Fermentation process

Information

  • Patent Grant
  • 11932672
  • Patent Number
    11,932,672
  • Date Filed
    Wednesday, December 19, 2018
    6 years ago
  • Date Issued
    Tuesday, March 19, 2024
    9 months ago
Abstract
Some embodiments relate to a method for producing a product of interest with a microbial host using an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers an advantage to the host, optionally wherein the genetic activity of said first nucleic acid molecule is controlled.
Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2018/085941, filed on Dec. 19, 2018, which claims the benefit of European Application No. EP17208600, filed Dec. 19, 2017. The content of each of the aforementioned applications is expressly incorporated herein by reference in its entirety.


FIELD

Embodiments herein relate to a method for producing a product of interest with a microbial host using an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers an advantage to the host, optionally wherein the genetic activity of said first nucleic acid molecule is controlled.


BACKGROUND

Antibiotics are widely used as selection agents for the production of a product of interest in microbial cells. However, there are several drawbacks associated with the use of antibiotics such as large-scale spreading of antibiotics in the environment. In addition the sequence coding for the resistance of the antibiotic in the DNA constructs represent an energetic burden for the cell and therefore negatively affects the yield of the product. This energetic burden is particularly relevant when the resistance-conferring gene is a large gene, when it is expressed at a high level and/or when it is expressed constitutively.


Therefore, there is still a need for an alternative and even improved method, which does not have all the drawbacks of existing methods.


DESCRIPTION

In a first aspect, there is provided a method for producing a product of interest with a microbial host, said method comprising the steps of:

    • a) Providing the microbial host comprising an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence, optionally wherein the genetic activity of said first nucleic acid sequence is controlled;
    • b) Optionally said auto-replicative extra-chromosomal nucleic acid molecule comprises a second nucleic acid sequence that is involved in the production of said product of interest, wherein the genetic activity of said second nucleic acid sequence is controlled independently from the first sequence;
    • c) Culturing said microbial host under conditions allowing said microbial host to express the first nucleic acid sequence to a given level to maintain the auto-replicative extra-chromosomal molecule into the growing microbial population and simultaneously genetically controlling the second sequence coding for said product of interest.


      Step a)


Step a) comprises providing a microbial host comprising an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers an advantage to the host, optionally wherein the genetic activity of said first nucleic acid sequence is controlled. The auto-replicative extra-chromosomal nucleic acid molecule can be provided in a microbial host (e.g., a microbial cell as described herein). For example, the host or a predecessor of the host may have been previously transformed with the auto-replicative extra-chromosomal nucleic acid molecule. As such, in some embodiments, step a) comprises providing a microbial cell host comprising an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers an advantage to the host, optionally wherein the genetic activity of said first nucleic acid sequence is controlled.


Optional Transforming Step


In some embodiments, the microbial host is transformed with the auto-replicative extra-chromosomal nucleic acid molecule under conditions allowing only host that has received said auto-replicative extra-chromosomal nucleic acid molecule to survive, thus providing a microbial host comprising an auto-replicative extra-chromosomal nucleic acid molecule. As such, in some embodiments, the method further comprises transforming the microbial host with said auto-replicative extra-chromosomal nucleic acid molecule prior to or during step a) under conditions allowing only host that has received said auto-replicative extra-chromosomal nucleic acid molecule to survive, thus providing the microbial host comprising the auto-replicative extra-chromosomal nucleic acid molecule.


The auto-replicative extra-chromosomal nucleic acid molecule transformed into the microbial host optionally comprises the second nucleic acid sequence of step b). The microbial host comprising the auto-replicative extra-chromosomal nucleic acid molecule can subsequently be cultured according to step c).


Within the context of methods, uses, compositions, hosts, and nucleic acids of embodiments herein, an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence is provided. An auto-replicative extra-chromosomal nucleic acid molecule can exist free of the genome and may be derived from or comprise, consist essentially of, or consist of a plasmid, or episome, minichromosome, or alike. This feature is attractive as a higher number (from one to hundreds of copies or from 10 to 50 copies depending on the plasmid used) of copies of such nucleic acid molecule can be introduced and maintained into the microbial cell host. In addition, any host can be used in the methods of embodiments herein. In some embodiments, there is no need to modify the genome of the host. The genetic elements needed to carry out the methods of embodiments herein are present in the auto-replicative extra-chromosomal nucleic acid molecule. Such an auto-replicative extra-chromosomal nucleic acid molecule usually comprises an origin of replication, a first nucleic acid sequence which is of interest and a regulatory region. In some embodiments, without being limited by theory, a first nucleic acid sequence encoding an immunity modulator acts as a selectable marker to maintain the presence and function of the auto-replicative extra-chromosomal nucleic acid in the host cell. In some embodiments, the first nucleic acid sequence encoding the immunity modulator maintains the presence of the auto-replicative extra-chromosomal nucleic acid so that a product can be produced. The product can alter the environment in which the host is present, for example by fermenting a substance in the environment to produce one or more new substances. In some embodiments, genetic drift is minimized by providing selective pressure against auto-replicative extra-chromosomal nucleic acids that have acquired mutations, and do not produce a functional immunity modulator, produce an immunity modulator with reduced function, and/or produced lower levels of immunity modulator than an auto-replicative extra-chromosomal nucleic acid that has not acquired the mutation(s).


Within the context of methods, uses compositions, hosts, and nucleic acids of embodiments herein, the first nucleic acid molecule represented by the first nucleic acid sequence is able to exhibit a genetic activity, said genetic activity confering an selective advantage to the microbial host cell wherein it is present and wherein this genetic activity is expressed. This genetic activity is provided by the product encoded by the first nucleic acid molecule. Moreover this genetic activity can be controlled or is expressed constitutively at a low level or is tunable or is under the control of a weak constitutive promoter. The control of said activity is believed to provide an advantage to limit the burden of energy for the host. Similarly, an advantage to limit the energy burden of the host may be obtained when the genetic activity is expressed constitutively at a low level or is tunable or is under the control of a weak constitutive promoter. Throughout the application text, the concept “conferring an advantage” may be replaced by “conferring immunity to a bacteriocin” or “conferring resistance to a bacteriocin”. In some embodiments, the first nucleic acid sequence encodes an immunity modulator as described herein, and thereby confers an advantage to the host.


A second nucleic acid sequence encodes directly or indirectly for a product of interest. The same description holds for the genetic activity of the second nucleic acid molecule described herein. In some embodiments, the product of interest comprises an enzyme that is useful in an industrial process, for example a fermentation process. The fermentation process can ferment at least one compound in the culture medium. In some embodiments, the product of interest comprises an industrially useful molecule, for example a carbohydrate, a lipid, an organic molecule, a nutrient, a fertilizer, a biofuel, a cosmetic (or precursor thereof), a pharmaceutical or biopharmaceutical product (or precursor thereof), or two or more of any of the listed items.


Within the context of methods, uses, compositions, hosts, and nucleic acids of embodiments herein, a genetic activity may mean any activity that is caused by or linked with the presence of the first nucleic acid molecule in a microbial host. The advantage of said activity may be the ability to survive or survive and grow under given conditions (pH, temperature, presence of a given molecule such as a bacteriocin or combination of two or more bacteriocins as described herein, . . . ). Accordingly the advantage of said activity may be assessed by determining the number of microbial cells/hosts comprising the auto-replicative extrachromosomal nucleic acid molecule. The assessment may be carried out at the end of and/or during the optional transforming step (but prior to culturing step c), or prior to steps a) and culturing step c)) and/or prior to culturing step c). In an embodiment, the number of microbial host cells comprising the auto-replicative extra-chromosomal nucleic acid molecule present has not been decreased and may be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more compared to the number of initial microbial cells/host when the cells are being cultured under conditions allowing the microbial host that has received said auto-replicative extra-chromosomal nucleic acid molecule to survive (e.g., by possessing immunity to one or more bacteriocins as described herein, and which are present in the given conditions). This assessment step may have a duration of at least 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours or more, including ranges between any two of the listed values.


Within the context of methods, uses, compositions, hosts, and nucleic acids of embodiments herein, the control of a genetic activity may mean either an increase or decrease of activity of a nucleic acid molecule (i.e. first and/or second nucleic acid molecule). Accordingly, the control of a genetic activity can be controlled or is expressed constitutively at a low level or is tunable or is under the control of a weak constitutive promoter. In some embodiments, the coding product for which genetic activity is regulated/controlled comprises, consists essentially of, or consists of an immunity modulator or is involved in the production of a product of interest. In some embodiments, genetic activity is regulated/controlled at the level of gene expression. In some embodiments, genetic activity is regulated at the transcriptional level, for example by activating or repressing a promoter. In some embodiments, promoters in this context are inducible promoters. In some embodiments, promoters in this context are weak promoters. Without being limited by theory, weak promoters of some embodiments can be amendable to up- or down-regulating the level of transcription so that the advantage conferred to the host (e g immunity modulator activity) is sensitive to changes in levels and/or activity of the gene product(s) under the control of the promoter. In some embodiments, the promoter comprises, consists of, or consists essentially of the P24 promoter represented by SEQ ID NO:707 and/or the ProC promoter represented by SEQ ID NO: 708 and/or the P24 LacO hybrid promoter. The P24LacO hybrid promoter is a tunable/controlled promoter. In some embodiments, gene activity is regulated/controlled at the post-transcriptional level, for example through regulation of RNA stability. In some embodiments, genetic activity is regulated/controlled at the translational level, for example through regulation of initiation of translation. In some embodiments, genetic activity is regulated/controlled at the post-translational level, for example through regulation of polypeptide stability, post-translational modifications to the polypeptide, or binding of an inhibitor to the polypeptide.


In some embodiments, genetic activity is increased. In some embodiments, activity of at least one of an immunity modulator and/or the coding product of the second nucleic acid molecule is involved in the production of a product of interest is increased. Conceptually, genetic activity can be increased by directly activating genetic activity, or by decreasing the activity of an inhibitor of genetic activity. In some embodiments, genetic activity is activated by at least one of: inducing promoter activity, inhibiting a transcriptional repressor, increasing RNA stability, inhibiting a post-transcriptional inhibitor (for example, inhibiting a ribozyme or antisense oligonucleotide), inducing translation (for example, via a regulatable tRNA), making a desired post-translational modification, or inhibiting a post-translational inhibitor (for example a protease directed to a polypeptide encoded by the gene). In some embodiments, a compound present in a desired environment induces a promoter. For example, the presence of iron in culture medium can induce transcription by an iron-sensitive promoter as described herein. In some embodiments, a compound present in a desired culture medium inhibits a transcriptional repressor. For example, the presence of tetracycline in an environment can inhibit the tet repressor, and thus allow activity from the tetO promoter. In some embodiments, a compound found only outside of a desired culture medium induces transcription.


In some embodiments, genetic activity is decreased. Conceptually, genetic activity can be decreased by directly inhibiting genetic activity, or by decreasing the activity of an activator of genetic activity. In some embodiments, genetic activity is reduced, but some level of activity remains. In some embodiments, genetic activity is fully inhibited. In some embodiments, genetic activity is decreased by at least one of inhibiting promoter activity, activating a transcriptional repressor, decreasing RNA stability, activating a post-transcriptional inhibitor (for example, expressing a ribozyme or antisense oligonucleotide), inhibiting translation (for example, via a regulatable tRNA), failing to make a required post-translational modification, inactivating a polypeptide (for example by binding an inhibitor or via a polypeptide-specific protease), or failing to properly localize a polypeptide. In some embodiments, genetic activity is decreased by removing a gene from a desired location, for example by excising a gene using a FLP-FRT or cre-lox cassette, homologous recombination or CRIPR-CAS9 activity or through loss or degradation of a plasmid. In some embodiments, a gene product (e.g. a polypeptide) or a product produced by a gene product (e.g. the product of an enzymatic reaction) inhibits further gene activity (e.g. a negative feedback loop).


In some embodiments, the advantage conferred to a microbial host by the genetic activity of the first nucleic acid molecule is the ability to survive or survive and grow in a medium comprising a bacteriocin (or a mix of bacteriocins). As used herein, “bacteriocin” encompasses a cell-free or chemically synthesized version of such a polypeptide. A “bacteriocin,” and variations of this root term, may also refer to a polypeptide that had been secreted by a host cell. A bacteriocin therefore encompasses a proteinaceous toxin produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are similar to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse. A bacteriocin also encompasses a synthetic variant of a bacteriocin secreted by a host cell. Synthetic variant of a bacteriocin may be derived from the bacteriocin secreted by a host cell in any way as long as the synthetic variant still exhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 901© of the activity of the corresponding bacteriocin secreted by a host cell A detailed description of an antibiotic is provided in the part dedicated to general descriptions at the end of the specification.


A “bacteriocin” can neutralize at least one cell other than the individual host cell in which the polypeptide is made, including cells clonally related to the host cell and other microbial cells.


A cell that expresses a particular “immunity modulator” (discussed in more detail herein) is immune to the neutralizing effects of a particular bacteriocin or group of bacteriocins. As such, bacteriocins can neutralize a cell producing the bacteriocin and/or other microbial cells, so long as these cells do not produce an appropriate immunity modulator. As such, a bacteriocin can exert cytotoxic or growth-inhibiting effects on a plurality of other microbial organisms. In an embodiment, a bacteriocin is produced by the translational machinery (e.g. a ribosome, etc.) of a microbial cell. In another embodiment, a bacteriocin is chemically synthesized. Some bacteriocins can be derived from a polypeptide precursor. The polypeptide precursor can undergo cleavage (for example processing by a protease) to yield the polypeptide of the bacteriocin itself. As such, in some embodiments, a bacteriocin is produced from a precursor polypeptide. In some embodiments, a bacteriocin comprises, consists essentially of, or consists of a polypeptide that has undergone post-translational modifications, for example cleavage, or the addition of one or more functional groups.


Neutralizing activity of bacteriocins can include arrest of microbial reproduction, or cytotoxicity. Some bacteriocins have cytotoxic activity (e.g. “bacteriocide” effects), and thus can kill microbial organisms, for example bacteria, yeast, algae, synthetic micoorganisms, and the like. Some bacteriocins can inhibit the reproduction of microbial organisms (e.g. “bacteriostatic” effects), for example bacteria, yeast, algae, synthetic micoorganisms, and the like, for example by arresting the cell cycle.


A number of bacteriocins have been identified and characterized (see tables 1.1 and 1.2.). Without being limited by any particular theory, exemplary bacteriocins can be classified as “class I” bacteriocins, which typically undergo post-translational modification, and “class II” bacteriocins, which are typically unmodified. Additionally, exemplary bacteriocins in each class can be categorized into various subgroups, as summarized in Table 1.1, which is adapted from Cotter, P. D. et al. “Bacteriocins—a viable alternative to antibiotics” Nature Reviews Microbiology 11: 95-105, hereby incorporated by reference in its entirety.


Without being limited by any particular theory, bacteriocins can effect neutralization of a target microbial cell in a variety of ways. For example, a bacteriocin can permeabilize a cell wall, thus depolarizing the cell wall and interfering with respiration. Table 1.1: Classification of Exemplary Bacteriocins.









TABLE 1.1







Classification of Exemplary Bacteriocins









Group
Distinctive feature
Examples










Class I (typically modified)









MccC7-C51-type
Is covalently attached to a carboxy-
MccC7-051


bacteriocins
terminal aspartic acid



Lasso peptides
Have a lasso structure
MccJ25


Linear azole- or
Possess heterocycles but not other
MccB17


azoline-containing
modifications



peptides




Lantibiotics
Possess lanthionine bridges
Nisin, planosporicin,




mersacidin, actagardine,




mutacin 1140


Linaridins
Have a linear structure and contain
Cypemycin



dehydrated amino acids



Proteusins
Contain multiple hydroxylations,
Polytheonamide A



epimerizations and methylations



Sactibiotics
Contain sulphur-α-carbon linkages
Subtilosin A, thuricin CD


Patellamide-like
Possess heterocycles and undergo
Patellamide A


cyanobactins
macrocyclization



Anacyclamide-like
Cyclic peptides consisting of
Anacyclamide A10


cyanobactins
proteinogenic amino acids with prenyl




attachments



Thiopeptides
Contain a central pyridine,
Thiostrepton, nocathiacin I,



dihydropyridine or piperidine ring as
GE2270 A, philipimycin



well as heterocycles



Bottromycins
Contain macrocyclic amidine, a
Bottromycin A2



decarboxylated carboxy-terminal thiazole




and carbon-methylated amino acids



Glycocins
Contain S-linked glycopeptides
Sublancin 168







Class II (typically unmodified or cyclic)









IIa peptides (pediocin
Possess a conserved YGNGV motif (in
Pediocin PA-1, enterocin


PA-1-like bacteriocins)
which N represents any amino acid)
CRL35, carnobacteriocin




BM1


IIb peptides
Two unmodified peptides are required for
ABP118, lactacin F



activity



IIc peptides
Cyclic peptides
Enterocin AS-48


IId peptides
Unmodified, linear, non-pediocin-like,
MccV, MccS, epidermicin



single-peptide bacteriocins
NI01, lactococcin A


IIe peptides
Contain a serine-rich carboxy-terminal
MccE492, MccM



region with a non-ribosomal siderophore-




type modification









A number of bacteriocins can be used in accordance with embodiments herein. Exemplary bacteriocins are shown in Table 1.2. In some embodiments, at least one bacteriocin comprising, consisting essentially of, or consisting of a polypeptide sequence of Table 1.2 is provided. As shown in Table 1.2, some bacteriocins function as pairs of molecules. As such, it will be understood that unless explicity stated otherwise, when a functional “bacteriocin” or “providing a bacteriocin,” or the like is discussed herein, functional bacteriocin pairs are included along with bacteriocins that function individually. With reference to Table 1.2, “organisms of origin” listed in parentheses indicate alternative names and/or strain information for organisms known to produce the indicated bacteriocin.


Embodiments herein also include peptides and proteins with identity to bacteriocins described in Table 1.2. The term “identity” is meant to include nucleic acid or protein sequence homology or three-dimensional homology. Several techniques exist to determine nucleic acid or polypeptide sequence homology and/or three-dimensional homology to polypeptides. These methods are routinely employed to discover the extent of identity that one sequence, domain, or model has to a target sequence, domain, or model. A vast range of functional bacteriocins can incorporate features of bacteriocins disclosed herein, thus providing for a vast degree of identity to the bacteriocins in Table 1.2. In some embodiments, a bacteriocin has at least 50% identity, for example, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the polypeptides of Table 1.2. Percent identity may be determined using the BLAST software (Altschul, S. F., et al. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410, accessible on the world wide web at blast.ncbi.nlm.nih.gov) with the default parameters.


While the bacteriocins in Table 1.2 are naturally-occurring, the skilled artisan will appreciate that variants of the bacteriocins of Table 1.2, naturally-occurring bacteriocins other than the bacteriocins of Table 1.2 or variants thereof, or synthetic bacteriocins can be used according to some embodiments herein. In some embodiments, such variants have enhanced or decreased levels of cytotoxic or growth inhibition activity on the same or a different microorganism or species of microorganism relative to the wild type protein. Several motifs have been recognized as characteristic of bacteriocins. For example, the motif YGXGV (SEQ ID NO: 2), wherein X is any amino acid residue, is a N-terminal consensus sequence characteristic of class Ila bacteriocins. Accordingly, in some embodiments, a synthetic bacteriocin comprises an N-terminal sequence with at least 50% identity to SEQ ID NO: 2, for example at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2. In some embodiments, a synthetic bacteriocin comprises a N-terminal sequence comprising SEQ ID NO: 2. Additionally, some class lib bacteriocins comprise a GxxxG motif (x means any amino acid). Without being limited by any particular theory, it is believed that the GxxxG motif can mediate association between helical proteins in the cell membrane, for example to facilitate bacteriocin-mediated neutralization through cell membrane interactions. As such, in some embodiments, the bacteriocin comprises a motif that facilitates interactions with the cell membrane. In some embodiments, the bacteriocin comprises a GxxxG motif. Optionally, the bacteriocin comprising a GxxxG motif can comprise a helical structure. In addition to structures described herein, “bacteriocin” as used herein also encompasses structures that have substantially the same effect on microbial cells as any of the bacteriocins explicitly provided herein.


It has been shown that fusion polypeptides comprising, consisting essentially of, or consisting of two or more bacteriocins or portions thereof can have neutralizing activity against a broader range of microbial organisms than either individual bacteriocin. For example, it has been shown that a hybrid bacteriocin, Ent35-MccV (GKYYGNGVSCNKKGCSVDWGRAIGIIGNNSAANLATGGAAGWKSGGGASGR DIAMAIGTLSGQFVAGGIGAAAGGVAGGAIYDYASTHKPNPAMSPSGLGGTIK QKPEGIPSE AWNYAAGRLCNWSPNNLSDVCL, SEQ ID NO: 3), displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria (Acuna et al. (2012), FEBS Open Bio, 2: 12-19). It is noted that that Ent35-MccV fusion bacteriocin comprises, from N-terminus to C-terminus, an N-terminal glycine, Enterocin CRL35, a linker comprising three glycines, and a C-terminal Microcin V. It is contemplated herein that bacteriocins can comprise fusions of two or more polypeptides having bacteriocin activity. In some embodiments, a fusion polypeptide of two or more bacteriocins is provided. In some embodiments, the two or more bacteriocins comprise, consist essentially of, or consist of polypeptides from Table 1.2, or modifications thereof. In some embodiments, the fusion polypeptide comprising of two or more bacteriocins has a broader spectrum of activity than either individual bacteriocin, for example having neutralizing activity against more microbial organisms, neutralizing activity under a broader range of environmental conditions, and/or a higher efficiency of neutralization activity. In some embodiments, a fusion of two or more bacteriocins is provided, for example two, three, four, five, six, seven, eight, nine, or ten bacteriocins. In some embodiments, two or more bacteriocin polypeptides are fused to each other via a covalent bond, for example a peptide linkage. In some embodiments, a linker is positioned between the two bacteriocin polypeptides. In some embodiments, the linker comprises, consists essentially of, or consists of one or more glycines, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glycines. In some embodiments, the linker is cleaved within the cell to produce the individual bacteriocins included in the fusion protein. In some embodiments, a bacteriocin as provided herein is modified to provide a desired spectrum of activity relative to the unmodified bacteriocin. For example, the modified bacteriocin may have enhanced or decreased activity against the same organisms as the unmodified bacteriocin. Alternatively, the modified bacteriocin may have enhanced activity against an organism against which the unmodified bacteriocin has less activity or no activity.









TABLE 1.2







Exemplary Bacteriocins











Poly-






peptide



Poly-


SEQ



nucleotide


ID



SEQ ID


NO:
Name
Class
Organism of origin
NO:














4
Acidocin 8912
Unclassified

Lactobacillus
acidophilus

5


6
Acidocin A
class IIA/YGNGV

Lactobacillus
acidophilus

7


8
Acidocin B
Unclassified

Lactobacillus
acidophilus

9



(AcdB)





10
Acidocin
Unclassified

Lactobacillus
gasseri

11



LF221B






(Gassericin K7






B)





12
Aureocin A53
Unclassified

Staphylococcus
aureus

13


14
Avicin A
class IIA/YGNGV

Enterococcus
avium (Streptococcus

15






avium)




16
Bacteriocin 31
Unclassified

Enterococcus
faecalis

17





(Streptococcusfaecalis)



18
Bacteriocin J46
Unclassified

Lactococcus
lactis

19


20
Bacteriocin T8
class IIa

Enterococcus
faecium

21





(Streptococcusfaecium)



22
Boticin B
Unclassified

Clostridium
botulinum

23


24
Bovicin HJ50
Lantibiotic

Streptococcus
equinus

25





(Streptococcusbovis)



26
Brochocin-c
Unclassified

Brochothrix
campestris

27


28
Butyrivibriocin
Unclassified

Butyrivibrio
fibrisolvens

29



AR10





30
Butyrivibriocin
Lantibiotic

Butyrivibrio
fibrisolvens

31



OR79





32
Carnobacteriocin
class IIA/YGNGV

Carnobacterium
maltaromaticum

33



B2 (Carnocin

(Carnobacteriumpiscicola)




CP52)





34
Carnobacteriocin
class IIA/YGNGV

Carnobacterium
maltaromaticum

35



BM1

(Carnobacteriumpiscicola)




(Carnobacterioc






in B1)





36
Carnobacteriocin-
class IIc, non

Carnobacterium
maltaromaticum

37



A (Piscicolin-
subgrouped
(Carnobacteriumpiscicola)




61)
bacteriocins






(problematic)




38
Carnocyclin-A
Unclassified

Carnobacterium
maltaromaticum

39





(Carnobacteriumpiscicola)



40
Carocin D
Unclassified

Pectobacterium
carotovorum subsp.

41






carotovorum (Erwiniacarotovora







subsp. carotovora)



42
Cerein 7B
Unclassified

Bacillus
cereus

43


44
Cinnamycin
Lantibiotic

Streptoverticillium

45



(Lanthiopeptin)


griseoverticillatum




46
Circularin A
Unclassified

Geobacillus
kaustophilus (strain

47





HTA426)



48
Closticin 574
Unclassified

Clostridium
tyrobutyricum

49


50
Coagulin A
Unclassified

Bacillus
coagulans

51


52
Colicin-10
Unclassified

Escherichia
coli

53


54
Colicin-E1
Unclassified

Escherichia
coli

55


56
Colicin-Ia
Unclassified

Escherichia
coli

57


58
Colicin-Ib
Unclassified

Escherichia
coli

59


60
Colicin-M
Unclassified

Escherichia
coli

61


62
Colicin-N
Unclassified

Escherichia
coli

63


64
Colicin-V
Unclassified

Escherichia
coli

65



(Microcin-V)





66
Columbicin A
Lantibiotic

Enterococcus
columbae

69


68
Curvacin-A
class IIA/YGNGV

Lactobacillus
curvatus

69


70
Cypemycin
Unclassified

Streptomyces sp.

71


72
Cytolysin
Lantibiotic

Bacillus
halodurans (strain ATCC







BAA-125/DSM 18197/FERM
73





7344/JCM 9153/C-125)



74
Divercin V41
class IIa/YGNGV

Carnobacterium
divergens

75





(Lactobacillusdivergens)



76
Divergicin 750
Unclassified

Carnobacterium
divergens

77





(Lactobacillusdivergens)



78
Divergicin A
Class IIc

Carnobacterium
divergens

79





(Lactobacillusdivergens)



80
Durancin Q
Unclassified

Enterococcus
durans

81


82
Durancin TW-
Unclassified

Enterococcus
durans

83



49M





84
Dysgalacticin
Unclassified

Streptococcus
dysgalactiae subsp.

85






equisimilis (Streptococcus








equisimilis)




86
Enterocin
Unclassified

Enterococcus
faecalis

87



1071A

(Streptococcusfaecalis)



88
Enterocin 7A
bacteriocins without

Enterococcus
faecalis

89



(Enterocin
sequence leader
(Streptococcusfaecalis)




L50A)





90
Enterocin 7B
Unclassified

Enterococcus
faecalis

91





(Streptococcusfaecalis)



92
Enterocin 96
Class II

Enterococcus
faecalis (strain ATCC

93





700802/V583)



94
Enterocin A
Class IIa, IIc

Enterococcus
faecium

95




(problematic)
(Streptococcusfaecium)



96
Enterocin AS-48
Unclassified

Enterococcus
faecalis

97



(BACTERIOCIN

(Streptococcusfaecalis)




AS-48)





98
Enterocin B
class IIc, non

Enterococcus
faecium

99




subgrouped
(Streptococcusfaecium)





bacteriocins






(problematic)




100
Enterocin
Class IIa

Enterococcus
mundtii

101



CRL35






(Mundticin KS)





102
Enterocin EJ97
Unclassified

Enterococcus
faecalis

103





(Streptococcusfaecalis)



104
Enterocin P
Class IIa, IIb and IIc

Enterococcus
faecium

105




(problematic)
(Streptococcusfaecium)



106
Enterocin Q
Class IIc

Enterococcus
faecium

107





(Streptococcusfaecium)



108
Enterocin SE-
Class IIa

Enterococcus
faecalis

109



K4

(Streptococcusfaecalis)



110
Enterocin W
Class IIb

Enterococcus
faecalis

111



alfa

(Streptococcusfaecalis)



112
Enterocin W
Class IIb

Enterococcus
faecalis

113



beta

(Streptococcusfaecalis)



114
Enterocin
Class IIb

Enterococcus
faecium

115



Xalpha

(Streptococcusfaecium)



116
Enterocin Xbeta
Class IIb

Enterococcus
faecium

117





(Streptococcusfaecium)



118
Enterolysin A
class III

Enterococcus
faecalis

119





(Streptococcusfaecalis)



120
Epicidin 280
Lantibiotic

Staphylococcus
epidermidis

121


122
Epidermicin
Unclassified

Staphylococcus
epidermidis

123



NI01





124
Epidermin
Lantibiotic

Staphylococcus
epidermidis

125


126
Epilancin K7
Lantibiotic

Staphylococcus
epidermidis

127


128
Gallidermin
Lantibiotic

Staphylococcus
gallinarum

129


130
Garvicin A
IId

Lactococcus
garvieae

131


132
Garvicin ML
Unclassified

Lactococcus
garvieae

133


134
Gassericin A
Unclassified

Lactobacillus
gasseri

135


136
Gassericin T
Unclassified

Lactobacillus
gasseri

137



(gassericin K7






B)





138
Glycocin F
Unclassified

Lactobacillus
plantarum

139


140
Halocin H4
Unclassified

Haloferax
mediterranei (strain







ATCC 33500/DSM 1411/JCM






8866/NBRC 14739/NCIMB
141





2177/R-4) (Halobacterium







mediterranei)




142
Halocin-S8
Unclassified

Haloarchaeon S8a

143


144
Helveticin-J
Unclassified

Lactobacillus
helveticus

145





(Lactobacillussuntoryeus)



146
Hiracin JM79
Class II sec-

Enterococcus
hirae

147




dependent




148
Lactacin-F
class IIB

Lactobacillus
johnsonii (strain

149



(lafA)

CNCM I-12250/La1/NCC 533)



150
Lactacin-F
class IIB

Lactobacillus
johnsonii (strain

151



(lafX)

CNCM I-12250/La1/NCC 533)



152
Lacticin 3147
Lantibiotic

Lactococcus
lactis subsp. lactis

153



A1

(Streptococcuslactis)



154
Lacticin 3147
Lantibiotic

Lactococcus
lactis subsp. lactis

155



A2

(Streptococcuslactis)



156
Lacticin 481
Lantibiotic

Lactococcus
lactis subsp. lactis

157



(Lactococcin

(Streptococcuslactis)




DR)





158
Lacticin Q
Unclassified

Lactococcus
lactis

159


160
Lacticin Z
Unclassified

Lactococcus
lactis

161


162
Lactobin-A
class IIB

Lactobacillus
amylovorus

163



(Amylovorin-






L471)





164
Lactocin-S
Lantibiotic

Lactobacillus
sakei L45

165


166
Lactococcin 972
Unclassified

Lactococcus
lactis subsp. lactis

167





(Streptococcuslactis)



168
Lactococcin-A
Unclassified

Lactococcus
lactis subsp. cremoris

169





(Streptococcuscremoris)



170
Lactococcin-B
Unclassified

Lactococcus
lactis subsp. cremoris

171





(Streptococcuscremoris)



172
Lactocyclicin Q
Unclassified

Lactococcus sp. QU 12

173


174
Laterosporulin
Unclassified

Brevibacillus sp. GI-9

175


176
Leucocin N
Class IId

Leuconostoc
pseudomesenteroides

177


178
Leucocin Q
Class IId

Leuconostoc
pseudomesenteroides

179


180
Leucocin-A
class IIA/YGNGV

Leuconostoc
gelidum

181



(Leucocin A-






UAL 187)





182
Leucocin-B
class IIA/YGNGV

Leuconostoc
carnosum

183



(Leucocin B-






Ta11a)





184
Leucocyclicin Q
Unclassified

Leuconostoc
mesenteroides

185


186
Lichenicidin A1
Lantibiotic (two-

Bacillus
licheniformis (strain DSM






peptide)
13/ATCC 14580)
187


188
Linocin M18
Unclassified

Brevibacterium
linens

189


190
Listeriocin
Class IIa

Listeria
innocua

191



743A





192
Mersacidin
Lantibiotic, type B

Bacillus sp. (strain HIL-

193





Y85/54728)



194
Mesentericin
class IIA/YGNGV

Leuconostoc
mesenteroides

195



Y105





196
Michiganin-A
Lantibiotic

Clavibacter
michiganensis subsp.

197






michiganensis




198
Microcin B17
Unclassified

Escherichia
coli

199



(MccB17)





200
Microcin C7
Unclassified

Escherichia
coli

201


202
Microcin E492
Unclassified

Klebsiella
pneumoniae

203


204
Microcin H47
Unclassified

Escherichia
coli

205


206
Microcin J25
Unclassified

Escherichia
coli

207


208
Microcin-24
Unclassified

Escherichia
coli

209


210
Mundticin KS
Unclassified

Enterococcus
mundtii

211


212
Mundticin L
class IIA/YGNGV

Enterococcus
mundtii

213


214
Mutacin 1140
Lantibiotic

Streptococcus
mutans

215



(Mutacin III)





216
Mutacin-2
Lantibiotic

Streptococcus
mutans

217


218
Nisin A
Lantibiotic

Lactococcus
lactis subsp. lactis

219





(Streptococcuslactis)



220
Nisin F
Lantibiotic

Lactococcus
lactis

221


222
Nisin Q
Lantibiotic

Lactococcus
lactis

223


224
Nisin U
Lantibiotic

Streptococcus
uberis

225


226
Nisin Z
Lantibiotic

Lactococcus
lactis subsp. lactis

227





(Streptococcuslactis)



228
Nukacin ISK-1
Lantibiotic

Staphylococcus
warneri

229


230
Paenicidin A
Lantibiotic

Paenibacillus
polymyxa (Bacillus

231






polymyxa)




232
Pediocin PA-1
class IIA/YGNGV

Pediococcus
acidilactici

233



(Pediocin ACH)





234
Penocin A
class IIA/YGNGV

Pediococcus
pentosaceus (strain

235





ATCC 25745/183-1w)



236
Pep5
Lantibiotic

Staphylococcus
epidermidis

237


238
Piscicolin 126
class IIA/YGNGV

Carnobacterium
maltaromaticum

239





(Carnobacteriumpiscicola)



240
Plantaricin 1.25
Unclassified

Lactobacillus
plantarum

241



β





242
Plantaricin 423
class IIa

Lactobacillus
plantarum

243


244
Plantaricin
Unclassified

Lactobacillus
plantarum

245



ASM1





246
Plantaricin E
Unclassified

Lactobacillus
plantarum

247


248
Plantaricin F
Class IIb

Lactobacillus
plantarum

249


250
Plantaricin J
Class IIb

Lactobacillus
plantarum

251


252
Plantaricin K
Unclassified

Lactobacillus
plantarum

253


254
Plantaricin NC8
Unclassified

Lactobacillus
plantarum

255



α





256
Plantaricin NC8
Unclassified

Lactobacillus
plantarum

257



β





258
Plantaricin S α
Unclassified

Lactobacillus
plantarum

259


260
Plantaricin S β
Unclassified

Lactobacillus
plantarum

261


262
Plantaricin W α
Lantibiotic (two-peptide)

Lactobacillus
plantarum

263


264
Plantaricin W β
Lantibiotic (two-peptide)

Lactobacillus
plantarum

265


266
Plantaricin-A
Unclassified

Lactobacillus
plantarum (strain







ATCC BAA-793/NCIMB 8826/
267





WCFS1)



268
Propionicin
Unclassified

Propionibacterium
jensenii

269



SM1





270
Propionicin T1
Unclassified

Propionibacterium
thoenii

271


272
Propionicin-F
Unclassified

Propionibacterium
freudenreichii

273





subsp. freudenreichii



274
Pyocin S1
Unclassified

Pseudomonas
aeruginosa

275


276
Pyocin S2
colicin/pyosin

Pseudomonas
aeruginosa (strain






nuclease family
ATCC 15692/PAO1/1C/PRS
277





101/LMG 12228)



278
Ruminococcin-A
Lantibiotic

Ruminococcus
gnavus

279


280
Sakacin G
Class IIa

Lactobacillus
sakei

281


282
Sakacin-A
class IIA/YGNGV

Lactobacillus
sakei

283


284
Sakacin-P
class IIA/YGNGV

Lactobacillus
sakei

285



(Sakacin 674)





286
Salivaricin 9
lantibiotic

Streptococcus
salivarius

287


288
Salivaricin A
Lantibiotic

Streptococcus
pyogenes serotype

289





M28 (strain MGAS6180)



290
Salivaricin A3
Lantibiotic

Streptococcus
salivarius

291


292
Salivaricin-A sa
Lantibiotic

Streptococcus
salivarius

293


294
Staphylococcin
Lantibiotic (two-peptide)

Staphylococcus
aureus

295



C55 alpha





296
Staphylococcin
Lantibiotic (two-peptide)

Staphylococcus
aureus

297



C55 beta





298
Streptin
lantibiotic

Streptococcus
pyogenes

299


300
Streptococcin
Lantibiotic

Streptococcus
pyogenes

301



A-FF22





302
Streptococcin
Lantibiotic

Streptococcus
pyogenes serotype

303



A-M49

M49



304
Sublancin 168
Lantibiotic

Bacillus
subtilis (strain 168)

305


306
Subtilin
Lantibiotic

Bacillus
subtilis

307


308
Subtilosin
Unclassified

Bacillus
subtilis (strain 168)

309


310
Subtilosin-A
Unclassified

Bacillus
subtilis (strain 168)

311


312
Thermophilin
Lantibiotic

Streptococcus
thermophilus

313



1277





314
Thermophilin 13
Unclassified

Streptococcus
thermophilus

315


316
Thermophilin A
Unclassified

Streptococcus
thermophilus

317


318
Thiocillin
Unclassified

Bacillus
cereus (strain ATCC

319



(Micrococcin

14579/DSM 31)




P1)






(Micrococcin






P2) (Thiocillin






I) (Thiocillin II)






(Thiocillin III)






(Thiocillin IV)






(Antibiotic YM-266183)






(Antibiotic YM-266184)





320
Thuricin CD
two-peptide

Bacillus
cereus 95/8201

321



alpha
lantibiotic




322
Thuricin CD
two-peptide

Bacillus
cereus 95/8201

323



beta
lantibiotic




324
Thuricin-17
Class IId

Bacillus
thuringiensis

325


326
Trifolitoxin
Unclassified

Rhizobium
leguminosarum bv.

327






trifolii




328
Ubericin A
Class IIa

Streptococcus
uberis

329


330
Uberoly sin
Unclassified

Streptococcus
uberis

331


332
UviB
Unclassified

Clostridium
perfringens

333


334
Variacin
Lantibiotic, Type A

Micrococcus
varians

335


336
Zoocin A
Unclassified

Streptococcus
equi subsp.

337






zooepidemicus




338
Fulvocin-C
Unclassified

Myxococcus
fulvus

339


340
Duramycin-C
Lantibiotic

Streptomyces
griseoluteus

341


342
Duramycin
Lantibiotic B

Streptoverticillium

343



(duramycin-B)


griseoverticillatum





(Leucopeptin)





344
Carnocin UI49
lantibiotic

Carnobacterium sp. (strain UI49)

345


346
Lactococcin-G α
Unclassified

Lactococcus
lactis subsp. lactis

347





(Streptococcuslactis)



348
Lactococcin-G β
Unclassified

Lactococcus
lactis subsp. lactis

349





(Streptococcuslactis)



350
Ancovenin
Lantibiotic

Streptomyces sp. (strain A647P-2)

351


352
Actagardine
Lantibiotic

Actinoplanes
liguriae

353



(Gardimycin)





354
Curvaticin FS47
Unclassified

Lactobacillus
curvatus

355


356
Bavaricin-MN
class IIA/YGNGV

Lactobacillus
sakei

357


358
Mutacin B-
Lantibiotic

Streptococcus
mutans

359



Ny266





360
Mundticin
class IIA/YGNGV

Enterococcus
mundtii

361


362
Bavaricin-A
class IIA/YGNGV

Lactobacillus
sakei

363


364
Lactocin-705
Class IIb

Lactobacillus
paracasei

365


366
Leucocin-B
Unclassified

Leuconostoc
mesenteroides

367


368
Leucocin C
class IIA/YGNGV

Leuconostoc
mesenteroides

369


370
LCI
Unclassified

Bacillus
subtilis

371


372
Lichenin
Unclassified

Bacillus
licheniformis

373


374
Lactococcin
class IIA/YGNGV

Lactococcus
lactis subsp. lactis

375



MMFII

(Streptococcuslactis)



376
Serracin-P
Phage-Tail-Like

Serratia
plymuthica

377


378
Halocin-C8
Unclassified

Halobacterium sp. (strain A57092)

379


380
Subpeptin JM4-
Unclassified

Bacillus
subtilis

381



B





382
Curvalicin-28a
Unclassified

Lactobacillus
curvatus

383


384
Curvalicin-28b
Unclassified

Lactobacillus
curvatus

385


386
Curvalicin-28c
Unclassified

Lactobacillus
curvatus

387


388
Thuricin-S
Unclassified

Bacillus
thuringiensis subsp.

389






entomocidus




390
Curvaticin L442
Unclassified

Lactobacillus
curvatus

391


392
Divergicin M35
class IIa/YGNGV

Carnobacterium
divergens

393





(Lactobacillusdivergens)



394
Enterocin E-760
class IIb

Enterococcus sp.

395


396
Bacteriocin
Unclassified

Enterococcus
faecium

397



E50-52

(Streptococcusfaecium)



398
Paenibacillin
Unclassified

Paenibacillus
polymyxa (Bacillus

399






polymyxa)




400
Epilancin 15x
Unclassified

Staphylococcus
epidermidis

401


402
Enterocin-HF
class IIa

Enterococcus
faecium

403





(Streptococcusfaecium)



404
Bacillocin 602
Class IIa

Paenibacillus
polymyxa (Bacillus

405






polymyxa)




406
Bacillocin 1580
Class IIa

Bacillus
circulans

407


408
Bacillocin B37
Unclassified

Paenibacillus
polymyxa (Bacillus

409






polymyxa)




410
Rhamnosin A
Unclassified

Lactobacillus
rhamnosus

411


412
Lichenicidin A2
Lantibiotic (two-

Bacillus
licheniformis (strain DSM






peptide)
13/ATCC 14580)
413


414
Plantaricin C19
Class IIa

Lactobacillus
plantarum

415


416
Acidocin J1132 β
Class IIb

Lactobacillus
acidophilus

417


418
factor with anti-
Unclassified

Enterococcus
faecalis

419




Candida activity






420
Ava_1098
Unclassified

Anabaena
variabilis ATCC 29413

421



(putative






heterocyst






differentiation






protein)





422
alr2818
Unclassified

Nostoc sp 7120

423



(putative






heterocyst






differentiation






protein)





424
Aazo_0724
Unclassified

Nostoc
azollae 0708

425



(putative






heterocyst






differentiation






protein)





426
AM1_4010
Unclassified

Acaryochloris
marina MBIC11017

427



(putative






heterocyst






differentiation






protein)





428
PCC8801_3266
Unclassified

Cyanothece PCC 8801

429



(putative






heterocyst






differentiation






protein)





430
Cyan8802_2855
Unclassified

Cyanothece PCC 8802

431



(putative






heterocyst






differentiation






protein)





432
PCC7424_3517
Unclassified

Cyanothece PCC 7424

433


434
cce_2677(putative
Unclassified

Cyanothece ATCC 51142





HetP protein)


435


436
CY0110_11572
Unclassified

Cyanothece CCY0110

437



(putative






heterocyst






differentiation






protein)





438
MC7420_4637
Unclassified

Microcoleus
chthonoplastes PCC

439





7420



440
asr1611
Unclassified

Nostoc sp 7120

441



(putative






DUF37 family






protein)





442
Ava_4222
Unclassified

Anabaena
variabilis ATCC 29413

443



(putative






DUF37 family






protein)





444
N9414_07129
Unclassified

Nodularia
spumigena CCY9414

445



(putative






DUF37 family






protein)





446
Aazo_0083
Unclassified

Nostoc
azollae 0708

447



(putative






DUF37 family






protein)





448
S7335_3409
Unclassified

Synechococcus PCC 7335

449



(putative






DUF37 family






protein)





450
P9303_21151
Unclassified

Prochlorococcus
marinus

451



(putative

MIT 9303




DUF37 family






protein)





720
Curvalicin-28c
Unclassified

Lactobacillus
curvatus

721


722
thruicin-S
Unclassified

Bacillus
thuringiensis

723


724
curvaticin L442
Unclassified

Lactobacillus
curvatus L442

725


726
Bacteriocin
P84962

Carnobacterium
divergens

727



divergicin M35

(Lactobacillusdivergens)



728
Lantibiotic
P85065

Microbispora sp. (strain 107891)

729



107891





730
Enterocin E-760
P85147

Enterococcus sp.

731



(Bacteriocin E-






760)





732
Bacteriocin
P85148

Enterococcus
faecium

733



E50-52

(Streptococcusfaecium)



734
Lantibiotic
P86013

Paenibacillus
polymyxa (Bacillus

735



paenibacillin


polymyxa)




736
Lantibiotic
P86047

Staphylococcus
epidermidis

737



epilancin 15X





738
Enterocin-HF
P86183

Enterococcus
faecium

739





(Streptococcusfaecium)



740
Bacteriocin
P86393

Paenibacillus
polymyxa (Bacillus

741



SRCAM 602


polymyxa)




742
Bacteriocin
P86394

Bacillus
circulans

743



SRCAM 1580





744
Bacteriocin
P86395

Paenibacillus
polymyxa (Bacillus

745



SRCAM 37


polymyxa)




746
Bacteriocin
P86526

Lactobacillus
rhamnosus

747



rhamno sin A






(Fragment)





748
Lantibiotic
P86720

Bacillus
licheniformis (strain

749



lichenicidin A2

ATCC 14580/DSM 13/JCM




(LchA2)

2505/NBRC 12200/NCIMB




(BliA2)

9375/NRRL NRS-1264/Gibson






46)



750
Pyocin-52 (EC


Pseudomonas
aeruginosa (strain

751



3.1.-.-) (Killer

ATCC 15692/DSM 22644/CIP




protein)

104116/JCM 14847/LMG 12228/






1C/PRS 101/PAO1)



752
Plantaricin C19


Lactobacillus
plantarum

753



(Fragment)





754
LsbB


Lactococcus
lactis subsp. lactis

755





(Streptococcuslactis)



756
ACIDOCIN


Lactobacillus
acidophilus

757



J1132 beta






peptide






(Fragment)





758
Uncharacterized


Lactobacillus
salivarius cp400

759



protein









For example, in some embodiments, an anti-fungal activity (such as anti-yeast activity) is desired. A number of bacteriocins with anti-fungal activity have been identified. For example, bacteriocins from Bacillus have been shown to have neutralizing activity against yeast strains {see Adetunji and Olaoye (2013) Malaysian Journal of Microbiology 9: 130-13, hereby incorporated by reference in its entirety), an Enterococcus faecalis peptide (WLPPAGLLGRCGRWFRPWLLWLQ SGAQY KWLGNLFGLGPK, SEQ ID NO: 1) has been shown to have neutralizing activity against Candida species {see Shekh and Roy (2012) BMC Microbiology 12: 132, hereby incorporated by reference in its entirety), and bacteriocins from Pseudomonas have been shown to have neutralizing activity against fungi such as Curvularia lunata, Fusarium species, Helminthosporium species, and Biopolaris species (Shalani and Srivastava (2008) The Internet Journal of Microbiology. Volume 5 Number 2. DOI: 10.5580/27dd—accessible on the worldwide web at archive, ispub.com/journal/the-internet-journal-of-micro biology/volume-5-number-2/screening-for-antifungal-activity-of-pseudomonas-fluorescens-against-phytopathogenic-fungi.html #sthash.d0Ys03UO. 1DKuT1US.dpuf, hereby incorporated by reference in its entirety). By way of example, botrycidin AJ1316 {see Zuber, P et al. (1993) Peptide Antibiotics. In Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics ed Sonenshein et al., pp. 897-916, American Society for Microbiology, hereby incorporated by reference in its entirety) and alirin B1 {see Shenin et al. (1995) Antibiot Khimioter 50: 3-7, hereby incorporated by reference in its entirety) from 5. subtilis have been shown to have antifungal activities. As such, in some embodiments, for example embodiments in which neutralization of a fungal microbial organism is desired, a bacteriocin comprises at least one of botrycidin AJ1316 or alirin B1.


For example, in some embodiments, bacteriocin activity in a culture of cyanobacteria is desirable. In some embodiments, bacteriocins are provided to neutralize cyanobacteria. In some embodiments, bacteriocins are provided to neutralize invading microbial organisms typically found in a cyanobacteria culture environment. Clusters of conserved bacteriocin polypeptides have been identified in a wide variety of cyanobacteria species. For example, at least 145 putative bacteriocin gene clusters have been identified in at least 43 cyanobacteria species, as reported in Wang et al. (2011), Genome Mining Demonstrates the Widespread Occurrence of Gene Clusters Encoding Bacteriocins in Cyanobacteria. PLoS ONE 6(7): e22384, hereby incorporated by reference in its entirety. Exemplary cyanobacteria bacteriocins are shown in Table 1.2 as SEQ ID NO's 420, 422, 424, 426, 428, 30, 432, 434, 436, 438, 440, 442, 444, 446, 448, and 450.


Within the context of methods, uses, compositions, hosts, and nucleic acids of embodiments herein, although a bacteriocin may work via different mechanisms on a microbial cell as explained herein, a bacteriocin may be said to be active when the number of microbial host has decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more compared to the number of initial microbial host when the microbial hosts are being cultured with a medium comprising a bacteriocin. This culture step may have a duration of at least 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours or more before assessing the activity of the bacteriocin by counting the number of microbial hosts present. The activity may be assessed by counting the cells under the microscope or by any known microbial techniques. In some embodiments, a bacteriocin is active when the growth has been arrested in at least a specified number or percentage of microbial hosts, for example at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the microbial hosts arrested compared to the initial population of microbial hosts when the microbial hosts are being cultured with a medium comprising a bacteriocin.


Within the context of methods, uses, compositions, hosts, and nucleic acids of some embodiments herein, the bacteriocin is B17 or C7 represented by an amino acid sequence comprising or consisting of SEQ ID NO: 198 or 200 respectively. B17 and C7 have been experimentally confirmed to be selection agents simple to produce, easy to use and stable in culture medium in accordance with some embodiments herein (See Example 1). Some of methods, uses, compositions, hosts, and nucleic acids of embodiments herein also encompass the use a bacteriocin having at least 50% identity to SEQ ID NO: 198 or 200, for example at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 198 or 200. Such variants of B17 or C7 may be used in methods, uses, compositions, hosts, and nucleic acids of embodiments herein as long as they exhibit at least a substantial activity of B17 or C7. In this context, “substantial” means, for example, at least 50%, at least 60%, at least 705, at least 80%, at least 90%, or at least 100% or more of the activity of B17 or C7 having SEQ ID NO: 198 or 200. The activity of a bacteriocin has been described earlier herein.


Within the context of methods, uses, compositions, hosts, and nucleic acids of embodiments herein, and depending on the microbial host targeted and the bacteriocin used, the skilled person will know which concentration of bacteriocin is to be used in a medium or in an agar petri plate. Using bacteriocin B17 or C7 inventors were able to prepare culture medium comprising said bacteriocin in a concentration which allows one to carry out the methods and uses of embodiments herein, i.e. to observe or visualize an advantage of the expression of said genetic activity. If an advantage of said activity is to allow the growth of the host comprising the auto-replicative extra-chromosomal nucleic acid molecule, then the quantity of bacteriocin in said medium or agar plate is such that the number of host that does not comprise said auto-replicative extra-chromosomal has been decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more compared to the number of initial microbial cells/host when the cells are being cultured under conditions allowing the microbial host that has received said auto-replicative extra-chromosomal nucleic acid molecule to survive and to grow. This assessment step may have a duration of at least 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours or more. Said culture medium may be sterilized without losing substantial bacteriocin activity. In this context “substantial” means, for example, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% of the bacteriocin activity present in the culture medium before sterilization.


First nucleic acid sequences suitable for methods, uses, compositions, hosts, and nucleic acids of some embodiments herein, and whose product provides immunity to a bacteriocin are shown in Table 2.









TABLE 2







Exemplary nucleic acid sequences whose provide immunity to a bacteriocin










Poly-peptide


Poly-nucleotide


SEQ ID NO:
Name
Organism of origin
SEQ ID NO:





452
Microcin H47 immunity modulator MchI

Escherichia coli

453


454
Colicin-E3 immunity modulator (Colicin-E3 chain B)

Escherichia coli

455



(ImmE3) (Microcin-E3 immunity modulator)




456
Colicin-E1 immunity modulator (ImmE1) (Microcin-

Escherichia coli

457



E1 immunity modulator)




458
Cloacin immunity modulator

Escherichia coli

459


460
Colicin-E2 immunity modulator (ImmE2) (Microcin-

Escherichia coli

461



E2 immunity modulator)




462
Colicin-A immunity modulator (Microcin-A

Citrobacter

463



immunity modulator)

freundii




464
Colicin-Ia immunity modulator

Escherichia coli

465


466
Colicin-Ib immunity modulator

Escherichia coli

467


468
Colicin-N immunity modulator (Microcin-N

Escherichia coli

469



immunity modulator)




470
Colicin-E8 immunity modulator (ImmE8) (Microcin-

Escherichia coli

471



E8 immunity modulator)




472
Lactococcin-A immunity modulator
Lactococcus lactis subsp. lactis
473




(Streptococcus lactis)



474
Lactococcin-A immunity modulator
Lactococcus lactis subsp. cremoris
475




(Streptococcus cremoris)



476
Colicin-D immunity modulator (Microcin-D

Escherichia coli

477



immunity modulator)




478
Colicin-E5 immunity modulator (ImmE5) (Microcin-

Escherichia coli

479



E5 immunity modulator)




480
Colicin-E6 immunity modulator (ImmE6) (Microcin-

Escherichia coli

481



E6 immunity modulator)




482
Colicin-E8 immunity modulator in ColE6

Escherichia coli

483



(E8Imm[E6])




484
Colicin-E9 immunity modulator (ImmE9) (Microcin-

Escherichia coli

485



E9 immunity modulator)




486
Colicin-M immunity modulator (Microcin-M

Escherichia coli

487



immunity modulator)




488
Colicin-B immunity modulator (Microcin-B

Escherichia coli

489



immunity modulator)




490
Colicin-V immunity modulator (Microcin-V

Escherichia coli

491



immunity modulator)




492
Colicin-E1* immunity modulator (ImmE1)
Shigella sonnei
493



(Microcin-E1* immunity modulator)




494
Colicin-E1 immunity modulator (ImmE1) (Microcin-

Escherichia coli

495



E1 immunity modulator)




496
Probable leucocin-A immunity modulator

Leuconostoc gelidum

497


498
Lactococcin-B immunity modulator
Lactococcus lactis subsp. cremoris
499




(Streptococcus cremoris)



500
Pediocin PA-1 immunity modulator (Pediocin

Pediococcus acidilactici

501



ACH immunity modulator)




502
Putative carnobacteriocin-BM1 immunity modulator
Carnobacterium maltaromaticum
503




(Carnobacterium piscicola)



504
Putative carnobacteriocin-B2 immunity modulator
Carnobacterium maltaromaticum
505



(Carnocin-CP52 immunity modulator)
(Carnobacterium piscicola)



506
Nisin immunity modulator
Lactococcus lactis subsp. lactis
507




(Streptococcus lactis)



508
Trifolitoxin immunity modulator
Rhizobium leguminosarum bv. trifolii
509


510
Antilisterial bacteriocin subtilosin biosynthesis

Bacillus subtilis (strain 168)

511



protein AlbD




512
Putative ABC transporter ATP-binding protein AlbC

Bacillus subtilis (strain 168)

513



(Antilisterial bacteriocin subtilosin biosynthesis





protein AlbC)




514
Antilisterial bacteriocin subtilosin biosynthesis

Bacillus subtilis (strain 168)

515



protein AlbB




516
Colicin-E7 immunity modulator (ImmE7) (Microcin-

Escherichia coli

517



E7 immunity modulator)




518
Pyocin-S1 immunity modulator

Pseudomonas aeruginosa

519


520
Pyocin-S2 immunity modulator

Pseudomonas aeruginosa (strain ATCC

521




15692 /PAO1 / 1C / PRS 101 / LMG 12228)



522
Hiracin-JM79 immunity factor

Enterococcus hirae

523


524
Probable mesentericin-Y105 immunity modulator

Leuconostoc mesenteroides

525


526
Microcin-24 immunity modulator

Escherichia coli

527


528
Colicin-K immunity modulator

Escherichia coli

529


530
Microcin C7 self-immunity modulator MccF

Escherichia coli

531


532
Sakacin-A immunity factor

Lactobacillus sakei

533


534
Colicin-E5 immunity modulator in ColE9

Escherichia coli

535



(E5Imm[E9])




536
Antilisterial bacteriocin subtilosin biosynthesis

Bacillus subtilis

537



protein AlbD




538
Microcin-J25 export ATP-binding/permease protein

Escherichia coli

539



McjD (Microcin-J25 immunity modulator)





(Microcin-J25 secretion ATP-binding protein McjD)




540
Microcin E492 immunity modulator

Klebsiella pneumoniae

541



McbG

Escherichia coli

699



MccE

Escherichia coli

700


706
C-terminal part of MccE
Derived from E. coli and considered as
701




artificial




Cvi

Escherichia coli

709



McbG-MccE
Derived from E. coli and considered as
715




artificial




McbG-Cter part MccE
Derived from E. coli and considered as
716



(Cter could be replaced by C-terminal)
artificial




Cvi-MccE
Derived from E. coli and considered as
717




artificial




Cvi-Cter part MccE
Derived from E. coli and considered as
718



(Cter could be replaced by C-terminal)
artificial









While the sequence providing immunity to a bacteriocin of Table 2 are naturally-occurring, the skilled artisan will appreciate that variants of such molecules, naturally-occurring molecules other than the ones of Table 2, or synthetic ones can be used according to some embodiments herein. In some embodiments, a particular molecule conferring immunity or particular combination of molecules conferring immunity to a particular bacteriocin, particular class or category of bacteriocins, or particular combination of bacteriocins. Exemplary bacteriocins to which molecules can confer immunity are identified in Table 2. While Table 2 identifies an “organism of origin” for a molecule conferring immunity, these molecules conferring immunity can readily be expressed in other naturally-occurring, genetically modified, or synthetic microorganisms to provide a desired bacteriocin immunity activity in accordance with some embodiments herein. As such, as used herein “immunity modulator” or “molecule conferring or providing immunity to a bacteriocin” encompasses not only to structures expressly provided herein, but also structures that have substantially the same effect as the “immunity modulator” structures described herein, including fully synthetic immunity modulators, and immunity modulators that provide immunity to bacteriocins that are functionally equivalent to the bacteriocins disclosed herein.


Exemplary polynucleotide sequences encoding the polypeptides of Table 2 are indicated in Table 2. The skilled artisan will readily understand that the genetic code is degenerate, and moreover, codon usage can vary based on the particular organism in which the gene product is being expressed, and as such, a particular polypeptide can be encoded by more than one polynucleotide. In some embodiments, a polynucleotide encoding a bacteriocin immunity modulator is selected based on the codon usage of the organism expressing the bacteriocin immunity modulator. In some embodiments, a polynucleotide encoding a bacteriocin immunity modulator is codon optimized based on the particular organism expressing the bacteriocin immunity modulator. A vast range of functional immunity modulators can incorporate features of immunity modulators disclosed herein, thus providing for a vast degree of identity to the immunity modulators in Table 2. In some embodiments, an immunity modulator has at least about 50% identity, for example, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the polypeptides of Table 2.


Within the context of methods, uses, compositions, hosts, and nucleic acids of embodiments herein, resistance or immunity to a bacteriocin may mean the number of microbial cells at the end of a culturing step with a bacteriocin has not been decreased, and in some embodiments has been increased of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more compared to the number of initial microbial cells when the cells are being cultured with a medium comprising a bacteriocin. This culture step may have a duration of at least 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours or more before assessing the activity of the bacteriocin by counting the number of microbial cells present.


A nucleic acid molecule suitable for methods, uses, compositions, hosts, and nucleic acids of some embodiments herein and whose encoding product confers immunity is McbG (Immunity to the bacteriocin B17), which is represented by SEQ ID NO: 699. McbG has been experimentally confirmed to be useful as a selectable marker either constitutively or inducibly in accordance with some embodiments herein (See Example 3). Another suitable nucleic acid is the MccE (Immunity to the bacteriocin C7) which is represented by SEQ ID NO: 700 or its c-terminal portion, represented by SEQ ID NO: 701. MccE had been used as a vector selection marker in strains sensitive to microcines/bacteriocins (See Example 2). Methods, uses, compositions, hosts, and nucleic acids of some embodiments also encompass the use of a nucleic acid molecule whose encoding product confers immunity to bacteriocin B17 and/or C7 and having at least 50% identity to SEQ ID NO: 699, 700, or 701, for example at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 699, 700 or 701. Such variants of McbG and/or MccE may be used in methods, uses, compositions, hosts, and nucleic acids of embodiments herein as long as they exhibit at least a substantial activity of McbG (respectively MccE). In this context, “substantial” means, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% or more of the activity of McbG (respectively MccE) having SEQ ID NO: 699, 700 or 701. The immunity conferred by the encoding product of McbG (respectively MccE) has been described earlier herein.


Surprisingly it has been found that the C-terminal part of MccE which is represented by SEQ ID NO: 701 is sufficient to confer resistance to bacteriocin C7. Part means in this context, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the original nucleic acid molecule. This is quite attractive and surprising that such a short nucleic acid molecule can confer resistance to a bacteriocin. It is expected that an auto-replicative extra-chromosomal nucleic acid molecule comprising such short nucleic acid molecule does not form any burden for the microbial cell.


A further suitable nucleic acid molecule for methods, uses, compositions, hosts, and nucleic acids of some embodiments herein, and whose product provides immunity to a bacteriocin is a single nucleic acid molecule whose single product provides immunity to at least two distinct bacteriocins. In some embodiments, such product of such nucleic acid molecule provides immunity to B17 and C7 or to ColV and C7 or to ColV and B17 or to B17, C7 and ColV. A nucleic acid encoding ColV is identified as SEQ ID NO: 65 and a corresponding coding amino acid sequence is identified as SEQ ID NO: 64.


In some embodiments, a nucleic acid molecule whose product provides immunity to B17 and C7 is represented by a sequence having at least 50% identity to SEQ ID NO: 715 or 716 for example at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 715 or 716. SEQ ID NO: 715 is a nucleic acid molecule of McbG fused to MccE. SEQ ID NO: 716 is a nucleic acid molecule of McbG fused to the C-terminal part of MccE as earlier described herein.


In some embodiments, a nucleic acid molecule whose product provides immunity to ColV and C7 is represented by a sequence having at least 50% identity to SEQ ID NO: 717 or 718 for example at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 717 or 718. SEQ ID NO: 717 is a nucleic acid molecule of Cvi fused to MccE. SEQ ID NO: 718 is a nucleic acid molecule of Cvi fused to the C-terminal part of MccE as earlier described herein.


Such identity variants of the core sequence may be used in methods, uses, compositions, hosts, and nucleic acids of embodiments herein as long as they exhibit at least a substantial activity of the molecule they derived from as earlier described herein.


In methods, uses, compositions, hosts, and nucleic acids of some embodiments herein, each of these nucleic acid molecules described herein whose product confers immunity to a single or to more than one or to at least two bacteriocins may be operably linked to a promoter as described herein. In some embodiments, the promoter is a weak promoter. In some embodiments, the weak promoter is the proC promoter represented by SEQ ID NO: 708 or the P24 promoter represented by SEQ ID NO: 707, which has been experimentally confirmed (See, e.g. Example 3).


Suitable constructs useful in methods, uses, compositions, hosts, and nucleic acids of embodiments herein can comprise a first nucleic acid molecule whose product confers immunity to a bacteriocin, and these constructs may comprise, consist essentially of, or consist of SEQ ID NO: 702, 703, 710, 711, 704, 705, 712, 713 or 714. Each of these constructs has been extensively described in the experimental part of the application, which notes that each of these constructs was actually constructed and confirmed to be suitable in accordance with some embodiments herein (See, e.g., Examples 1 and 2 and 3).


In a method of some embodiments, the bacteriocin added to the culture medium is a B17 and/or a C7 and/or a ColV as identified herein


The method may allow the production of any product of interest. In a method of some embodiments, the product of interest is a microbial biomass, the auto-replicative extra-chromosomal nucleic acid molecule, the transcript of said second nucleic sequence, a polypeptide encoded by said second sequence or a metabolite produced directly or indirectly by said polypeptide.


In a method of some embodiments, the product of interest is purified at the end of the culturing step c). This may be carried out using techniques known to the skilled person. Since the energetic burden associated with the presence of the auto-replicative extra-chromosomal nucleic acid molecule has been minimized, the yield of the product of interest is expected to be optimal.


The method may use any suitable microbial cells, for example as hosts. Suitable microbial cells are listed in the part of the specification entitled general descriptions. Suitable microbial cells per se and for use in methods, uses, compositions, and hosts of embodiments herein include, but are not limited to: a bacterium (for example, a Gram negative bacterium, for example an E. coli species), a yeast, a filamentous fungus or an algae. In some embodiments, the microbial cell is a synthetic microbial cell.


In a method, the first nucleic acid sequence present on the auto-replicative extra-chromosomal nucleic acid molecule may be operably linked to a promoter. In some embodiments, said promoter is a weak promoter. In some embodiments, said promoter is a constitutive promoter. In some embodiments, said promoter is inducible. In some embodiments, said promoter is a weak constitutive promoter. In some embodiments, said promoter is a weak inducible promoter. The inducibility of said promoter is a way of controlling the presence of the genetic activity of the first nucleic acid sequence. Promoters are well known in the art. A detailed description is provided in the part of the specification dedicated to the general descriptions. A promoter can be used to drive the transcription of one or more coding sequences. Optionally said auto-replicative extra-chromosomal nucleic acid molecule comprises a second nucleic acid sequence that is involved in the production of a product of interest, wherein the genetic activity of said second nucleic acid sequence is controlled independently from the one of the first sequence.


In an embodiment, the control of the genetic activity of said second nucleic acid sequence is not independent from the control of the genetic activity of the first sequence.


In some embodiments, a second promoter drives expression of said second nucleic acid sequence being involved in the production of a product of interest as described herein. In an embodiment, a first promoter drives expression of an immunity modulator polynucleotide as described herein.


A promoter that could be used herein may be not native to a nucleic acid molecule to which it is operably linked, i.e. a promoter that is heterologous to the nucleic acid molecule (coding sequence) to which it is operably linked. Although a promoter of some embodiments is heterologous to a coding sequence to which it is operably linked, in some embodiments, a promoter is homologous, e.g., endogenous to a microbial cell. In some embodiments, a heterologous promoter (to the nucleotide sequence) is capable of producing a higher steady state level of a transcript comprising a coding sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is a promoter that is native to a coding sequence. Some promoters can drive transcription at all times (“constitutive promoters”). Some promoters can drive transcription under only select circumstances (“conditional promoters” or “inducible promoter”), for example depending on the presence or absence of an environmental condition, chemical compound, gene product, stage of the cell cycle, or the like.


The skilled artisan will appreciate that depending on the desired expression activity, an appropriate promoter can be selected, and placed in cis (i.e. or is operably linked with) with a sequence to be expressed. Exemplary promoters with exemplary activities are provided in Table 3.1-3.11 herein. The skilled artisan will appreciate that some promoters are compatible with particular transcriptional machinery (e.g. RNA polymerases, general transcription factors, and the like). As such, while compatible “species” are identified for some promoters described herein, it is contemplated that according to some embodiments herein, these promoters can readily function in microorganisms other than the identified species, for example in species with compatible endogenous transcriptional machinery, genetically modified species comprising compatible transcriptional machinery, or fully synthetic microbial organisms comprising compatible transcriptional machinery.


The promoters of Tables 3.1-3.11 herein are publicly available from the Biobricks foundation. It is noted that the Biobricks foundation encourages use of these promoters in accordance with BioBrick™ Public Agreement (BPA).


It should be appreciated that any of the “coding” polynucleotides described herein (for example a first nucleic acid sequence and/or a second nucleic acid sequence involved in the production of a product of interest) is generally amenable to being expressed under the control of a desired promoter. In an embodiment, a first nucleic acid sequence is under the control of a first promoter. In an embodiment, a second nucleic acid sequence involved in the production of a product of interest is under the control of a second promoter.


Generally, translation initiation for a particular transcript is regulated by particular sequences at 5′ end of the coding sequence of a transcript. For example, a coding sequence can begin with a start codon configured to pair with an initiator tRNA. While naturally-occurring translation systems typically use Met (AUG) as a start codon, it will be readily appreciated that an initiator tRNA can be engineered to bind to any desired triplet or triplets, and accordingly, triplets other than AUG can also function as start codons in certain embodiments. Additionally, sequences near the start codon can facilitate ribosomal assembly, for example a Kozak sequence ((gcc)gccNccAUGG, SEQ ID NO: 542, in which N represents “A” or “G”) or Internal Ribosome Entry Site (IRES) in typical eukaryotic translational systems, or a Shine-Dalgarno sequence (GGAGGU, SEQ ID NO: 543) in typical prokaryotic translation systems. As such in some embodiments, a transcript comprising a “coding” polynucleotide sequence, for example a first nucleic acid sequence, or second nucleic acid sequence involved in the production of a fermentation product, comprises an appropriate start codon and translational initiation sequence. In some embodiments, for example if two or more “coding” polynucleotide sequences are positioned in cis on a transcript, each polynucleotide sequence comprises an appropriate start codon and translational initiation sequence(s).


In some embodiments, for example if two or more “coding” polynucleotide sequences are positioned in cis on a transcript, the two sequences are under control of a single translation initiation sequence, and either provide a single polypeptide that can function with both encoded polypeptides in cis, or provide a means for separating two polypeptides encoded in cis, for example a 2A sequence or the like. In some embodiments, a translational intiator tRNA is regulatable, so as to regulate initiation of translation of an immunity modulator or industrially useful molecule.









TABLE 3.1







Exemplary Metal-Sensitive Promoters









SEQ ID NO:
Name
Description





544
BBa_I721001
Lead Promoter


545
BBa_I731004
FecA promoter


546
BBa_I760005
Cu-sensitive promoter


547
BBa_I765000
Fe promoter


548
BBa_I765007
Fe and UV promoters


549
BBa_J3902
PrFe (PI + PII rus operon)
















TABLE 3.2







Exemplary Cell-Signaling-Responsive Promoters









SEQ ID NO:
Name
Description





550
BBa_I1051
Lux cassette right promoter


551
BBa_I14015
P(Las) TetO


552
BBa_I14016
P(Las) CIO


553
BBa_I14017
P(Rhl)


554
BBa_I739105
Double Promoter (LuxR/HSL, positive / cI, negative)


555
BBa_I746104
P2 promoter in agr operon from S. aureus


556
BBa_I751501
plux-cI hybrid promoter


557
BBa_I751502
plux-lac hybrid promoter


558
BBa_I761011
CinR, CinL and glucose controlled promotor


559
BBa_J06403
RhIR promoter repressible by CI


560
BBa_J102001
Reverse Lux Promoter


561
BBa_J64000
rhlI promoter


562
BBa_J64010
lasI promoter


563
BBa_J64067
LuxR + 3OC6HSL independent R0065


564
BBa_J64712
LasR/LasI Inducible & RHLR/RHLI repressible Promoter


565
BBa_K091107
pLux/cI Hybrid Promoter


566
BBa_K091117
pLas promoter


567
BBa_K091143
pLas/cI Hybrid Promoter


568
BBa_K091146
pLas/Lux Hybrid Promoter


569
BBa_K091156
pLux


570
BBa_K091157
pLux/Las Hybrid Promoter


571
BBa_K145150
Hybrid promoter: HSL-LuxR activated, P22 C2 repressed


572
BBa_K266000
PAI + LasR → LuxI (AI)


573
BBa_K266005
PAI + LasR → LasI & AI + LuxR --| LasI


574
BBa_K266006
PAI + LasR → LasI + GFP & AI + LuxR --| LasI + GFP


575
BBa_K266007
Complex QS → LuxI & LasI circuit


576
BBa_K658006
position 3 mutated promoter lux pR-3 (luxR & HSL regulated)


577
BBa_K658007
position 5 mutated promoter lux pR-5 (luxR & HSL regulated)


578
BBa_K658008
position 3&5 mutated promoter lux pR-3/5 (luxR & HSL regulated)


579
BBa_R0061
Promoter (HSL-mediated luxR repressor)


580
BBa_R0062
Promoter (luxR & HSL regulated -- lux pR)


581
BBa_R0063
Promoter (luxR & HSL regulated -- lux pL)


582
BBa_R0071
Promoter (Rh1R & C4-HSL regulated)


583
BBa_R0078
Promoter (cinR and HSL regulated)


584
BBa_R0079
Promoter (LasR & PAI regulated)


585
BBa_R1062
Promoter, Standard (luxR and HSL regulated -- lux pR)
















TABLE 3.3







Exemplary Constitutive E. coli σ70 Promoters









SEQ ID NO:
Name
Description





586
BBa_I14018
P(Bla)


587
BBa_I14033
P(Cat)


588
BBa_I14034
P(Kat)


589
BBa_I732021
Template for Building Primer Family Member


590
BBa_I742126
Reverse lambda cI-regulated promoter


591
BBa_J01006
Key Promoter absorbs 3


592
BBa_J23100
constitutive promoter family member


593
BBa_J23101
constitutive promoter family member


594
BBa_J23102
constitutive promoter family member


595
BBa_J23103
constitutive promoter family member


596
BBa_J23104
constitutive promoter family member


597
BBa_J23105
constitutive promoter family member


598
BBa_J23106
constitutive promoter family member


599
BBa_J23107
constitutive promoter family member


600
BBa_J23108
constitutive promoter family member


601
BBa_J23109
constitutive promoter family member


602
BBa_J23110
constitutive promoter family member


603
BBa_J23111
constitutive promoter family member


604
BBa_J23112
constitutive promoter family member


605
BBa_J23113
constitutive promoter family member


606
BBa_J23114
constitutive promoter family member


607
BBa_J23115
constitutive promoter family member


608
BBa_J23116
constitutive promoter family member


609
BBa_J23117
constitutive promoter family member


610
BBa_J23118
constitutive promoter family member


611
BBa_J23119
constitutive promoter family member


612
BBa_J23150
1bp mutant from J23107


613
BBa_J23151
1bp mutant from J23114


614
BBa_J44002
pBAD reverse


615
BBa_J48104
NikR promoter, a protein of the ribbon helix-helix family of




trancription factors that repress expre


616
BBa_J54200
lacq_Promoter


617
BBa_J56015
lacIQ - promoter sequence


618
BBa_J64951

E. Coli CreABCD phosphate sensing operon promoter



619
BBa_K088007
GlnRS promoter


620
BBa_K119000
Constitutive weak promoter of lacZ


621
BBa_K119001
Mutated LacZ promoter


622
BBa_K137029
constitutive promoter with (TA)10 between −10 and −35 elements


623
BBa_K137030
constitutive promoter with (TA)9 between −10 and −35 elements


624
BBa_K137031
constitutive promoter with (C)10 between −10 and −35 elements


625
BBa_K137032
constitutive promoter with (C)12 between −10 and −35 elements


626
BBa_K137085
optimized (TA) repeat constitutive promoter with 13 bp between




−10 and −35 elements


627
BBa_K137086
optimized (TA) repeat constitutive promoter with 15 bp between




−10 and −35 elements


628
BBa_K137087
optimized (TA) repeat constitutive promoter with 17 bp between




−10 and −35 elements


629
BBa_K137088
optimized (TA) repeat constitutive promoter with 19 bp between




−10 and −35 elements


630
BBa_K137089
optimized (TA) repeat constitutive promoter with 21 bp between




−10 and −35 elements


631
BBa_K137090
optimized (A) repeat constitutive promoter with 17 bp between




−10 and −35 elements


632
BBa_K137091
optimized (A) repeat constitutive promoter with 18 bp between




−10 and −35 elements


633
BBa_K256002
J23101:GFP


634
BBa_K256018
J23119:IFP


635
BBa_K256020
J23119:HO1


636
BBa_K256033
Infrared signal reporter (J23119:IFP:J23119:HO1)


637
BBa_K292000
Double terminator + constitutive promoter


638
BBa_K292001
Double terminator + Constitutive promoter + Strong RBS


639
BBa_K418000
IPTG inducible Lac promoter cassette


640
BBa_K418002
IPTG inducible Lac promoter cassette


641
BBa_K418003
IPTG inducible Lac promoter cassette


642
BBa_M13101
M13K07 gene I promoter


643
BBa_M13102
M13K07 gene II promoter


644
BBa_M13103
M13K07 gene III promoter


645
BBa_M13104
M13K07 gene IV promoter


646
BBa_M13105
M13K07 gene V promoter


647
BBa_M13106
M13K07 gene VI promoter


648
BBa_M13108
M13K07 gene VIII promoter


649
BBa_M13110
M13110


650
BBa_M31519
Modified promoter sequence of g3.


651
BBa_R1074
Constitutive Promoter I


652
BBa_R1075
Constitutive Promoter II


653
BBa_S03331
Constitutive promoter
















TABLE 3.4







Exemplary Constitutive E. coli σ8 Promoters









SEQ ID NO:
Name
Description





654
BBa_J45992
Full-length stationary phase osmY promoter










655
BBa_J45993

Minimal stationary phase osmY promoter
















TABLE 3.5







Exemplary Constitutive E. coli σ32 Promoters









SEQ ID NO:
Name
Description





656
BBa_J45504
htpG Heat Shock Promoter
















TABLE 3.6







Exemplary Constitutive B. subtilis σA Promoters









SEQ ID NO:
Name
Description





657
BBa_K143012
Promoter veg a constitutive




promoter for B. subtilis


658
BBa_K143013
Promoter 43 a constitutive




promoter for B. subtilis


659
BBa_K780003
Strong constitutive promoter




for Bacillus subtilis


660
BBa_K823000
PliaG


661
BBa_K823002
PlepA


662
BBa_K823003
Pveg
















TABLE 3.7







Exemplary Constitutive B. subtilis σB Promoters









SEQ ID NO:
Name
Description





663
BBa_K143010
Promoter etc for B. subtilis


664
BBa_K143011
Promoter gsiB for B. subtilis


665
BBa_K143013
Promoter 43 a constitutive




promoter for B. subtilis
















TABLE 3.8







Exemplary Constitutive Promoters from


miscellaneous prokaryotes









SEQ ID NO:
Name
Description





666
a_K112706
Pspv2 from Salmonella


667
BBa_K112707
Pspv from Salmonella
















TABLE 3.9







Exemplary Constitutive Promoters from bacteriophage T7











SEQ ID NO:
Name
Description







668
BBa_I712074
T7 promoter (strong promoter





from T7 bacteriophage)



669
BBa_I719005
T7 Promoter



670
BBa_J34814
T7 Promoter



671
BBa_J64997
T7 consensus −10 and rest



672
BBa_K113010
overlapping T7 promoter



673
BBa_K113011
more overlapping T7 promoter



674
BBa_K113012
weaken overlapping T7 promoter



675
BBa_R0085
T7 Consensus Promoter Sequence



676
BBa_R0180
T7 RNAP promoter



677
BBa_R0181
T7 RNAP promoter



678
BBa_R0182
T7 RNAP promoter



679
BBa_R0183
T7 RNAP promoter



680
BBa_Z0251
T7 strong promoter



681
BBa_Z0252
T7 weak binding and processivity



682
BBa_Z0253
T7 weak binding promoter

















TABLE 3.10







Exemplary Constitutive Promoters from yeast











SEQ ID NO:
Name
Description







683
BBa_I766555
pCyc (Medium) Promoter



684
BBa_I766556
pAdh (Strong) Promoter



685
BBa_I766557
pSte5 (Weak) Promoter



686
BBa_J63005
yeast ADH1 promoter



687
BBa_K105027
cyc100 minimal promoter



688
BBa_K105028
cyc70 minimal promoter



689
BBa_K105029
cyc43 minimal promoter



690
BBa_K105030
cyc28 minimal promoter



691
BBa_K105031
cyc16 minimal promoter



692
BBa_K122000
pPGK1



693
BBa_K124000
pCYC Yeast Promoter



694
BBa_K124002
Yeast GPD (TDH3) Promoter



695
BBa_K319005
yeast mid-length ADH1 promoter



696
BBa_M31201
Yeast CLB1 promoter region,





G2/M cell cycle specific

















TABLE 3.11







Exemplary Constitutive Promoters from miscellaneous eukaryotes









SEQ ID NO:
Name
Description





697
BBa_I712004
CMV promoter


698
BBa_K076017
Ubc Promoter









The above-referenced promoters are provided by way of non-limiting example only. A promoter may be a synthetic promoter. Suitable promoters for methods, uses, compositions, hosts, and nucleic acids of some embodiments herein have been described earlier herein e.g., proC represented by SEQ ID NO: 708 which has been experimentally confirmed in accordance with some embodiments herein (See Example 2) and P24 represented by SEQ ID NO: 707 which has been experimentally confirmed in accordance with some embodiments herein (See Example 3). In some embodiments, a suitable inducible promoter is the P24 LacO hybrid promoter, which is repressed in the presence of Lad and active in presence of IPTG. This promoter has been experimentally confirmed in accordance with some embodiments herein (See Example 3).


The skilled artisan will readily recognize that many variants of the above-referenced promoters, and many other promoters (including promoters isolated from naturally existing organisms, variations thereof, and fully synthetic or engineered promoters) can readily be used in accordance with some embodiments herein. A variant, fully synthetic or synthetic or engineer promoter is said to be active or functional and can therefore be used in methods, uses, compositions, hosts, and nucleic acids of embodiments herein when tested in a control or reference plasmid being operably linked with a nucleic acid molecule encoding a transcript, a detectable amount of said transcript molecule is present when said plasmid is present in a cell. A variant, fully synthetic or synthetic or engineer promoter may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the activity of the promoter it derives from.


Optionally, the method comprises transforming said microbial host with said auto-replicative extra-chromosomal nucleic acid molecule under conditions allowing the host that has received said auto-replicative extra-chromosomal nucleic acid molecule to survive. It is noted that in some embodiments, the auto-replicative extra-chromosomal nucleic acid molecule can be provided in a microbial cell (e.g., if the microbial cell, or a predecessor thereof was transformed with the auto-replicative extra-chromosomal nucleic acid molecule), and as such, in some embodiments, the transformation step is not needed in the method. The transforming step can be performed prior to the culturing of step c). In some embodiments, the transforming step is provided prior to step a) so as to provide the host cell comprising the auto-replicative extra-chromosomal nucleic acid molecule. In some embodiments, the auto-replicative extra-chromosomal nucleic acid molecule used in the transforming step further comprises the second nucleic acid of optional step b).


Techniques of genetically modifying microbial organisms are well known in the art (for example see Molecular Cloning Fourth edition, 2012 Cold Spring Harbor Laboratory Press, A laboratory manual, by M. R. Green and J Sambrook, which is herein incorporated by reference in its entirety). In some embodiments, a microorganism is genetically modified to comprise said auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence and optionally a molecule involved in the production of a product of interest. Polynucleotides or nucleic acid molecules can be delivered to microorganisms.


In an embodiment a microbial cell is positively selected for by the genetic activity of the first nucleic acid sequence corresponding to at least one given condition allowing the cell that has received the said auto-replicative extra-chromosomal nucleic acid molecule to survive and said conditions can be environmental conditions. Environmental conditions may be a culture medium.


It can be useful to flexibly genetically modify a microbial cell, for example to engineer or reengineer a microbial cell to have a desired type and/or spectrum of genetic activities. In some embodiments, a cassette for inserting one or more desired distinct first nucleic acid sequences is provided. Exemplary cassettes include, but are not limited to, a Cre/lox cassette or FLP/FRT cassette.


In an embodiment, a microbial cell comprises more than one (more than two, more than three, . . . ) different auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence as described herein, meaning that said cell can exhibit more than one (more than two, more than three, . . . ) genetic activity, each genetic activity conferring an advantage to the cell. If a first promoter is present in each of the different auto-replicative extra-chromosomal nucleic acid molecule, each of said first promoters may be different or identical. It is therefore within the scope of the of methods, uses, compositions, hosts, and nucleic acids of embodiments herein to use one, two, three, four or more distinct bacteriocins in a method for producing a product of interest wherein the microbial host comprises one, two, three, four or more distinct extra-chromosomal nucleic acid molecule, each conferring a distinct genetic activity to said microbial host. Alternatively, it is within the scope of methods, uses, compositions, hosts, and nucleic acids of embodiments herein that a single nucleic acid molecule whose product provides immunity to at least distinct bacteriocins is used. Such a nucleic acid molecule has been described herein.


In some embodiments, plasmid conjugation can be used to introduce a desired plasmid from a “donor” microbial cell to a recipient microbial cell. Goni-Moreno, et al. (2013) Multicellular Computing Using Conjugation for Wiring. PLoS ONE 8(6): e65986, hereby incorporated by reference in its entirety. In some embodiments, plasmid conjugation can genetically modify a recipient microbial cell by introducing a conjugation plasmid from a donor microbial cell to a recipient microbial cell. Without being limited by any particular theory, conjugation plasmids that comprise the same or functionally same set of replication genes typically cannot coexist in the same microbial cell. As such, in some embodiments, plasmid conjugation “reprograms” a recipient microbial cell by introducing a new conjugation plasmid to supplant another conjugation plasmid that was present in the recipient cell. In some embodiments, plasmid conjugation is used to engineer (or reengineer) a microbial cell with a particular combination of first nucleic acid molecules (which can code for immunity modulators in some embodiments). According to some embodiments, a variety of conjugation plasmids comprising different combinations of first acid sequence (which can code for immunity modulators in some embodiments) is provided. The plasmids can comprise additional genetic elements as described herein, for example promoters, translational initiation sites, and the like. In some embodiments the variety of conjugation plasmids is provided in a collection of donor cells, so that a donor cell comprising the desired plasmid can be selected for plasmid conjugation. In some embodiments, a particular combination of immunity modulators is selected, and an appropriate donor cell is conjugated with a microbial cell of interest to introduce a conjugation plasmid comprising that combination into a recipient cell. In some embodiments, the recipient cell is a “newly engineered” cell, for example to be introduced into or for initiating a culture.


Step b)


In addition to step a), in some embodiments the method further comprises optional step b) wherein said auto-replicative extra-chromosomal nucleic acid molecule comprises a second nucleic acid sequence that is involved in the production of said product of interest, wherein the genetic activity of said second nucleic acid sequence is controlled independently from the one of the first sequence.


In the context of methods, uses, compositions, hosts, and nucleic acids of embodiments herein, the expression “controlled independently” has its customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, including meaning that distinct ways are used for controlling the genetic activity of the first and the second nucleic acid sequences. Ways of controlling the genetic activity of a nucleic acid sequence have been already described in detail herein.


Step c)


In some embodiments, step a) (which optionally includes transforming as described herein) and optional step b) is followed by step c), which comprises culturing said transformed microbial host under conditions allowing said transformed microbial host to express the first nucleic acid sequence to a given level to maintain the auto-replicative extra-chromosomal molecule into the growing microbial population. In some embodiments, optionally controlling the second sequence coding for said product of interest.


In a method of some embodiments, at least part of step c) conditions are such that the first nucleic acid sequence does not exhibit said genetic activity. “Part of step c)” means, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or up to 100% of the duration of step c). This embodiment of the method is quite attractive as part of step c) is carried out without the presence of the genetic activity of the first nucleic acid sequence. The presence of said genetic activity forms an energetic burden for the microbial host cell and is not always needed in order to keep a suitable production level of a product of interest. It is envisaged in some embodiments to have part of step c) without genetic activity of the first nucleic acid sequence followed by a part with said activity. These two parts may be repeated one or more time during step c).


A microbial cell may be cultured in any suitable microbial culture environment. Microbial culture environments can comprise a wide variety of culture media, for example feedstocks. The selection of a particular culture medium can depend upon the desired application. Conditions of a culture medium include not only chemical composition, but also temperature, amounts of light, pH, CO2 levels, and the like. The culture medium can comprise a bacteriocin. In an embodiment, a compound that induces the activity of the bacteriocin is present outside of the feedstock, but not in the feedstock.


In an embodiment, a genetically engineered or transformed microorganism as described herein is added to a culture medium that comprises at least one feedstock. In an embodiment, the culture medium comprises a compound that induces the activity or expression of an immunity modulator.


The term “feedstock” has is customary and ordinary meaning as understood by one of skill in the art in view of this disclosure, and encompasses material that can be consumed, fermented, purified, modified, or otherwise processed by microbial organisms, for example in the context of industrial processes. As such, “feedstock” is not limited to food or food products. As used herein a “feedstock” is a category of culture medium. Accordingly, as used herein “culture medium” includes, but it is not limited to feedstock. As such, whenever a “culture medium” is referred to herein, feedstocks are also expressly contemplated.


Before culturing a transformed microbial cell, it can be useful to determine the effects, if any, or optimize the conditions allowing the host that has received said auto-replicative extra-chromosomal nucleic acid molecule to survive and optionally to grow.


In some embodiments, a microbial cell or microbial host or microbial host cell or synthetic microbial host cell comprising an auto-replicative extra-chromosomal nucleic acid molecule is provided, comprising a first nucleic acid sequence whose genetic activity confers an advantage to a microbial host wherein the genetic activity of said first nucleic acid sequence is controlled, and optionally comprising a second nucleic acid sequence that is directly or indirectly involved in the production of a product of interest.


In some embodiments, there is provided an auto-replicative extra-chromosomal nucleic acid molecule, comprising a first nucleic acid sequence whose genetic activity confers an advantage to a microbial host wherein the genetic activity of said first nucleic acid sequence is controlled, and optionally comprising a second nucleic acid sequence that is directly or indirectly involved in the production of a product of interest.


Each feature of this microbial host and of this auto-replicative extra-chromosomal nucleic acid molecule have already been described herein.


General Descriptions


The terms used herein have the customary and ordinary meaning understood by one of skill in the art when read in view of this disclosure, and can include the following general descriptions.


Microorganism


As used herein, “microbial organism,” “microorganism,” “microbial cell” or “microbial host” and variations of these root terms (such as pluralizations and the like) have their customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, including any naturally-occurring species or synthetic or fully synthetic prokaryotic or eukaryotic unicellular organism, as well as Archae species. Thus, this expression can refer to cells of bacterial species, fungal species, and algae. Exemplary microorganisms that can be used in accordance with embodiments herein include, but are not limited to, bacteria, yeast, filamentous fungi, and algae, for example photosynthetic microalgae. Furthermore, fully synthetic microorganism genomes can be synthesized and transplanted into single microbial cells, to produce synthetic microorganisms capable of continuous self-replication (see Gibson et al. (2010), “Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome,” Science 329: 52-56, hereby incorporated by reference in its entirety). As such, in some embodiments, the microorganism is fully synthetic. A desired combination of genetic elements, including elements that regulate gene expression, and elements encoding gene products (for example immunity modulators, poison, antidote, and industrially useful molecules also called product of interest) can be assembled on a desired chassis into a partially or fully synthetic microorganism. Description of genetically engineered microbial organisms for industrial applications can also be found in Wright, et al. (2013) “Building-in biosafety for synthetic biology” Microbiology 159: 1221-1235. Suitable embodiments of genetic elements will be described later herein.


A variety of bacterial species and strains can be used in accordance with embodiments herein, and genetically modified variants, or synthetic bacteria based on a “chassis” of a known species can be provided. Exemplary bacteria with industrially applicable characteristics, which can be used in accordance with embodiments herein include, but are not limited to, Bacillus species (for example Bacillus coagulans, Bacillus subtilis, and Bacillus licheniformis), Paenibacillus species, Streptomyces species, Micrococcus species, Corynebacterium species, Acetobacter species, Cyanobacteria species, Salmonella species, Rhodococcus species, Pseudomonas species, Lactobacillus species, Enterococcus species, Alcaligenes species, Klebsiella species, Paenibacillus species, Arthrobacter species, Corynebacterium species, Brevibacterium species, Thermus aquaticus, Pseudomonas stutzeri, Clostridium thermocellus, and Escherichia coli. A variety of yeast species and strains can be used in accordance with embodiments herein, and genetically modified variants, or synthetic yeast based on a “chassis” of a known species can be provided. Exemplary yeast with industrially applicable characteristics, which can be used in accordance with embodiments herein include, but are not limited to Saccharomyces species (for example, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces boulardii), Candida species (for example, Candida utilis, Candida krusei), Schizosaccharomyces species (for example Schizosaccharomyces pombe, Schizosaccharomyces japonicas), Pichia or Hansenula species (for example, Pichia pastoris or Hansenula polymorphd) species, and Brettanomyces species (for example, Brettanomyces claussenii).


A variety of algae species and strains can be used in accordance with embodiments herein, and genetically modified variants, or synthetic algae based on a “chassis” of a known species can be created. In some embodiments, the algae comprises, consists essentially of, or consists of photosynthetic microalgae. Exemplary algae species that can be useful for biofuels, and can be used in accordance with embodiments herein, include Botryococcus braunii, Chlorella species, Dunaliella tertiolecta, Gracilaria species, Pleurochrysis carterae, and Sargassum species. Additionally, many algaes can be useful for food products, fertilizer products, waste neutralization, environmental remediation, and carbohydrate manufacturing (for example, biofuels).


A variety of filamentous fungal species and strains can be used in accordance with embodiments herein, and genetically modified variants, or synthetic filamentous fungi based on a “chassis” of a known species can be provided. Exemplary filamentous fungi with industrially applicable characteristics, which can be used in accordance with embodiments herein include, but are not limited to an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocaffimastix, Neurospora, Paecilomyces, Peniciffium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria.


Species include Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zona turn, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenaturn, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.


Antibiotic


“Antibiotic,” and variations of this root term, have their customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, including a metabolite, or an intermediate of a metabolic pathway which can kill or arrest the growth of at least one microbial cell. Some antibiotics can be produced by microbial cells, for example bacteria. Some antibiotics can be synthesized chemically. It is understood that bacteriocins are distinct from antibiotics, at least in that bacteriocins refer to gene products (which, in some embodiments, undergo additional post-translational processing) or synthetic analogs of the same, while antibiotics refer to intermediates or products of metabolic pathways or synthetic analogs of the same.


Sequence Identity and Similarity


Sequence identity has its customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, including a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, “identity” can also refer to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. “Similarity” between two amino acid sequences can be determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. “Identity” and “similarity” can be readily calculated by various methods, known to those skilled in the art. In some embodiments, sequence identity is determined by comparing the whole length of the sequences as identified herein.


Some methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Computer program methods to determine identity and similarity between two sequences include e.g. the BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990), publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894). An algorithm used can be EMBOSS (accessible on the world wide web at www(dot)ebi(dot)ac(dot)uk/emboss/align). Parameters for amino acid sequences comparison using EMBOSS can include gap open 10.0, gap extend 0.5, Blosum 62 matrix. Parameters for nucleic acid sequences comparison using EMBOSS can include gap open 10.0, gap extend 0.5, DNA full matrix (DNA identity matrix).


Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called “conservative” amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Suitable conservative amino acids substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. In some embodiments, the amino acid change is conservative. Suitable conservative substitutions for each of the naturally occurring amino acids include: Ala to ser; Arg to lys; Asn to gln or his; Asp to glu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asn or gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.


Homologous


The term “homologous” has its customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, including when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, it can be understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, optionally of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically be operably linked to another promoter sequence than in its natural environment. When used to indicate the relatedness of two nucleic acid sequences the term “homologous” has its customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, and can refer to one single-stranded nucleic acid sequence that may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as earlier presented. The region of identity can be greater than about 5 bp, the region of identity can be greater than 10 bp. In some embodiments, two nucleic acid or polypeptides sequences are said to be homologous when they have more than 80% identity.


Heterologous


The term “heterologous” has its customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, including when used with respect to a nucleic acid (DNA or RNA) or protein, it can refer to a nucleic acid or protein (also named polypeptide or enzyme) that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but has been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.


Operably Linked


As used herein, the term “operably linked” has its customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, and can refer to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence or nucleic acid molecule) in a functional relationship. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the nucleic acid sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.


Promoter


As used herein, the term “promoter” has its customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, and can refer to a nucleic acid fragment that functions to control the transcription of one or more nucleic acid molecules, located upstream with respect to the direction of transcription of the transcription initiation site of the nucleic acid molecule, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate/control the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation.


In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that an auto-replicative extra-chromosomal nucleic acid molecule, a microbial host (or a method) as defined herein may comprise additional component(s) (or additional steps) than the ones specifically identified, said additional component(s) (or additional steps) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.


All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way


SEQUENCE LISTING (DNA UNLESS OTHERWISE INDICATED)

SEQ ID NO: 1 Enterococcus faecalis peptide


SEQ ID NO: 2 motif characteristic of bacteriocin


SEQ ID NO: 3 hybrid bacteriocin, Ent35-MccV


SEQ ID NO: 4-698, 720-759: Sequences in tables 1-3 (half DNA/half protein as indicated in the tables)


SEQ ID NO: 699 McbG


SEQ ID NO: 700 MccE


SEQ ID NO: 701 C terminal part of MccE


SEQ ID NO: 702 pSyn2-McbG


SEQ ID NO: 703 pSyn2-McbE/F


SEQ ID NO: 704 pMcbG1.0


SEQ ID NO: 705 pMcbG.1.1.


SEQ ID NO: 706 C-terminal part of MccE (amino acid)


SEQ ID NO: 707 P24 promoter


SEQ ID NO: 708 proC promoter


SEQ ID NO: 709 Cvi


SEQ ID NO: 710 proC-McbG-CterMccE(proc) (Cter could be replaced by C-terminal)


SEQ ID NO: 711 proC-Cvi-Cter-MccE (proc) (Cter could be replaced by C-terminal)


SEQ ID NO: 712 pBACT5.0


SEQ ID NO: 713 pBACT2.0


SEQ ID NO: 714 pBACT5.0-mcherry


SEQ ID NO: 715 McbG-MccE


SEQ ID NO: 716 McbG-Cter part MccE (Cter could be replaced by C-terminal)


SEQ ID NO: 717 Cvi-MccE


SEQ ID NO: 718 Cvi-Cter part MccE (Cter could be replaced by C-terminal)


SEQ ID NO: 719: vector pUC-ColV





DESCRIPTION OF THE FIGURES


FIG. 1: Construction: pSyn2-McbE/F: containing the gene McbE and F under Ptac.



FIG. 2: Construction: pSyn2-McbG: containing the gene McbG under Ptac.



FIG. 3: Construction: pMcbG 1.1: containing the gene McbG under P24.



FIG. 4: Construction: pMcbG 1.0: containing the gene McbG under P24 LacO.



FIG. 5: pBACT5.0 vector



FIG. 6: pBACT2.0 vector



FIG. 7: pBACT5.0-mcherry vector



FIG. 8: Tuning promoter. In the absence of inductor (upper part), repressor can bind to operator and prevent expression of selection gene. In the presence of inductor (lower part), repressor cannot bind to operator allowing expression of selection gene.



FIG. 9: Comparison of overexpression of protein X in E. coli with KanR (pKan-pLac) and with 2 immunities against microcines C7 and ColV (pBACT6.0-pLac). 5 mg of total extract was analysed in SDS-PAGE.



FIG. 10: Comparison of overexpression of iota-carrageenase protein in E. coli with KanR (pKan-T7prom) and with 2 immunities against microcines C7 and ColV (pBACT5.0-T7prom). 5 mg of total extract was analysed in SDS-PAGE.



FIG. 11: Comparison of overexpression of lambda-carrageenase protein in E. coli with KanR (pKan-T7prom) and with 2 immunities against microcines C7 and ColV (pBACT5.0-T7prom). 5 mg of total extract was analysed in SDS-PAGE.





EXAMPLES
Example 1: Use of Bacteriocin B17 and C7 as Selection Agent

1. Production of Bacteriocin B17, C7 and ColV


Strain used: C600: F tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1λ


Described in Appleyard Genetics 39 (1954), 440-452.


The vector used for producing Mic B17 is described in the table below.









TABLE X





Vector used for producing Mic B17
















Construct used
pACYC184 containing the mccB17-producing genes


pCID909
(mcbABCDEFG), chloramphenicol resistance









These constructs were described in detail in Rodriguez-Sainz, M. C., C. et al. 1990. Mol. Microbiol. 4:1921-1932.


The vector used for producing Mic C7 is Pp70. This vector is based on pBR322 and bears a ˜6000 bp DNA fragment with the mcc gene cluster (as described in Zukher I et al, Nucleic Acids Research, 2014, Vol. 42, No. 19 11891-11902).


The vector used for producing ColV is pUC-ColV (SEQ ID NO: 719). This vector is based on pUC57 and bear a ˜5000 bp DNA fragment with the ColV gene cluster. The strains harbouring these recombinant vectors were grown in LB medium at 37° C.


After an overnight culture the fermented medium was centrifuged and the supernatant flit red on a 0.2 micron filter.


The bacteriocin activity present in the supernatant was estimated by the size of the diffusion inhibition growth on a plate containing a sensitive strain.


2. Results


We demonstrated that we can use the supernatants that exhibit B17, C7 or ColV activities as classical antibiotics such as Amp, Kan or Chlo added in culture medium. Supernatant presenting such a bacteriocin activity were stored for several months (at least 12 months) at −20° C. and we did not observe a significant decrease of activity. Petri plates containing medium with such a bacteriocin activity were stored at +4° C. for several weeks (at least 4 weeks). We did not observe a decrease of activity. Therefore we demonstrated that such B17, C7 or ColV activities as present in culture medium are stable.


3. Conclusion


Bacteriocins B17, C7 and ColV produced by fermentation in laboratory are selection agents simple to produce, easy to use and stable in culture medium. These properties are similar to the ones of antibiotics used as classical selection agent.


Example 2: Identification of the Minimum Genetic Elements Necessary to Confer Resistance to C7 and B17

1. Construction of Needed Vectors


The literature has made it possible to determine the elements necessary for the production of the host against the production of its own bacteriocin, also in the case of B17 bacteriocin: McbG for B17, represented by SEQ ID NO: 699 and pumps (McbE and McbF for B17, represented by SEQ ID NO: 703). These genes are known to be necessary (or more precisely involved in protection against the action of bacteriocin B17). The literature for the B17 locus does not identify which is or is the sufficient element to give resistance.


We have separated genes from B17 immunity structures and cloned these into vectors behind an inducible promoter (Ptac).


Construction: pSyn2-McbG (FIG. 2, SEQ ID NO: 702): containing the gene McbG under Ptac


Construction: pSyn2-McbE/F (FIG. 1, SEQ ID NO: 703): containing the gene McbE and F under Ptac


We have separated the genes from B17 immunity structures and cloned them into vectors behind an inducible promoter (Ptac).


We have shown that low McbG expression (Ptac not induced) is sufficient to give the phenotype of resistance to the strain on the other hand the presence of McbE/F is toxic and did not allow to give a response As to the protection provided in relation to the presence of B17.


2. Results


Surprisingly it has been found that the C-terminal part of MccE which is represented by SEQ ID NO: 701 is sufficient to confer resistance to bacteriocin C7.


We have demonstrated that expression from a plasmid of the McbG and MccE genes (or truncated MccE, represented by SEQ ID NO: 701) are capable when cloned into a vector to give resistance to B17 and C7 respectively and that these proteins can be used as a vector selection marker in strains sensitive to these microcines/bacteriocins. The vector used is pBACT2.0 (FIG. 6, SEQ ID NO: 713).


SEQ ID NO: 710 represents the construct proC-McbG-CterMccE


We have demonstrated that expression from a plasmid of the Cvi and C-terminal part of MccE genes are capable when cloned into a vector to give resistance to ColV and C7 respectively and that these proteins can be used as a vector selection marker in strains sensitive to these microcines/bacteriocins. The vector used is pBACT5.0 (FIG. 5, SEQ ID NO: 712).


SEQ ID NO: 711 represents the construct proC-Cvi-CterMccE


3. Conclusion


It is therefore possible to use a single segment of small size represented by SEQ ID NO: 701 as a selection marker against C7.


Example 3: Can we Generate a Selectable Marker Using Little or No Energy from the Bacteria from McbG?

1. Vectors Constructed


To answer this question the McbG gene was cloned under a weak promoter P24 (SEQ ID NO: 707). The P24 promoter was described in Braatsch S et al, Biotechniques. 2008 Sep.; 45(3):335-7.


We inserted the B17 McbG immunity structure gene and cloned the latter into vectors behind the weak constitutive promoter (P24). A second construct was generated with a P24 LacO hybrid promoter which is an inducible promoter repressed in the presence of lad and active in presence of IPTG.


Construction: pMcbG 1.0 (FIG. 4, SEQ ID NO: 704) containing the gene McbG under P24 LacO.


Construction: pMcbG 1.1 (FIG. 3, SEQ ID NO: 705) containing the gene McbG under P24.


The strains used are the following:


BL21(DE3): fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS


λ DE3=λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5


The BL21(DE3) strain was transformed with vector pMcbG1.0 or pMcbG1.1 (see FIG. 3 or 4). After transformation, transformants were selected on plates containing B17. Isolated colonies were re-grown and the plasmid they contained was analyzed by gel electrophoresis after treatment with relevant restriction enzymes.


2. Results


We showed that a weak transcription of McbG is sufficient to give the resistance to B17. In addition, we have shown that this selection marker is inducible via the P24 LacO promoter and that the vectors containing this gene gives the phenotype of resistance only in presence of IPTG.


3. Conclusions


It is possible to use the McbG gene as a selectable marker either constitutively or inducibly. Thus, it is shown that constitutive expression at a low level and inducible expression according to the need during the process, allows to reduce the energy burden for the producing cell, without loss of the plasmid from the producing cell.


Example 4: Production of m-Cherry Protein

SEQ ID NO: 714 represents the construct used for producing the m-cherry protein. This construct is depicted in FIG. 7.


The m-cherry protein was produced and visualised as the bacterial colony turns red on petri dish in the presence of IPTG.


Example 5: Tuning Promoter

We have prepared vectors with immunity selection that is tunable (see FIG. 8). Tuning the promoter allows us to switch the selection “on” or “off”. For the first time this allows to adapt the selective pressure to the need during the fermentation process, for example according to the loss by the host of the recombinant plasmid. This will provide an advantage by limiting the burden of energy for the host. Thus, such vectors improve the industrial outcome (recombinant product). Moreover, they are easy to use in any strain (no requirement for a special feature in the host genome) and require no antibiotics.


Example 6: Comparison of Antibiotic (Kanamycin) Selection with Immunity Selection

We have applied immunity selection on different recombinant proteins.



FIG. 9 shows the comparison of overexpression of protein X in E. coli with KanR (pKan-pLac) and with 2 immunities against microcines C7 and ColV (pBACT6.0-pLac). 5 mg of total extract was analysed in SDS-PAGE.



FIG. 10 shows the comparison of overexpression of iota-carrageenase protein in E. coli with KanR (pKan-T7prom) and with 2 immunities against microcines C7 and ColV (pBACT5.0-T7prom). 5 mg of total extract was analysed in SDS-PAGE. The vector used is based on the pBACT5.0 vector (SEQ ID NO: 712).



FIG. 11 shows the comparison of overexpression of lambda-carrageenase protein in E. coli with KanR (pKan-T7prom) and with 2 immunities against microcines C7 and ColV (pBACT5.0-T7prom). 5 mg of total extract was analysed in SDS-PAGE. The vector used is based on the pBACT5.0 vector (SEQ ID NO: 712).


The weak constitutive proC promoter used in this example allows to reduce the energy burden for the host.

Claims
  • 1. A method for producing a product of interest with a microbial host, said method comprising the steps of: a) providing the microbial host comprising i) an auto-replicative extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers immunity or resistance to a bacteriocin to the microbial host, wherein the genetic activity of said first nucleic acid sequence is controlled and ii) a second nucleic acid sequence coding for said product of interest; andb) culturing said transformed microbial host under conditions allowing said transformed microbial host to express the first nucleic acid sequence to a given level such that the genetic activity of the first nucleic acid sequence confers a selective advantage to the microbial host during the culturing, to thereby maintain the auto-replicative extra-chromosomal molecule in the growing microbial population, and simultaneously genetically controlling the second nucleic acid sequence to produce said product of interest,wherein during at least a portion of the culturing of step b) conditions are such that the first nucleic acid sequence does not exhibit said genetic activity thereby reducing the energetic burden for the microbial host cell during the production of the product of interest.
  • 2. The method of claim 1, further comprising transforming the microbial host with said auto-replicative extra-chromosomal nucleic acid molecule prior to or during step a), thereby providing the microbial host comprising the auto-replicative extra-chromosomal nucleic acid molecule.
  • 3. The method of claim 1, wherein the product of interest is purified at the end of the culturing step b).
  • 4. The method of claim 1, wherein the product of interest is a microbial biomass, the auto-replicative extra-chromosomal nucleic acid molecule, the transcript of said second nucleic sequence, a polypeptide encoded by said second sequence or a metabolite produced directly or indirectly by said polypeptide.
  • 5. The method of claim 1, wherein the microbial host is a bacterium, yeast, filamentous fungus or an algae.
  • 6. The method of claim 1, wherein the first nucleic acid sequence is operably linked to an inducible promoter.
  • 7. The method of claim 1, wherein the first nucleic acid sequence comprises an immunity gene whose expression confers to its microbial host a resistance to the presence of a specific bacteriocin in the medium.
  • 8. The method of claim 7, wherein the sequence encoded by the first nucleic acid sequence confers to its microbial host a resistance to the presence of at least two distinct bacteriocins in the medium.
  • 9. The method of claim 8, wherein the bacteriocin is B17, C7 or Colicin-V (ColV) and the immunity conferring resistance to a B17 is McbG, to C7 is either MccE or C-terminal MccE and to a ColV is Colicin-V immunity modulator (Cvi).
  • 10. The method of claim 1, wherein the auto-replicative extra-chromosomal nucleic acid molecule is a plasmid.
  • 11. The method of claim 1, wherein the genetic activity of said second nucleic acid sequence is controlled independently from the genetic activity of said first nucleic acid sequence.
  • 12. The method of claim 1, wherein said auto-replicative extra-chromosomal nucleic acid molecule comprises said second nucleic acid sequence.
  • 13. The method of claim 2, wherein the auto-replicative extra-chromosomal nucleic acid molecule comprises said second nucleic acid sequence.
  • 14. The method of claim 1, wherein the first nucleic acid sequence is operably linked to a weak constitutive promoter.
  • 15. The method of claim 1, wherein the method is a fermentation method.
  • 16. The method of claim 1, wherein the product of interest comprises an industrially useful molecule.
  • 17. The method of claim 16, wherein the industrially useful molecule is a carbohydrate, a lipid, an organic molecule, a nutrient, a biofuel, or precursor thereof, a pharmaceutical or biopharmaceutical product or precursor thereof, or two or more of said molecules.
Priority Claims (1)
Number Date Country Kind
17208600 Dec 2017 EP regional
PCT Information
Filing Document Filing Date Country Kind
PCT/EP2018/085941 12/19/2018 WO
Publishing Document Publishing Date Country Kind
WO2019/121983 6/27/2019 WO A
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