Genes and a Cytochrome P450 Monooxygenase
The following strains of Escherichia coli used in the example are listed below.
The heme pathway genes, hemALBCDEGH and either hemN or hemF as well as BM3* mutant gene derived from Bacillus megaterium, cloned into 3 plasmids (see table 88), are transformed into a derivative of an E. coli parent strain comprising either wild-type (ΔyfeX-LdhA-Pta) or H107Y mutant IscR protein (ΔyfeX-LdhA-Pta H107Y IscR), as shown in Table 8A.
Cells of each transformed strain are then cultivated in biotin-supplemented mMOPS medium supplemented with 100 μg/mL ampicillin, 50 μg/mL spectinomycin and 50 μg/mL kanamycin at 37° C., including IPTG-induction of the heme pathway genes and BM3*. Indole and NADPH are added as a substrate and cofactor of the P450 for a biotransformation. Indigo production by an E. coli strain over-expressing the monooxygenase gene BM3* and the genes of the heme pathway (i.e. hemALBCDEGH and hemN), and lacking yfeX-LdhA-Pta genes, is enhanced when the host strain expresses a gene encoding a mutant form of the IscR protein (IscR protein having an H107Y substitution) as compared to a strain expressing a gene encoding a native, wild-type IscR protein.
4.01 Enhanced Growth and Quinolinate Synthase Activity in an IscR H107Y Mutant Strain Overexpressing nadA Genes.
The following strains of Escherichia coli used in the example are listed below.
The following plasmids used in the example are listed below.
IPTG-inducible genes encoding NadA (quinolate synthase) was cloned to give plasmid pBS1167; and an empty plasmid pBS1259 having a p15A backbone was used as a control. pBS1167 or pBS1259 were introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strains BS2382 and BS2630, respectively. Similarly, the plasmids pBS1167 and pBS1259 were introduced into E. coli strain BS1011 carrying a wild-type version of the iscR gene, resulting in strains BS2379 and BS2629, respectively.
Cells of each strain (Table 9) are cultivated 24 h at 37° C. in parallel cultures comprising mMOPS medium (as described in Example 1.03) supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin and with 0 to 1 mM IPTG in deep culture plates. Cell growth was monitored over 24 h by measuring cell density at OD620 nm (
The catalytic activity of NadA expressed in IscR wild type (BS1011) and mutant (BS1353) E. coli strains transformed with pBS1651 or pBS1259 is determined by measuring quinolate, an intermediate in the NR pathway. Cells of each strain are grown in 50 ml mMOPS medium, with or without aspartic acid, supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin, at 37° C. and 250 rpm until the OD600nm reaches 0.6. IPTG is then added to each culture to a final concentration of 0.064 mM, that are then incubated for a further 6 h. Every hour, a 1 mL aliquot, sampled from each culture, is normalized to the same cell density (OD600 nm) and then pelleted with centrifugation at 17000 g for 5 min. Pellets, after washing in ice-cold PBS (phosphate buffer saline) solution, are re-suspended, and then lysed by sonication and finally centrifuged at 17000 g for 5 min. Quinolate, present in the recovered lysed cell supernatant, is measured by LC-MS using a 1290 Infinity series UHPLC coupled to a 6470 triple quadrupole from Agilent Technologies (Santa Clara, USA) as described by Ollagnier-de Choudens et. al., (2005). Alternatively HPLC can applied in stead af LC-MS. Quinolate production is also quantified in the supernatant by HPLC. Cells of each strain are grown in 50 ml mMOPS medium, without aspartic acid, supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin, and IPTG concentration between 0-1 mM and incubated at 37° C. for 24 h.
4.02 Enhanced Nicotinamide Riboside (NR) Production in an IscR H107Y Mutant Strain Overexpressing nadABC, nadE* and aphA Genes
The parent E. coli strain BS1011, and its mutant derivative expressing IscR H107Y, are transformed with a plasmid (pBS_NR) comprising the genes nadABCE*aphA operatively linked an IPTG inducible promoter, or with a control empty plasmid. The genes expressed in plasmid pBS_NAM include: E. coli nadA gene encodes quinolate synthase (NadA); the E. coli nadB encodes L-aspartate oxidase (NadB); nadC encodes Nicotinate-nucleotide pyrophosphorylase (NadC); aphA encodes a Class B acid phosphatase (AphA); and the Mannheimia succiniciproducens nadE gene encodes a polypeptide with nicotinic acid mononucleotide amidating activity (NadE*). Cells of each strain are grown in 50 ml mMOPS medium, supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin, at 37° C. and 250 rpm until the OD600 nm reaches 0.6. IPTG is then added to each culture to a final concentration of 0.064 mM that are then incubated for a further 6 h, and subsequently lysed.
NR, present in the recovered lysed cell supernatant, is measured by LC-MS using a 1290 Infinity series UHPLC coupled to a 6470 triple quadrupole from Agilent Technologies (Santa Clara, USA) (Ollagnier-de Choudens et. al., 2005). NR production by an E. coli strain expressing the genes of the NR pathway (i.e. nadABCE*aphA), is enhanced when the host strain expresses a gene encoding a mutant form of the IscR protein (IscR protein having an H107Y substitution) as compared to a strain expressing a gene encoding a native, wild-type IscR protein.
Cells of each strain are grown in 50 ml mMOPS medium, supplemented with 1 nmol/L biotin, 100 g/mL ampicillin, at 37° C. and 250 rpm until the OD600 nm reaches 0.6. IPTG is then added to each culture to a final concentration of 0.064 mM that are then incubated for a further 6 h, and subsequently lysed.
NR, present in the recovered lysed cell supernatant, is measured by LC-MS using a 1290 Infinity series UHPLC coupled to a 6470 triple quadrupole from Agilent Technologies (Santa Clara, USA) (Ollagnier-de Choudens et. al., 2005). NR production by an E. coli strain expressing the genes of the NR pathway (i.e. nadABCE*aphA), is enhanced when the host strain expresses a gene encoding a mutant form of the IscR protein (IscR protein having an H107Y substitution) as compared to a strain expressing a gene encoding a native, wild-type IscR protein.
4.03 Enhanced Nicotinamide (NAM) Production in an IscR H107Y Mutant Strains Overexpressing nadABC, nadE* and NMN Nucleosidase (Chi) Genes.
For nicotinamide NAM production E. coli strain BS1011, and its mutant derivative expressing IscR H107Y, are transformed with a plasmid (pBS_NAM) comprising the genes nadABCE* and chi operatively linked an IPTG inducible promoter, or with a control empty plasmid. The genes expressed in plasmid pBS_NAM include: E. coli nadA gene encodes quinolate synthase (NadA); the E. coli nadB encodes L-aspartate oxidase (NadB); nadC encodes Nicotinate-nucleotide pyrophosphorylase (NadC); chi encodes NMN nucleosidase (chi) and the Mannheimia succiniciproducens nadE* gene encodes a polypeptide with nicotinic acid mononucleotide amidating activity.
5.01 Enhanced Precorrin-3B Synthase Activity in an IscR H107Y Mutant Strain Overexpressing a cobG Gene.
The following strains of Escherichia coli used in the example are listed below.
The following plasmids used in the example are listed below.
IPTG-inducible gene encoding CobG (precorrin-3B synthase) was cloned into plasmid pBS1288; and an empty plasmid pBS1259 having a p15A backbone was used as a control. A first operon encoding CobIMF and a second operon encoding CobKHLJ was cloned on a further plasmid pBS1637. The plasmid pBS1288 (alone) or in combination with pBS1637; or pBS1259 alone were introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strains BS4139, BS4138 and BS2630, respectively. Additionally, pBS1288 (alone), or in combination with pBS1637, or pBS1259 alone were introduced into E. coli strain BS1011 carrying a wild-type version of the iscR gene, resulting in strains BS4140, B43137 and BS2629, respectively.
Cells of each strain (Table 11) are cultivated aerobically at 37° C. in parallel cultures comprising mMOPS medium (as described in Example 1.03) supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin (and additionally 50 μg/ml kanamycin for strains BS4137 and BS4138 or 50 μg/mL spectomycin for BS4141 and BS4142). When each culture reaches a cell density of 0.8-1.0 OD600nm, the culture medium is supplemented with 10 mM aminolaevulinic acid and 0.1 mM IPTG to induce cobG transgene expression, and incubated for at 28° C. for 24-48 h.
The catalytic activity of CobG transgenically expressed in the mutant E. coli strain (BS4139) as compared its expression in the IscR wild type (BS4140) is shown to be enhanced when determined by measuring hydrogenobyrinic acid (HBA) production, an intermediate in the Cob pathway, as follows. The cultured cells, harvested by centrifugation (17000 g, 5 min), are resuspended in IEX-Buffer A (20 mM Tris-HCl, pH 7.4, 100 mM NaCl); sonicated, centrifuged (17000 g 5 min), and the supernatant is then used for detection of HBA via LC-MS using a 1290 Infinity series UHPLC coupled to a 6470 triple quadrupole from Agilent Technologies (Santa Clara, USA).
The production of the intermediate, HBA, is further enhanced in the IscR mutant strain, BS4138, where the transgene encoding CobG and the transgenes encoding CobIMF and CobKHLJ are co-expressed in the cells, as compared to the parent host E. coli strain expressing wild type IscR (BS4137).
Cobalamin is produced by E. coli cells expressing an IPTG inducible transgene encoding CobG and a constitutively expressing transgenes encoding transgenes encoding CobHIJLFK, CobMNST, CobCDTPduX, CobROQBtuR and CobUSCbiB; and where the host E. coli cells further comprise the transgenes cbiNQOM inserted into their genome. E. coli strains BS4141 and BS4142 are cultured as described in 5.01 above. Cobalamin produced by the cultures is measured as follows: 2.5 mL of NaNO2 8% (w/v) and 2.5 mL of glacial acetic acid are added to 25 mL samples of each culture; which are then boiled for 30 min, and the resulting mixture filtered. Then 20 μL NaCN 10% (w/v) is added to 1 mL of aqueous phase; and 20 μL of resulting upper aqueous phase is injected into an HP1100 HPLC system (Agilent). NH2 column (4.6×250 mm2, 5 um) is employed for HPLC analysis with a flow rate of 1.7 ml/min and a wavelength of 360 nm, using a mobile phase of 250 mM phosphoric acid/acetonitrile (30/70, v/v). The production of cobalamin is enhanced when said host E. coli cells comprise the mutant iscR gene (BS4142) as compared to host E. coli cells comprising the WT iscR gene (BS4141).
6.01 Enhanced Growth and Dihydroxy-Acid Dehydratase Activity in an IscR H107Y Mutant Strain Overexpressing ilvD Gene.
The following strains of Escherichia coli used in the example are listed below.
The following plasmids used in the example are listed below.
IPTG-inducible gene encoding IlvD (dihydroxy-acid dehydratase) was cloned to give plasmid pBS1140; and an empty plasmid pBS1259 having a p15A backbone was used as a control. pBS1140 or pBS1259 were introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strains BS2381 and BS2630, respectively. Similarly, the plasmids pBS1140 or pBS1259 were introduced into E. coli strain BS1011 carrying a wild-type version of the iscR gene, resulting in strains BS2378 and BS2629, respectively.
Cells of each strain (Table 13) are cultivated 24 h at 37° C. in parallel cultures comprising mMOPS medium (as described in Example 1.03) supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin and with 0 to 1 mM IPTG in deep culture plates. Cell growth was monitored over 24 h by measuring cell density at OD620 (
The catalytic activity of IlvD expressed in IscR wild type (BS1011) and mutant (BS1353) E. coli strains transformed with pBS1140 or pBS1259 is determined by measuring 3-methyl-2-oxobutanoate, produced from 2,3-dihydroxy-3-methylbutanoate by dihydroxy-acid dehydratase. Cells of each strain are grown as described above; the cultures are normalized for cell density, and then subsequently pelleted at 17,000 g, where the supernatant is passed through a cation separation column and the eluant is then subsequently analysed for 3-methyl-2-oxobutanoate, amino acid and pantothenic acid content by LC-MS using a 1290 Infinity series UHPLC coupled to a 6470 triple quadrupole from Agilent Technologies (Santa Clara, USA) as described by Ollagnier-de Choudens et. al., (2005).
6.02 Enhanced Valine Production in an IscR H107Y Mutant Strain Overexpressing ilvD, ilvC, ilvB and ilvN Genes
The parent E. coli strain BS1011, and its mutant derivative expressing IscR H107Y BS1353, are transformed with a plasmid (pBS1652) comprising the genes ilvC, ilvB and ilvN operably linked a constitutive promoter, and pBS1140 comprising the ilvD gene, or only the control empty plasmid pBS1259. The E. coli ilvC gene in plasmid pBS1652 encodes ketol-acid reductoisomerase (NADP+); E. coli ilvB encodes acetolactate synthase isozyme 1 large subunit; and E. coli ilvN encodes acetolactate synthase isozyme 1 small subunit.
Cells of each strain are cultivated aerobically in 50 mL of NM1 Medium (NM1 composition: glucose, 20 g; (NH4)2SO4, 20 g; KH2PO4, 2.0 g; MgSO4, 7H2O, 0.4 g; NaCl, 1.6 g; yeast extract, 2 g; trace metal solution, supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin, 50 μg/mL kanamycin, at 31° C. and 250 rpm until reaching a cell density of OD600 reaches 0.4. IPTG is then added to the medium at a concentration of 0,028 mM and cells are further incubated for 24 h. 3-methyl-2-oxobutanoate produced in the culture medium by each strain, following normalization for cell density, is measured as described above.
The production of 3-methyl-2-oxobutanoate is enhanced in the IscR mutant strain, BS2632, when co-expressing the transgene encoding ilvD and the transgenes encoding ilvCBN, as compared to their co-expression in the parent host E. coli strain expressing wild type IscR (BS2631).
6.03 Enhanced L-Valine Production in an IscR H107Y Mutant Strain Overexpressing ilvD, ilvE, ilvC, ilvB and ilvNbis as Well as ygaZH and IrP.
The parent E. coli strain BS1011, and its mutant derivative expressing IscR H107Y B51353, are first genomically engineered to yield the strains BS3313 and BS3314. BS3313 and BS3314 are transformed with a plasmid (pBS1767) comprising the genes ilvD operatively linked to a T5 LacO promoter, and ilvE, ilvC, ilvB and ilvNbis operatively linked a constitutive promoter, and pBS1768 comprising the ygaZH and IrP gene. The E. coli ilvC gene in plasmid pBS1652 encodes ketol-acid reductoisomerase (NADP+); E. coli ilvB encodes acetolactate synthase isozyme 1 large subunit; E. coli ilvNbis encodes a mutated acetolactate synthase isozyme 1 small subunit. E. coli ilvE encodes a branched-chain-amino-acid aminotransferase; E. coli ygaH encodes a valine transporter; E. coli ygaz encodes a inner membrane protein; and E. coli IrP encodes a leucine-responsive regulatory protein.
Cells of each strain are cultivated aerobically in 50 mL of NM1 Medium (NM1 composition: glucose, 20 g; (NH4)2SO4, 20 g; KH2PO4, 2.0 g; MgSO4, 7H2O, 0.4 g; NaCl, 1.6 g; yeast extract, 2 g; trace metal, solution, supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin, 50 μg/ml kanamycin, at 31° C. and 250 rpm until reaching a cell density of OD600nm reaches 0.4. IPTG is then added to the medium at a concentration of 0,028 mM and cells are further incubated for 24 h. L-valine produced in the culture medium by each strain, following normalization for cell density, is measured as described in 6.01.
L-valine production is enhanced in the IscR mutant strain, BS3316, when co-expressing the transgene encoding ilvD and the transgenes encoding ilvE, ilvC, ilvB and ilvNbis as well as ygaZH and IrP as compared to expression in a host E. coli strain expressing wild type IscR (BS3315).
7.01 Enhanced Isoprenoid Precursor Synthesis Activity in an IscR H107Y Mutant Strain Overexpressing ispG and ispH Genes.
The following strains of Escherichia coli used in the example are listed below.
The following plasmids used in the example are listed below.
IPTG-inducible genes encoding IspG (4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase) and IspH (4-hydroxy-3-methylbut-2-enyl diphosphate reductase) were cloned to give plasmid pBS1139; and an empty plasmid pBS1259 having a p15A backbone was used as a control. pBS1139 or pBS1259 were introduced into the E. coli host strain (BS3318) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strains BS3141 and BS3143, respectively. Similarly, the plasmids pBS1139 or pBS1259 were introduced into E. coli strain BS3317 carrying a wild-type version of the iscR gene, resulting in strains BS3140 and BS3142, respectively. The E. coli host strains BS3317 and BS3318 (comprising the native iscR and mutant iscR genes respectively) are genetically modified to upregulate expression of the genes dxs, rpoS, idi and dxr; and to delete the gene ytjC. The native ribosomal binding site (RBS), upstream of these genes, is modification being the substituted with an optimized RBS sequence [dxsRBS, SEQ ID NO: 280]; [rpoSRBS, SEQ ID NO:282]; [idiRBS, SEQ ID NO: 284]; [dyrRBS, SEQ ID NO: 286]. Cells of each strain (Table 15) are cultivated 24 h at 30° C. in parallel cultures comprising LB medium supplemented with 100 μg/mL ampicillin at 250 rpm until reaching a cell density of OD600nm of 0.6. IPTG is then added to the medium at a concentration of 0.1 mM and cells are further incubated for 24 h. Next, a 6 mL cell suspension aliquot having an OD600 nm of 1.0 is centrifuged for 5 min (17000×g), and re-suspended in 10 mL acetonitrile/methanol/water 40:40:20 plus 0.1 M formic acid intracellular metabolite extraction. The sample is incubated at −20° C. for 60 min with periodic shaking. Following centrifugation as before, the supernatant is purified through a LC-NH2 resin and analyzed for the isoprenoid precursors, IPP and DMAPP, using a 1290 Infinity series UHPLC coupled to a 6470 triple quadrupole from Agilent Technologies (Santa Clara, USA), as described by Zhou K et al., 2012.
The catalytic activity of IspG and IspH expressed in IscR mutant (BS3140) E. coli strains transformed with pBS1139, as compared to Isc WT (BS3139) E. coli strain transformed with pBS1139 or pBS1259 is increased based on the increase in detected levels of the precursors IPP and DMAPP produced.
8.01 Enhanced L-Glutamic Acid Synthesis Activity in an IscR H107Y Mutant Strain Overexpressing gltB and gltD Genes.
The following strains of Escherichia coli used in the example are listed below.
The following plasmids used in the example are listed below.
IPTG-inducible genes encoding GltB (Glutamate synthase [NADPH] large chain) and GltD (Glutamate synthase [NADPH] small chain) were individually cloned to give plasmids pBS_gltB and pBS_glD respectively, as well as the two genes being cloned together to give plasmid pBS_gltBD (encoding both polypeptides of GOGAT); and an empty plasmid pBS1259 having a p15A backbone was used as a control. Each of the 3 plasmids (pBS_gltB; pBS_glD; pBS_gltBD and pBS1259) were individually introduced into the E. coli host strain (BS3149) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strains pBS_glt04, pBS_glt06, pBS_glt02 and BS2630, respectively. Similarly, these plasmids were introduced into E. coli strain BS1353 carrying a wild-type version of the iscR gene, resulting in strains BS_glt03, BS_glt05, BS_glt01, BS2629 respectively.
Overnight 500 uL seed cultures of each strain (Table 17) are cultivated in parallel at 37° C. in 50 mL LB medium supplemented with 100 μ/mL ampicillin and 5 g/l of NH4(SO4) as nitrogen source at 200 rpm until the cell density reaches an OD600nm of 0.8-1.0. IPTG is then added to the medium at a concentration of 0.1 mM of each culture, together with glutamine at a range of concentrations; and the cell cultures are further incubated at 28° C. for 24-48 h. Cells from each culture are then harvested by centrifugation (17000 g, 5 min). The supernatant is collected and used for detection of extracellular glutamate by colorimetric assay (#K629-100, Biovision, Milpitas, Calif., USA). Additionally, L-Glutamate is quantified via LC-MS using a 1290 Infinity series UHPLC coupled to a 6470 triple quadrupole from Agilent Technologies (Santa Clara, USA).
The catalytic activity of GOGAT, expressed in the IscR mutant E. coli strain transformed with pBS_gltBD (strain BS_glt02), as compared GOGAT expressed in Isc WT E. coli strain transformed with pBS_gltBD (BS_glt01), or either E. coli strain transformed with the control empty plasmid or plasmids pBS_gltB or pBS_gltD is increased, based on an increase in detected levels of L-glutamine produced.
8.02 Enhanced Aminolevulonic Acid Synthesis Activity in an IscR H107Y Mutant Strain Overexpressing gltB and gltD Genes Alongside Gltx, hemA and hemL Genes.
IPTG-inducible genes encoding GltDB (Glutamate synthase) and constitutively expressed GltX (Glutamate-tRNA ligase), HemA (Glutamyl-tRNA reductase), HemL (Glutamate-1-semialdehyde 2,1-aminomutase) were cloned to give the plasmid pBS1769. pBS1769 was introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strain BS3327. Similarly, pBS1769 was introduced into E. coli strain BS1011 carrying a wild-type version of the iscR gene, resulting in strain BS3326.
Overnight 500 μL seed cultures of each strain (Table 17) are cultivated in parallel at 37° C. in 50 mL LB medium supplemented with 100 μg/mL ampicillin and 5 g/l of NH4(SO4) as nitrogen source at 200 rpm until the cell density reaches an OD600nm of 0.8-1.0. IPTG is then added to the medium at a concentration of 0.1 mM of each culture, together with glutamine at a range of concentrations; and the cell cultures are further incubated at 28° C. for 24-48 h. Cells from each culture are then harvested by centrifugation (17000 g, 5 min). The supernatant is collected and used for detection of extracellular aminolaevulinic acid (ALA) using an Ehrlich reagent. ALA is then quantified by spectrometric analysis at 550 nm.
The catalytic activity of GltDB, expressed in the IscR mutant E. coli strain transformed with pBS1769, is increased as compared GltDB expressed in IscR WT E. coli strain transformed with pBS1769.
9.01 Enhanced PqqA Peptide Cyclase Activity in an IscR H107Y Mutant Strain Overexpressing a pqqA Gene.
The following strains of Escherichia coli used in the example are listed below.
The following plasmids used in the example are listed below.
IPTG-inducible operon comprising genes pqqA, pqqB, pqqc, pqqD, pqqE and pqqF genes derived from the pQQABCDEF operon of Klebsiella pneumoniae (ATCC 19606) encoding PqqABCDEF was cloned to create plasmid pBS_PQQ; and an empty plasmid pBS1259 having a p15A backbone was used as a control. The plasmid pBS_PQQ or the control pBS1259 were introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strain BS_PQQ2 and BS2630, respectively. Additionally, the plasmids pBS_PQQ and pBS1259 were introduced into E. coli strain BS1011 carrying a wild-type version of the iscR gene, resulting in strains BS_PQQ1 and BS2629, respectively.
Overnight 500 uL seed cultures of each strain (Table 19) are cultivated in parallel at 37° C. in 50 ml LB medium supplemented with 1.0 nM biotin, 100 μg/mL ampicillin at 200 rpm until the cell density reaches an OD600 nm of 0.8-1.0. IPTG is then added to the medium; and the cell cultures are further incubated at 28° C. for 24-48 h. Cells from each culture are then harvested by centrifugation (17000 g, 5 min). The supernatant is collected and used for detection of extracellular PQQ is quantified via LC-MS using a 1290 Infinity series UHPLC coupled to a 6470 triple quadrupole from Agilent Technologies (Santa Clara, USA) as described by Noji, et al., 2007.
The catalytic activity of PqqA peptide cyclase activity (PqqE), expressed in the IscR mutant E. coli strain transformed with pBS_PQQ (strain BS_PQQ2), as compared with its expression in Isc WT E. coli strain transformed with pBS_PQQ (strain BS_PQQ2), or either E. coli strain transformed with the control empty plasmid or plasmids pBS_PQQ is increased, based on an increase in detected levels of PQQ produced.
10.01 Enhanced Growth and Nitrogenase Activity in an IscR H107Y Mutant Strain Overexpressing nifB Gene.
The following strains of Escherichia coli used in the example are listed below.
The following plasmids used in the example are listed below.
IPTG-inducible gene encoding NifB (Nitrogenase iron-molybdenum cofactor biosynthesis protein) was cloned to give plasmid pBS1169; and an empty plasmid pBS1259 having a p15A backbone was used as a control. pBS1169 or pBS1259 were introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strains BS2472 and BS2630, respectively. Similarly, the plasmids pBS1169 or pBS1259 were introduced into E. coli strain BS1011 carrying a wild-type version of the iscR gene, resulting in strains BS2378 and BS2629, respectively.
Cells of strains in Table 21 were cultivated 24 h at 37° C. in parallel cultures comprising mMOPS medium (as described in Example 1.03) supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin and with 0 or 1 mM IPTG in deep culture plates. Cell growth was monitored over 24 h by measuring cell density at OD620 nm (
10.02 Enhanced Nitrogenase Activity and Nitrogen Fixation in an IscR H107Y Mutant Strain Overexpressing nifB, or Co-Overexpressing NifB and Additional Nitrogenase Pathway Genes
Additionally, E. coli strains having the wild type (BS1011) and mutant IscR gene (BS1353) were transformed with both pBS1169 (comprising the NifB gene) as well as pBS1653 plasmid comprising genes encoding NifHDKENXVHesA; and pBS1654 plasmid comprising genes encoding NifU, NifS and FldA. The catalytic activity of NifB expressed in the respective E. coli strains (Table 21) is determined as follows. Cells of each of the strains are cultured in mMOPS medium (as described in Example 1.03) supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin at 30 degrees Celsius for 16 h, then centrifuged (4000 g for 5 min) and washed three times in 1 mL water. Cells from each culture are then re-suspended in nitrogen-deficient medium (10.4 g Na2HPO4, 3.4 g KH2PO4, 26 mg CaCl2.2H2O, 30 mg MgSO4, 0.3 mg MnSO4, 36 mg Ferric citrate, 7.6 mg Na2MoO4.2H2O, 10 μg p-aminobenzoic acid, 5 μg biotin and 4 g glucose per liter and supplemented with 2 mM glutamate as nitrogen source. An optimal IPTG concentration for nifB gene expression was added. When OD600nm 0.4 is reached, 1 ml of each culture is transferred to an oxygen isolated tube by using argon gas to fill headspace. After 8 h incubation at 30° C., the obtained cells are assayed for nitrogenase activity by incubating the cells with acetylene (10% tube headspace volume) for 3 additional hours. The incubated samples are then analysed for ethylene levels, resulting from acetylene reduction, by gas chromatography, therefore giving a measure of nitrogenase activity in the strains.
Nitrogenase activity is enhanced in the IscR mutant E. coli strain, B52472, expressing the transgene encoding nifB alone, and is further enhanced in IscR mutant E. coli strain, B52474, by co-expression of transgenes encoding NifHDKENXVHesA and NifUSFIdA, as compared to their co-expression in the parent host E. coli strain expressing wild type IscR (BS2473).
The following strains of Escherichia coli used in the example are listed below.
The following plasmids used in the example are listed below.
IPTG-inducible operon comprising the naphthalene dioxygenase NdoBC and reductase NdoRA genes derived from the Pseudomonas putida were cloned to create plasmid pBS_NdoBCRA; and an empty plasmid pBS1259 having a p15A backbone was used as a control. The plasmid pBS_NdoBCRA and the control plasmid pBS1259 were introduced into the E. coli host strain (BS1353) in which the native iscR gene was substituted by a mutant iscR gene encoding an IscR protein having an H107Y substitution (as described in Example 1) resulting in the strain BS_NdoBCRA2 and BS2630, respectively. Additionally, the plasmids pBS_NdoBCRA and pBS1259 were introduced into E. coli strain BS1011 carrying a wild-type version of the iscR gene, resulting in strains BS_NdoBCRA1 and BS2629, respectively.
Overnight 500 uL seed cultures of each strain (Table 19) are cultivated in parallel at 37° C. in 50 mL LB medium supplemented with 1.0 nM biotin and 100 μg/mL ampicillin, at 200 rpm until the cell density reaches an OD600nm of 0.6. IPTG (0.5 mM) is then added to the medium; and the cell cultures are further incubated at 27° C. for 24-48 h. The cells are then harvested, mixed with an equal volume of N,N dimethylformamide to solubilize and extract the indigo, and following centrifugation to remove biomass, the absorbance of the extract is read at OD620. Indigo concentrations are calculated by comparison to a standard curve constructed using synthetic indigo.
The catalytic activity of the naphthalene dioxygenase NdoBC and reductase NdoRA complex expressed in the IscR mutant E. coli strain transformed with pBS_NdoBCRA (strain BS_NdoBCRA2), as compared with its expression in Isc WT E. coli strain transformed with pBS_NdoBCRA (strain BS_NdoBCRA1), or either E. coli strain transformed with the control empty plasmid is increased, based on an Increase in detected levels of Indigo pigment produced.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19152181.4 | Jan 2019 | EP | regional |
This is a national phase application of International Application No. PCT/EP2020/050950, filed 15 Jan. 2020, which claims the benefit of European Patent Application No. 19152181.4, filed 16 Jan. 2019, the disclosures of which are incorporated, in their entireties, by this reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/050950 | 1/15/2020 | WO | 00 |
