Fermentation process
阅读说明：本技术 发酵工艺 (Fermentation process ) 是由 菲利普·加班特 ***·尔·巴克库里 劳伦塞·范·梅尔德伦 于 2018-12-19 设计创作，主要内容包括：一些实施方式涉及使用包含第一核酸序列的自复制染色体外核酸分子、用微生物宿主生产目标产物的方法,该第一核酸序列的遗传活性向宿主赋予优势,可选地其中所述第一核酸分子的遗传活性是受控的。(Some embodiments relate to a method of producing a product of interest with a microbial host using a self-replicating extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers a advantage to the host, optionally wherein the genetic activity of the first nucleic acid molecule is controlled.)
1. A method for producing a product of interest with a microbial host, said method comprising the steps of:
a) providing a microbial host comprising a self-replicating extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers a advantage to the host, wherein the genetic activity of the first nucleic acid sequence is controlled;
b) optionally, the self-replicating extra-chromosomal nucleic acid molecule comprises a second nucleic acid sequence involved in production of the target product, wherein the genetic activity of the second nucleic acid sequence is controlled independently of the genetic activity of the first sequence;
c) culturing a transformed microbial host under conditions that allow the transformed microbial host to express the first nucleic acid sequence to a given level to maintain the self-replicating extra-chromosomal molecule in a growing microbial population while genetically controlling a second sequence encoding the product of interest.
2. The method of claim 1, further comprising transforming the microbial host with the self-replicating extrachromosomal nucleic acid molecule prior to or during step a), wherein the self-replicating extrachromosomal nucleic acid molecule optionally comprises the second nucleic acid sequence of step b), thereby providing a microbial host comprising the self-replicating extrachromosomal nucleic acid molecule.
3. The method of claim 1 or 2, wherein at least a portion of the conditions of step c) are such that the first nucleic acid sequence does not exhibit the genetic activity.
4. The method according to any one of claims 1 to 3, wherein the target product is purified at the end of the cultivation step c).
5. The method of any one of claims 1 to 4, wherein the target product is microbial biomass, the self-replicating extra-chromosomal nucleic acid molecule, a transcript of the second nucleic acid sequence, a polypeptide encoded by the second sequence, or a metabolite produced directly or indirectly by the polypeptide.
6. The method of any one of claims 1 to 5, wherein the microbial host is a bacterium, yeast, filamentous fungus, or algae.
7. The method of any one of claims 1 to 6, wherein the first nucleic acid sequence is operably linked to an inducible promoter.
8. The method of any one of claims 1 to 7, wherein said first nucleic acid sequence comprises a sequence encoding an immune gene whose expression confers resistance to the particular bacteriocin present in the culture medium to its host.
9. The method of claim 8, wherein the sequence encoded by the first nucleic acid sequence confers resistance to at least two different bacteriocins present in the culture medium to its host.
10. The method of claim 9 wherein the bacteriocin is B17, C7 or ColV, and the immunity conferring resistance to B17 is McbG, the immunity conferring resistance to C7 is MccE or C-terminal MccE, and the immunity conferring resistance to ColV is Cvi.
11. The method of any one of claims 1 to 10, wherein the self-replicating extra-chromosomal nucleic acid molecule is a plasmid.
12. A self-replicating extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence, the genetic activity of which confers a advantage to a microbial host, wherein the genetic activity of said first nucleic acid sequence is controlled,
and optionally a second nucleic acid sequence involved directly or indirectly in the production of the product of interest.
13. The self-replicating extra-chromosomal nucleic acid molecule of claim 12, wherein the first nucleic acid sequence is operably linked to an inducible promoter.
14. The self-replicating extra-chromosomal nucleic acid molecule of claim 12 or 13, which is a plasmid.
15. The self-replicating extra-chromosomal nucleic acid molecule of any of claims 12-14, wherein the first nucleic acid sequence comprises a sequence encoding an immune gene whose expression confers resistance to its host against a particular bacteriocin present in the culture medium.
16. The self-replicating extra-chromosomal nucleic acid molecule of any of claims 15, wherein the sequence encoded by the first nucleic acid sequence confers resistance to its host against at least two different bacteriocins present in the culture medium.
17. The self-replicating extra-chromosomal nucleic acid molecule of claim 16, wherein the bacteriocin is B17, C7, or ColV, and the immunomodulator that confers resistance to B17 is McbG, the immunomodulator that confers resistance to C7 is MccE or C-terminal MccE, and the immunomodulator that confers resistance to ColV is Cvi.
18. A microbial host comprising the self-replicating extra-chromosomal nucleic acid molecule of any of claims 11-16, optionally wherein the microbial cell is a bacterium, a yeast, a filamentous fungus, or an algae.
Technical Field
Embodiments herein relate to a method of producing a product of interest with a microbial host using a self-replicating extra-chromosomal nucleic acid molecule (auto-replicating extra-chromosomal nucleic acid molecule) comprising a first nucleic acid sequence whose genetic activity confers a advantage to the host, optionally wherein the genetic activity of the first nucleic acid molecule is controlled.
Background
Antibiotics are widely used as selective agents for the production of target products in microbial cells. However, there are a number of disadvantages associated with the use of antibiotics, such as the large-scale spread of antibiotics in the environment. In addition, the sequences encoding antibiotic resistance in the DNA construct represent an energy burden on the cell and therefore negatively affect the yield of the product. This energy burden is particularly important when the gene conferring resistance is a large gene, when it is expressed at high levels and/or when it is constitutively expressed.
Thus, there remains a need for an alternative and even improved method that does not suffer from all of the disadvantages of the prior art methods.
Disclosure of Invention
In a first aspect, there is provided a method of producing a product of interest with a microbial host, the method comprising the steps of:
a) providing a microbial host comprising a self-replicating extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence, optionally wherein the genetic activity of the first nucleic acid sequence is controlled;
b) optionally, the self-replicating extra-chromosomal nucleic acid molecule comprises a second nucleic acid sequence involved in production of the target product, wherein the genetic activity of the second nucleic acid sequence is controlled independently of the first sequence;
c) culturing the microbial host under conditions that allow the microbial host to express the first nucleic acid sequence to a given level to maintain a self-replicating extra-chromosomal molecule in the growing microbial population while genetically controlling a second sequence encoding the product of interest.
Step a)
Step a) comprises providing a microbial host comprising a self-replicating extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence, the genetic activity of which confers a advantage to the host, optionally wherein the genetic activity of the first nucleic acid sequence is controlled. The self-replicating extra-chromosomal nucleic acid molecule may be provided in a microbial host (e.g., a microbial cell as described herein). For example, the host or a precursor of the host may have been previously transformed using a self-replicating extrachromosomal nucleic acid molecule. Thus, in some embodiments, step a) comprises providing a microbial cell host comprising a self-replicating extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence whose genetic activity confers a advantage to the host, optionally wherein the genetic activity of the first nucleic acid sequence is controlled.
Optional transformation step
In some embodiments, a microbial host is transformed with a self-replicating extrachromosomal nucleic acid molecule under conditions that allow survival of only the host that has received the self-replicating extrachromosomal nucleic acid molecule, thereby providing a microbial host comprising the self-replicating extrachromosomal nucleic acid molecule. Thus, in some embodiments, the method further comprises, prior to or during step a), transforming the microbial host with the self-replicating extrachromosomal nucleic acid molecule under conditions that allow only the host that has received the self-replicating extrachromosomal nucleic acid molecule to survive, thereby providing a microbial host comprising the self-replicating extrachromosomal nucleic acid molecule.
The self-replicating extra-chromosomal nucleic acid molecule transformed into the microbial host optionally comprises the second nucleic acid sequence of step b). Subsequently, a microbial host comprising the self-replicating extra-chromosomal nucleic acid molecule may be cultured according to step c).
In the context of the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, there is provided a self-replicating extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence. The self-replicating extra-chromosomal nucleic acid molecule may be present without a genome and may be derived from or comprise, or consist essentially of, or consist of, a plasmid or episome, a minichromosome, or the like. This feature is attractive because higher numbers (from one to several hundred copies or from 10 to 50 copies depending on the plasmid used) of copies of such nucleic acid molecules can be introduced and maintained in the microbial cell host. Furthermore, any host may be used in the methods of the embodiments herein. In some embodiments, the genome of the host need not be modified. The genetic elements required to perform the methods of the embodiments herein are present in self-replicating extra-chromosomal nucleic acid molecules. Such self-replicating extra-chromosomal nucleic acid molecules typically comprise an origin of replication, a first nucleic acid sequence as a target, and a regulatory region. In some embodiments, without being limited by theory, the first nucleic acid sequence encoding an immunomodulatory agent serves as a selectable marker to maintain the presence and function of self-replicating extra-chromosomal nucleic acid in the host cell. In some embodiments, the first nucleic acid sequence encoding the immunomodulator is maintained in the presence of a self-replicating extra-chromosomal nucleic acid, such that a product can be produced. The product may alter the environment in which the host is located, for example, by fermenting a substance in the environment to produce one or more novel substances. In some embodiments, genetic drift is minimized by providing selective pressure against self-replicating extra-chromosomal nucleic acids that have acquired a mutation and do not produce a functional immunomodulator, produce an immunomodulator with reduced function, and/or produce lower levels of an immunomodulator than self-replicating extra-chromosomal nucleic acids that have not acquired a mutation.
In the context of the methods, uses, compositions, hosts and nucleic acids of the embodiments herein, a first nucleic acid molecule of the first nucleic acid sequence is capable of exhibiting genetic activity that confers a selective advantage to a microbial host in which the first nucleic acid molecule is present and expresses such genetic activity. The genetic activity is provided by a product encoded by the first nucleic acid molecule. Furthermore, such genetic activity may be controlled or constitutively expressed at low levels, either regulatable or under the control of a weak constitutive promoter. It is believed that control of the activity provides the advantage of limiting the energy burden on the host. Similarly, the advantage of limiting the energy burden on the host can be obtained when the genetic activity is expressed constitutively at low levels or is regulatable or under the control of a weak constitutive promoter. Throughout the application text, the concept of "conferring advantage" may be replaced by "conferring immunity to bacteriocins" or "conferring resistance to bacteriocins". In some embodiments, the first nucleic acid sequence encodes an immunomodulator described herein, and thereby confers an advantage to a host.
The second nucleic acid sequence encodes, directly or indirectly, a product of interest. The same description applies to the genetic activity of the second nucleic acid molecule described herein. In some embodiments, the target product comprises an enzyme useful in an industrial process (e.g., a fermentation process). The fermentation process may ferment at least one compound in a medium. In some embodiments, the product of interest comprises an industrially useful molecule, such as a carbohydrate, lipid, organic molecule, nutrient, fertilizer, biofuel, cosmetic (or precursor thereof), pharmaceutical or biopharmaceutical product (or precursor thereof), or two or more of the listed items.
In the context of the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, genetic activity may refer to any activity caused by or associated with the presence of the first nucleic acid molecule in a microbial host. An advantage of such 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 a combination of two or more bacteriocins as described herein). Thus, the advantage of the activity can be assessed by determining the number of microbial cells/hosts comprising the self-replicating extrachromosomal nucleic acid molecule. The evaluation may be performed at the end and/or during the optional transformation step (but before the culturing step c), or before step a) and the culturing step c)) and/or before the culturing step c). In one embodiment, the number of microbial host cells comprising the self-replicating extra-chromosomal nucleic acid molecule present is not reduced and can 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/hosts when the cells are cultured under conditions that allow survival of the microbial host that has received the self-replicating extra-chromosomal nucleic acid molecule (e.g., by having immunity to one or more bacteriocins as described herein and present under the given conditions). The evaluating step can last 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.
In the context of the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, control of genetic activity may refer to an increase or decrease in the activity of the nucleic acid molecule (i.e., the first and/or second nucleic acid molecule). Thus, the control of genetic activity may be controlled or constitutively expressed at low levels, either regulatable or under the control of a weak constitutive promoter. In some embodiments, the encoded product that is modulated/controlled for genetic activity comprises, consists essentially of, or consists of an immunomodulator and is involved in the production of a product of interest. In some embodiments, the genetic activity is modulated/controlled at the level of gene expression. In some embodiments, the genetic activity is regulated at the transcriptional level, for example by activating or repressing a promoter. In some embodiments, in this case, the promoter is an inducible promoter. In some embodiments, in this case, the promoter is a weak promoter. Without being limited by theory, the weak promoters of some embodiments may be modified to up-or down-regulate transcription levels, thereby conferring advantages to the host (e.g., immunomodulatory activity) sensitive to changes in the level and/or activity of the gene product under the control of the promoter. In some embodiments, the promoter comprises 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 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, 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 P24 LacO hybrid promoter is a regulated/controlled promoter. In some embodiments, gene activity is regulated/controlled at the post-transcriptional level, for example by modulating the stability of the RNA. In some embodiments, the genetic activity is regulated/controlled at the translational level, e.g., by regulating the initiation of translation. In some embodiments, the genetic activity is modulated/controlled at the post-translational level, for example by modulating the stability of the polypeptide, post-translationally modifying the polypeptide, or binding an inhibitor to the polypeptide.
In some embodiments, the genetic activity is increased. In some embodiments, the activity of at least one of an immunomodulator involved in producing a product of interest and/or an encoded product of a second nucleic acid molecule is increased. Conceptually, genetic activity can be increased by direct activation of genetic activity, or by decreasing the activity of an inhibitor of genetic activity. In some embodiments, the genetic activity is activated by at least one of: inducing promoter activity, repressing a transcriptional repressor, increasing RNA stability, repressing a post-transcriptional inhibitor (e.g., repressing a ribozyme or antisense oligonucleotide), inducing translation (e.g., by a regulatable tRNA), performing a desired post-translational modification, or repressing a post-translational inhibitor (e.g., a protease directed against 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 the culture medium can induce transcription by an iron-sensitive promoter as described herein. In some embodiments, the compound present in the desired medium inhibits the transcription repressor. For example, the presence of tetracycline in the environment can inhibit the tet repressor, thereby allowing for the activity of the tetO promoter. In some embodiments, only compounds found outside the desired medium induce transcription.
In some embodiments, the genetic activity is reduced. Conceptually, the genetic activity can be reduced by directly inhibiting the genetic activity, or by reducing the activity of an activator of genetic activity. In some embodiments, the genetic activity is reduced, but still retains a certain level of activity. In some embodiments, the genetic activity is completely inhibited. In some embodiments, genetic activity is reduced by at least one of inhibiting promoter activity, activating a transcriptional repressor, reducing RNA stability, activating a post-transcriptional suppressor (e.g., expressing a ribozyme or antisense oligonucleotide), inhibiting translation (e.g., by a regulatable tRNA), failing to perform desired post-translational modifications, inactivating a polypeptide (e.g., by binding an suppressor or by a polypeptide-specific protease), or failing to properly localize a polypeptide. In some embodiments, genetic activity is reduced by removing the gene from the desired location (e.g., by excision of the gene using FLP-FRT or cre-lox cassettes), homologous recombination or CRIPR-CAS9 activity, or by loss or degradation of the plasmid. In some embodiments, a gene product (e.g., a polypeptide) or a product produced from a gene product (e.g., a product of an enzymatic reaction) further inhibits gene activity (e.g., a negative feedback loop).
In some embodiments, the advantage conferred to the 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 mixture of bacteriocins). As used herein, "bacteriocins" encompass cell-free or chemically synthesized versions of such polypeptides. "bacteriocins" and variations of their roots, may also refer to polypeptides that have been secreted by the host cell. Thus, bacteriocins encompass protein toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains. They are similar to the killing factors of yeast and paramecium and are diverse in structure, function and ecology. Bacteriocins also encompass synthetic variants of bacteriocins secreted by the host cell. Synthetic variants of a bacteriocin may be derived from a bacteriocin secreted by a host cell in any manner as long as the synthetic variants still exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the activity of the corresponding bacteriocin secreted by the host cell. Detailed descriptions of antibiotics are provided in the general description section at the end of the specification.
"bacteriocins" can neutralize at least one cell other than the single host cell that produces the polypeptide, including cells that are clonally related to the host cell and other microbial cells.
Cells expressing a particular "immunomodulator" (discussed in more detail herein) are immune to the neutralization of a particular bacteriocin or group of bacteriocins. Thus, the bacteriocin can neutralize bacteriocin-producing cells and/or other microbial cells, provided that the cells do not produce the appropriate immunomodulator. As such, bacteriocins can exert cytotoxic or growth inhibitory effects on a variety of other microorganisms. In one embodiment, the bacteriocin is produced by the translation machinery (e.g., ribosomes, etc.) of the microbial cell. In another embodiment, the bacteriocin is chemically synthesized. Some bacteriocins may be derived from polypeptide precursors. The polypeptide precursor may undergo cleavage (e.g., by protease treatment) to produce the polypeptide of the bacteriocin itself. As such, in some embodiments, the bacteriocin is produced from a precursor polypeptide. In some embodiments, the bacteriocin comprises, consists essentially of, or consists of a polypeptide that has been post-translationally modified (e.g., cleaved or added with one or more functional groups).
The neutralizing activity of the bacteriocin may include preventing microbial proliferation or cytotoxicity. Some bacteriocins have cytotoxic activity (e.g., "bacteriocidal" action) and can therefore kill microorganisms, such as bacteria, yeasts, algae, synthetic microorganisms, and the like. Some bacteriocins can inhibit the proliferation (e.g., "bacteriostatic" effects) of microorganisms (e.g., bacteria, yeasts, algae, synthetic microorganisms, etc.), for example, by preventing the cell cycle.
A number of bacteriocins have been identified and characterized (see table 1.1 and table 1.2.). Without being bound by any particular theory, exemplary bacteriocins may be classified as "class I" bacteriocins, which typically undergo post-translational modifications, and "class II" bacteriocins, which typically are unmodified. In addition, exemplary Bacteriocins in each class can be divided into different subgroups, as shown 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, herein incorporated by reference in its entirety.
Without being bound by any particular theory, bacteriocins may neutralize the target microbial cells in a variety of ways. For example, bacteriocins can penetrate the cell wall, thereby depolarizing the cell wall and interfering with respiration. Table 1.1: classification of exemplary bacteriocins.
Table 1.1: classification of exemplary bacteriocins
According to embodiments herein, a number of bacteriocins may be used. Table 1.2 shows exemplary bacteriocins. In some embodiments, there is provided at least one bacteriocin comprising, consisting essentially of, or consisting of the polypeptide sequence of table 1.2. As shown in table 1.2, some bacteriocins act as molecular pairs. As such, it is understood that, unless specifically stated otherwise, when functional "bacteriocins" or "providing bacteriocins" or the like are discussed herein, functional pairs of bacteriocins are included as well as bacteriocins that can function alone. Referring to table 1.2, the "originating organism" listed in parentheses indicates an alternative name and/or strain information for an organism known to produce the indicated bacteriocin.
Embodiments herein also include peptides and proteins having identity to the bacteriocins described in table 1.2. The term "identity" is meant to include nucleic acid or protein sequence homology or three-dimensional homology. There are a variety of techniques to determine nucleic acid or polypeptide sequence homology and/or three-dimensional homology to a polypeptide. These methods are typically used to find the degree of identity of a sequence, domain or model to a target sequence, domain or model. A wide variety of functional bacteriocins can incorporate the features of the bacteriocins disclosed herein, thereby providing various degrees of identity relative to the bacteriocins in table 1.2. In some embodiments, the bacteriocin has at least 50% identity, e.g., 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 in table 1.2. Percent identity can be determined using BLAST software (Altschul, S.F., et al (1990) "Basic localization search tool," J.Mol.biol.215: 403-.
While the bacteriocins in table 1.2 are naturally occurring, it will be understood by those skilled in the art 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 may be used according to some embodiments herein. In some embodiments, such variants have enhanced or reduced levels of cytotoxic or growth inhibitory activity against the same or different microorganism or species of microorganism relative to the wild-type protein. Multiple motifs have been recognized as characteristic of bacteriocins. For example, the motif YGXGV (SEQ ID NO:2) (where X is any amino acid residue) is an N-terminal consensus sequence characteristic of class Ila bacteriocins. Thus, in some embodiments, the synthetic bacteriocin comprises an N-terminal sequence that is at least 50% identical to SEQ ID No. 2, e.g., 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% identical to SEQ ID No. 2. In some embodiments, the synthetic bacteriocin comprises an N-terminal sequence comprising SEQ ID NO 2. In addition, some of the lib-like bacteriocins contain the GxxxG motif (x refers to any amino acid). Without being bound by any particular theory, it is believed that the GxxxG motif can mediate associations between helices in cell membranes, for example, by cell membrane interactions to promote bacteriocin-mediated neutralization. Thus, in some embodiments, the bacteriocin comprises a motif that facilitates interaction with cell membranes. In some embodiments, the bacteriocin comprises a GxxxG motif. Alternatively, the bacteriocin comprising the GxxxG motif may comprise a helical structure. In addition to the structures described herein, "bacteriocins" as used herein also include structures that have substantially the same effect on microbial cells as any bacteriocins explicitly provided herein.
It has been demonstrated that a fusion polypeptide comprising, consisting essentially of, or consisting of two or more bacteriocins or portions thereof can have neutralizing activity against a broader range of microorganisms than any individual bacteriocin. For example, the hybrid bacteriocin Ent35-mccV (GKYYGNGVSCNKKGCSVDWGRAIGIIGNNSAANLATGGAAGWKSGGGASGRDIAMAIGTLSGQFVAGGIGAAAGGVAGGAIYDYASTHKPNPAMSPSGLGGTIKQKPEGIPSEAWNYAAGRLCNWSPNNLSDVCL, SEQ ID NO:3) has been shown to have antibacterial activity against pathogenic gram-positive and gram-negative bacteria (Acuna et al (2012), FEBS Open Bio,2: 12-19). Notably, the Ent35-MccV fusion bacteriocin comprises, from N-terminus to C-terminus, an N-terminal glycine, enteromycin CRL35, a linker comprising three glycines, and a C-terminal microcin V. It is contemplated herein that the bacteriocin may comprise a fusion of two or more polypeptides having bacteriocin activity. In some embodiments, fusion polypeptides of two or more bacteriocins are provided. In some embodiments, the two or more bacteriocins comprise, consist essentially of, or consist of the polypeptide in table 1.2 or modifications thereof. In some embodiments, a fusion polypeptide comprising two or more bacteriocins has a broader spectrum of activity than any single bacteriocin, e.g., has neutralizing activity against more microorganisms, neutralizing activity under a wider range of environmental conditions, and/or more efficient neutralizing activity. In some embodiments, a fusion of two or more bacteriocins (e.g., two, three, four, five, six, seven, eight, nine, or ten bacteriocins) is provided. In some embodiments, the two or more bacteriocin polypeptides are fused to each other by covalent bonds (e.g., peptide bonds). In some embodiments, the linker is located between two bacteriocin polypeptides. In some embodiments, the linker comprises, consists essentially of, or consists of one or more glycines, e.g., 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 single bacteriocin contained in the fusion protein. In some embodiments, a bacteriocin as provided herein is modified to provide a desired activity profile relative to an unmodified bacteriocin. For example, a modified bacteriocin may have increased or decreased activity against the same organism relative to an unmodified bacteriocin. Alternatively, the modified bacteriocin may have enhanced activity against organisms in which the unmodified bacteriocin has little or no activity.
Table 1.2: exemplary bacteriocins
For example, in some embodiments, antifungal activity (such as anti-yeast activity) is desired. A number of bacteriocins have been identified which have antifungal activity. For example, bacteriocins of bacillus have been shown to have neutralizing activity against yeast strains { see Adetunji and olaoyye (2013) Malaysian Journal of Microbiology 9:130-13, incorporated herein by reference in its entirety), enterococcus faecalis peptide (WLPPAGLLGRCGRWFRPWLLWLQ SGAQY KWLGNLFGLGPK, SEQ ID NO:1) has been shown to have neutralizing activity against candida { see Shekh and Roy (2012) BMCMicrobiology 12:132, incorporated herein by reference in its entirety), and bacteriocins of pseudomonas have been shown to have neutralizing activity against fungi (such as campylobacter crescentis, fusarium species, helminthosporium species, and biopolar species) (sharani and Srivastava (2008) The Internet Journal of Microbiology. volume5number 2.DOI:10.5580/27 dd-accessible from The following websites: "ceramic", "metal. For example, the staurosporines AJ1316 from Bacillus subtilis (see Zuber, P et al (1993) peptide antibiotics. in Bacillus subtilis and other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics in et al, pp.897-916, American Society for Microbiology, incorporated herein by reference in its entirety) and irinin Bl (see Shenin et al (1995) antibiotic Khimotor 50:3-7, incorporated herein by reference in its entirety) have been demonstrated to have antifungal activity. Thus, in some embodiments, for example, where it is desired to neutralize a fungal microorganism, the bacteriocin comprises one of the staurosporidine AJ1316 or alirin B1.
For example, in some embodiments, bacteriocin activity in cyanobacterial cultures is desired. In some embodiments, a bacteriocin is provided to neutralize cyanobacteria. In some embodiments, a bacteriocin is provided to neutralize invading microorganisms typically found in cyanobacterial culture environments. Conserved bacteriocin polypeptide clusters have been identified in a wide variety of cyanobacterial species. For example, at least 145 putative bacteriocin Gene Clusters have been identified in at least 43 cyanobacteria species as reported by Wang et al (2011), Genome Mining strategies the widespray Occurence of Gene Clusters Encoding bacteria in the cyanobacteria ONE 6(7): e22384, incorporated herein by reference in its entirety). Exemplary cyanobacterial bacteriocins are shown in Table 1.2 as SEQ ID NOs 420, 422, 424, 426, 428, 30, 432, 434, 436, 438, 440, 442, 444, 446, 448 and 450.
In the context of the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, a bacteriocin may be said to be active when the number of microbial hosts is reduced 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, when the microbial hosts are cultured with a medium comprising the bacteriocin, although the bacteriocin may act on the microbial cells by different mechanisms as described herein. This culturing step may last for at least 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours or more before the activity of the bacteriocin is assessed by counting the number of microbial hosts present. Activity can be assessed by counting cells under a microscope or by any known microbiological technique. In some embodiments, the bacteriocin is active when the microbial host is cultured with a medium comprising the bacteriocin when at least a specified number or percentage of microbial hosts are prevented from growing, e.g., 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 are prevented, as compared to the starting population of microbial hosts.
In the context of some of the methods, uses, compositions, hosts and nucleic acids of the embodiments herein, the bacterium is also given B17 or C7 represented by an amino acid sequence comprising or consisting of SEQ ID NO:198 or 200, respectively. According to some embodiments herein, it has been experimentally confirmed that B17 and C7 are selection agents that are easy to produce, easy to use, and stable in culture (see example 1). Some methods, uses, compositions, hosts, and nucleic acids of the embodiments herein also encompass the use of bacteriocins having at least 50% identity to SEQ ID No. 198 or 200, e.g., 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 the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, so long as they exhibit at least substantial activity of B17 or C7. In this context, "substantially" 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 B17 or C7 having SEQ ID NO:198 or 200. The activity of bacteriocins has been described previously herein.
In the context of the methods, uses, compositions, hosts and nucleic acids of the embodiments herein, and depending on the microorganism of interest and the bacteriocin used, the skilled artisan will appreciate which concentration of bacteriocin is used in the culture medium or agar plate. Using bacteriocins B17 or C7, the inventors were able to prepare a medium comprising said bacteriocins at a concentration which enables the implementation of the methods and uses of the embodiments herein, i.e. the observation or visualization of the advantages of the expression of said genetic activity. If the advantage of the activity is to allow growth of a host comprising the self-replicating extrachromosomal nucleic acid molecule, the amount of bacteriocin in the medium or agar plate has been such that the number of hosts that do not comprise the self-replicating extrachromosomal nucleic acid molecule is reduced 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/hosts when the cells are cultured under conditions that allow survival and growth of the microbial host that has received the self-replicating extrachromosomal nucleic acid molecule. The duration of the evaluating step can be 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. The medium can be sterilized without loss of 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 medium prior to sterilization.
A first nucleic acid sequence suitable for use in the methods, uses, compositions, hosts, and nucleic acids of some embodiments herein and the products of which provide immunity to bacteriocins is shown in table 2.
Table 2: exemplary nucleic acid sequences whose products provide immunity to bacteriocins
Although the sequences that provide immunity to the bacteriocins of table 2 are naturally occurring, those skilled in the art will appreciate that variants of these molecules, natural molecules other than those listed in table 2, or synthetic molecules may be used according to some embodiments herein. In some embodiments, a particular molecule or a particular combination of molecules confers immunity to a particular bacteriocin, a particular class or species of bacteriocin, or a particular combination of bacteriocins. Table 2 lists typical bacteriocins that the molecule can confer immunity. Although table 2 identifies the "organism of origin" of the immunity-conferring molecules, according to some embodiments herein, these immunity-conferring molecules can be readily expressed in other naturally-occurring, genetically modified, or synthetic microorganisms to provide the desired bacteriocin immunological activity. Thus, as used herein, an "immunomodulator" or "molecule conferring or providing immunity to a bacteriocin" encompasses not only the structures explicitly provided herein, but also structures that have substantially the same effect as the "immunomodulator" structures described herein, including fully synthetic immunomodulators, as well as immunomodulators that provide immunity to a bacteriocin that is functionally equivalent to a bacteriocin disclosed herein.
Exemplary polynucleotide sequences encoding the polypeptides of table 2 are shown in table 2. One skilled in the art will readily appreciate that the genetic code is degenerate and, in addition, the codon usage may vary based on the particular organism in which the gene product is expressed, and thus, a particular polypeptide may be encoded by more than one polynucleotide. In some embodiments, the polynucleotide encoding the bacteriocin immunomodulator is used based on the codon usage of the organism expressing the bacteriocin immunomodulator. In some embodiments, the polynucleotide encoding the bacteriocin immunomodulator is codon optimized based on the particular organism expressing the bacteriocin immunomodulator. A wide range of functional immunomodulators can combine the features of the immunomodulators disclosed herein, thereby providing a broad degree of identity relative to the immunomodulators in table 2. In some embodiments, an immunomodulatory agent is at least about 50% identical to any one of the polypeptides of table 2, e.g., 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% identical.
In the context of the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, resistance or immunity to a bacteriocin may refer to the number of microbial cells that has not been reduced at the end of a culturing step in which the bacteriocin is used, and in some embodiments, has 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, when the cells are cultured with a bacteriocin-containing medium, as compared to the initial microbial cell number. This culturing step may last for 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 the activity of the bacteriocin is assessed by counting the number of microbial cells present.
The nucleic acid molecule suitable for use in the methods, uses, compositions, hosts and nucleic acids of some embodiments herein and encoding a product conferring immunity is McbG (immune to bacteriocin B17), represented by SEQ ID NO: 699. According to some embodiments herein, it has been experimentally demonstrated that McbG can be used as a selection marker constitutively or inducibly (see example 3). Another suitable nucleic acid is MccE (having immunity to bacteriocin C7) represented by SEQ ID NO:700 or the C-terminal portion thereof represented by SEQ ID NO: 701. MccE has been used as a vector selection marker in strains sensitive to microcin/bacteriocin (see example 2). The methods, uses, compositions, hosts, and nucleic acids of some embodiments also encompass the use of such nucleic acid molecules, the encoded product thereof confers immunity to bacteriocins B17 and/or C7 and which is similar to SEQ ID NO: 699. 700 or 701 have at least 50% identity, for example, to SEQ ID NO: 699. 700 or 701 has 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. Such variants of McbG and/or MccE may be used in the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, so long as they exhibit at least substantial activity of McbG (MccE, respectively). In this context, "substantial" means 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, for example, McbG (MccE, respectively) having SEQ ID NO:699, 700 or 701. The immunity conferred by the encoded product of McbG (MccE, respectively) has been previously described herein.
It has surprisingly been found that the C-terminal part of MccE, represented by SEQ ID NO:701, is sufficient to confer resistance to bacteriocin C7. In this context, part means, 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. Such short nucleic acid molecules can confer resistance to bacteriocins, which is very attractive and surprising. It is expected that self-replicating extra-chromosomal nucleic acid molecules comprising such short nucleic acid molecules do not impose any burden on the microbial cells.
Further suitable nucleic acid molecules for use in the methods, uses, compositions, hosts and nucleic acids of some embodiments herein and whose products provide immunity to bacteriocins are single nucleic acid molecules, the single product of which provides immunity to at least two different bacteriocins. In some embodiments, such products of such nucleic acid molecules provide immunity to B17 and C7 or to ColV and C7 or to ColV and B17 or to B17, C7, and ColV. The nucleic acid encoding ColV was identified as SEQ ID NO 65 and the corresponding encoding amino acid sequence was identified as SEQ ID NO 64.
In some embodiments, the 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, e.g., 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. 715 is a nucleic acid molecule of McbG fused to MccE. 716 is a nucleic acid molecule of McbG fused to the C-terminal portion of MccE, as previously described herein.
In some embodiments, the 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, e.g., 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% sequence identity to SEQ ID No. 717 or 718. 717 is a nucleic acid molecule fused to Cvi of MccE. 718 is a nucleic acid molecule of Cvi fused to the C-terminal portion of MccE, as previously described herein.
Such variants of the identity of the core sequence may be used in the methods, uses, compositions, hosts and nucleic acids of the embodiments herein, as long as they exhibit at least substantial activity of the molecule from which they are derived as previously described herein. In some of the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, each of these nucleic acid molecules described herein whose products confer immunity to a single species 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).
Constructs suitable for use in the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein may comprise a first nucleic acid molecule whose product confers immunity to bacteriocins, and these constructs may comprise, consist essentially of, or consist of seq id NOs 702, 703, 710, 711, 704, 705, 712, 713, or 714. Each of these constructs has been extensively described in the experimental section of the present application, which indicates that each of these constructs has been actually constructed and proven to be suitable according to some embodiments herein (see, e.g., examples 1 and 2 and 3).
In the methods of some embodiments, the bacteriocin added to the culture medium is B17 and/or C7 and/or ColV as described herein.
The process may allow the production of any desired product. In the methods of some embodiments, the product of interest is a microbial biomass, a self-replicating extra-chromosomal nucleic acid molecule, a transcript of the second nucleic acid sequence, a polypeptide encoded by the second sequence, or a metabolite produced directly or indirectly by the polypeptide.
In some embodiments of the method, the target product is purified at the end of the culturing step c). This can be done using techniques known to the skilled person. Since the energy burden associated with the presence of self-replicating extrachromosomal nucleic acid molecules has been minimized, it is expected that yields of the desired product are optimal.
The method may use any suitable microbial cell, for example as a host. Suitable microbial cells are listed in the section of the specification entitled "general description". Suitable microbial cells per se and for use in the methods, uses, compositions and hosts of the embodiments herein include, but are not limited to: bacteria (e.g., gram negative bacteria, such as E.coli), yeast, filamentous fungi, or algae. In some embodiments, the microbial cell is a synthetic microbial cell.
In one method, a first nucleic acid sequence present on a self-replicating extra-chromosomal nucleic acid molecule may be operably linked to a promoter. In some embodiments, the promoter is a weak promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is inducible. In some embodiments, the promoter is a weak constitutive promoter. In some embodiments, the promoter is a weakly inducible promoter. Inducibility of the promoter is one way to control the presence of genetic activity of the first nucleic acid sequence. Promoters are well known in the art. The detailed description is provided in a section of the specification dedicated to the general description. Promoters may be used to drive the transcription of one or more coding sequences. Optionally, the self-replicating extra-chromosomal nucleic acid molecule comprises a second nucleic acid sequence involved in the production of the product of interest, wherein the genetic activity of the second nucleic acid sequence is controlled independently of the genetic activity of the first sequence.
In one embodiment, the control of the genetic activity of the second nucleic acid sequence is not independent of the control of the genetic activity of the first sequence.
In some embodiments, a second promoter drives expression of the second nucleic acid sequence involved in production of a product of interest described herein. In one embodiment, the first promoter drives expression of an immunomodulatory agent polynucleotide as described herein.
A promoter as used herein may not be native to the nucleic acid molecule to which it is operably linked, i.e., the promoter is heterologous to the nucleic acid molecule (coding sequence) to which it is operably linked. Although the promoter of some embodiments is heterologous to the coding sequence to which it is operably linked, in some embodiments the promoter is homologous, e.g., endogenous, to the microbial cell. In some embodiments, a heterologous promoter (for a nucleotide sequence) is capable of producing a transcript that comprises a higher steady state level of the coding sequence (or is capable of producing more transcript molecules, i.e., mRNA molecules, per unit time) than the promoter native to the coding sequence. Some promoters can drive transcription at any time ("constitutive promoters"). Some promoters drive transcription only under selected circumstances (| "conditional promoters" or "inducible promoters"), e.g., depending on the presence or absence of environmental conditions, compounds, gene products, stages of the cell cycle, etc.
It will be appreciated by those skilled in the art that depending on the desired expression activity, an appropriate promoter may be selected and placed in cis with (i.e., or operably linked to) the sequence to be expressed. Exemplary promoters with exemplary activities are provided in tables 3.1-3.11 herein. It will be appreciated by those skilled in the art that some promoters are compatible with specific transcription mechanisms (e.g., RNA polymerase, general transcription factors, etc.). Thus, while some compatible "species" of the promoters described herein have been identified, it is contemplated that, according to some embodiments herein, these promoters may readily function in microorganisms other than the identified species, for example, in species having compatible endogenous transcription mechanisms, transgenic species containing compatible transcription mechanisms, or fully synthetic microorganisms containing compatible transcription mechanisms.
The promoters of tables 3.1-3.11 herein are publicly available from the Biofricks Foundation. Notably, the Biofricks Foundation encourages the use of these promoters in accordance with the BioBrick public protocol (BPA).
It will be understood that any "encoding" polynucleotide described herein (e.g., a first nucleic acid sequence and/or a second nucleic acid sequence involved in the production of a product of interest) is generally suitable for expression under the control of a desired promoter. In one embodiment, the first nucleic acid sequence is under the control of a first promoter. In one embodiment, the second nucleic acid sequence involved in the production of the product of interest is under the control of a second promoter.
Typically, translation initiation of a particular transcript is regulated by a particular sequence 5' to the coding sequence of the transcript. For example, the coding sequence can begin with an initiation codon that is configured to pair with the initiator tRNA. While naturally occurring translation systems typically use met (AUG) as the initiation codon, it will be readily appreciated that the initiator tRNA may be designed to bind to any desired triplet or triplets, and thus triplets other than AUG may also function as initiation codons in certain embodiments. In addition, sequences near the start codon can facilitate ribosome assembly, such as the Kozak sequence ((gcc) gccnCCAUGG, SEQ ID NO:542, where N represents "A" or "G") or the Internal Ribosome Entry Site (IRES) in typical eukaryotic translation systems, or the Shine-Dalgarno sequence (GGAGGU, SEQ ID NO:543) in typical prokaryotic translation systems. Thus, in some embodiments, a transcript comprising an "encoding" polynucleotide sequence (e.g., a first nucleic acid sequence or a second nucleic acid sequence involved in the production of a hairalcohol product) comprises an appropriate initiation codon and translation initiation sequence. In some embodiments, for example, if two or more "encoding" polynucleotide sequences are located in cis positions on a transcript, each polynucleotide sequence comprises an appropriate initiation codon and translation initiation sequence.
In some embodiments, for example, if two or more "encoding" polynucleotide sequences are located in cis positions on a transcript, then the two sequences are under the control of a single translation initiation sequence and provide a single polypeptide that can function simultaneously with two cis-encoded polypeptides, or provide a means for separating two cis-encoded polypeptides, e.g., a 2A sequence, or the like. In some embodiments, the translation initiator tRNA is regulatable, thereby modulating the initiation of translation of the immunomodulator or industrially useful molecule.
Table 3.1: exemplary Metal-sensitive promoters
SEQ ID NO:
Name (R)
Description of the invention
544
BBa_I721001
Leader promoters
545
BBa_I731004
FecA promoter
546
BBa_I760005
Cu-sensitive promoters
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 Signal responsive promoters
70Table 3.3: exemplary constitutive E.coli sigma promoter
8Table 3.4: exemplary constitutive E.coli sigma promoter
SEQ ID NO:
Name (R)
Description of the invention
654
BBa_J45992
Full-length stationary phase osmY promoter
655
BBa_J45993
Minimal stationary phase osmY promoter
32Table 3.5: exemplary constitutive E.coli sigma promoter
SEQ ID NO:
Name (R)
Description of the invention
656
BBa_J45504
htpG heat shock promoter
ATable 3.6: exemplary constitutive Bacillus subtilis sigma promoter
SEQ ID NO:
Name (R)
Description of the invention
657
BBa_K143012
Promoter veg, constitutive promoter for Bacillus subtilis
658
BBa_K143013
Promoter 43 for Bacillus subtilis groupShaped promoters
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 Bacillus subtilis σBPromoters
SEQ ID NO:
Name (R)
Description of the invention
663
BBa_K143010
Promoter ctc for Bacillus subtilis
664
BBa_K143011
Promoter gsiB for Bacillus subtilis
665
BBa_K143013
Promoter 43 constitutive promoter for Bacillus subtilis
Table 3.8: exemplary constitutive promoters from Heteroprokaryotes
Table 3.9: exemplary constitutive promoter from bacteriophage T7
SEQ ID NO:
Name (R)
Description of the invention
668
BBa_I712074
T7 promoter (Strong promoter from T7 phage)
669
BBa_I719005
T7 promoter
670
BBa_J34814
T7 promoter
671
BBa_J64997
T7 has a total of-10 and others
672
BBa_K113010
Overlapping T7 promoter
673
BBa_K113011
More overlapping T7 promoters
674
BBa_K113012
Attenuating 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 progressive Properties
682
BBa_Z0253
T7 weakly bound promoter
Table 3.10: exemplary constitutive promoters from Yeast
Table 3.11: exemplary constitutive promoters from heteroeukaryotes
SEQ ID NO:
Name (R)
Description of the invention
697
BBa_I712004
CMV promoters
698
BBa_K076017
Ubc promoter
The above promoters are provided by way of non-limiting example only. The promoter may be a synthetic promoter. Promoters suitable for use in the methods, uses, compositions, hosts and nucleic acids of some embodiments herein have been previously described herein, such as proC represented by SEQ ID NO:708 (see example 2) which has been experimentally confirmed according to some embodiments herein and P24 represented by SEQ ID NO:707 (see example 3) which has been experimentally confirmed according to some embodiments herein. In some embodiments, a suitable inducible promoter is the P24 LacO hybrid promoter, which is repressed in the presence of LacI and active in the presence of IPTG. This promoter has been experimentally confirmed according to some embodiments herein (see example 3).
One skilled in the art can readily recognize that many variants of the above promoters, as well as many other promoters (including promoters isolated from naturally occurring organisms, variants thereof, and fully synthetic or engineered promoters) can be readily used according to some embodiments herein. When tested in a control or reference plasmid operably linked to a nucleic acid molecule encoding a transcript, a variant, fully synthetic or engineered promoter is said to be active or functional when said plasmid is present in a cell in detectable amounts of said transcript molecule and thus may be used in the methods, uses, compositions, hosts and nucleic acids of the embodiments herein. A variant, fully synthetic or engineered 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 from which it is derived.
Optionally, the method comprises transforming the microbial host with the self-replicating extrachromosomal nucleic acid molecule under conditions that allow survival of the host that has received the self-replicating extrachromosomal nucleic acid molecule. It is noted that in some embodiments, a self-replicating extra-chromosomal nucleic acid molecule may be provided in a microbial cell (e.g., if the microbial cell or its precursor has been transformed by a self-replicating extra-chromosomal nucleic acid molecule), and thus in some embodiments, no transformation step is required in the method. The transformation step may be performed prior to the culturing of step c). In some embodiments, a transformation step is provided prior to step a), thereby providing a host cell comprising a self-replicating extra-chromosomal nucleic acid molecule. In some embodiments, the self-replicating extra-chromosomal nucleic acid molecule used in the transforming step further comprises the second nucleic acid of optional step b).
Techniques for genetically engineering microorganisms are well known in the art (see, for example, Molecular cloning source edition,2012Cold Spring Harbor Laboratory Press, a Laboratory manual, by m.r. green and J Sambrook, which is incorporated herein by reference in its entirety). In some embodiments, the microorganism is genetically engineered to comprise a self-replicating extrachromosomal nucleic acid molecule comprising a first nucleic acid sequence and optionally a molecule involved in the production of a product of interest. The polynucleotide or nucleic acid molecule can be delivered to a microorganism.
In one embodiment, the microbial cell is positively selected by the genetic activity of said first nucleic acid sequence corresponding to at least one given condition that allows survival of a cell that has received a self-replicating extra-chromosomal nucleic acid molecule, and said condition may be an environmental condition. The environmental condition may be a culture medium.
It may be useful to flexibly genetically engineer microbial cells, e.g., to engineer or re-engineer microbial cells to have a desired type and/or spectrum of genetic activity. In some embodiments, a cassette for insertion of one or more desired unique first nucleic acid sequences is provided. Exemplary cassettes include, but are not limited to, Cre/lox cassettes or FLP/FRT cassettes.
In one embodiment, a microbial cell comprises more than one (more than two, more than three …) different self-replicating extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence as described herein, meaning that the cell can exhibit more than one (more than two, more than three …) genetic activities, each genetic activity conferring a advantage to the cell. If a first promoter is present in each of the different self-replicating extra-chromosomal nucleic acid molecules, each of the first promoters may be different or the same. Thus, the use of one, two, three, four or more different bacteriocins in a method for producing a product of interest is also within the scope of the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, wherein a microbial host comprises one, two, three, four or more different extrachromosomal nucleic acid molecules, each conferring a different genetic activity to said microbial host. Alternatively, the use of a single nucleic acid molecule whose product provides immunity to at least different bacteriocins is also within the scope of the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein. Such nucleic acid molecules have been described herein.
In some embodiments, plasmid conjugation may be used to introduce a desired plasmid from a | "donor" microbial cell into a recipient microbial cell (Goni-Moreno, et al (2013) multicell Computing using conjugation for wiring. plos ONE 8(6): e65986, incorporated herein by reference in its entirety). In some embodiments, plasmid conjugation can genetically modify a recipient microbial cell by introducing a conjugative plasmid from a donor microbial cell into the recipient microbial cell. Without being bound by any particular theory, conjugative plasmids comprising identical or functionally identical sets of replicating genes typically do not co-exist in the same microbial cell. Thus, in some embodiments, plasmid conjugation "reprograms" recipient microbial cells by introducing a new conjugative plasmid to replace another conjugative plasmid present in the recipient cells. In some embodiments, plasmid conjugation is used to engineer (or re-engineer) biological cells with a particular combination of first nucleic acid molecules (which may encode, in some embodiments, an immunomodulator). According to some embodiments, various conjugative plasmids comprising different combinations of first sequences (which may encode, in some embodiments, immunomodulators) are provided. The plasmid may contain other genetic elements described herein, such as promoters, translation start sites, and the like. In some embodiments, the plurality of conjugative plasmids is provided in a collection of donor cells, such that donor cells containing a desired plasmid can be selected for plasmid conjugation. In some embodiments, a particular combination of immunomodulators is selected, and a suitable donor cell is conjugated to a target microbial cell to introduce a conjugative plasmid comprising the combination into a recipient cell. In some embodiments, the recipient cell is a "newly engineered" cell, e.g., to be introduced or used to initiate culture.
Step b)
In addition to step a), in some embodiments, the method further comprises an optional step b), wherein the self-replicating extra-chromosomal nucleic acid molecule comprises a second nucleic acid sequence involved in the production of the target product, wherein the genetic activity of the second nucleic acid sequence is controlled independently of the genetic activity of the first sequence.
In the context of the methods, uses, compositions, hosts, and nucleic acids of the embodiments herein, expression "independently controlled" has its customary and ordinary meaning as understood by those of skill in the art in view of this disclosure, including meaning that a different means is used to control the genetic activity of the first and second nucleic acid sequences. The manner in which the genetic activity of a nucleic acid sequence is controlled has been described in detail herein.
Step c)
In some embodiments, step a) (which optionally includes transformation as described herein) and optional step b) is followed by step c) comprising culturing the transformed microbial host under conditions that allow the transformed microbial host to express the first nucleic acid sequence to a given level to maintain entry of the replicating extra-chromosomal molecule from the replicating extra-chromosomal molecule into a growing microbial population. In some embodiments, the second sequence encoding the target product is optionally controlled.
In the methods of some embodiments, at least a portion of the conditions of step c) are such that the first nucleic acid sequence does not exhibit the genetic activity. By "part of step c)" is meant, 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 because part of step c) is performed in the absence of the genetic activity of the first nucleic acid sequence. The presence of said genetic activity constitutes an energy burden on the microbial host cell and is not always required to maintain a suitable production level of the product of interest. In some embodiments, it is envisaged that a part of step c) is rendered free of genetic activity of the first nucleic acid sequence, followed by a part having said activity. During step c), the two parts may be repeated one or more times.
The microbial cells can be cultured in any suitable microbial culture environment. The microbial cultivation environment may comprise a variety of media, such as feedstock. The choice of a particular medium may depend on the desired application. The conditions of the medium include not only chemical composition but also temperature, light amount, pH, and CO2Level, etc. The culture medium may comprise a bacteriocin. In one embodiment, the compound that induces bacteriocin activity is present outside the feedstock, and is not present in the feedstock.
In one embodiment, the genetically engineered or transformed microorganism described herein is added to a culture medium comprising at least one feedstock. In one embodiment, the medium comprises a compound that induces activity or expression of an immunomodulator.
The term "feedstock" has its customary and ordinary meaning as understood by those skilled in the art in view of this disclosure, and encompasses materials that may be consumed, fermented, purified, modified, or otherwise processed by microorganisms, for example, in a process production. Thus, "raw material" is not limited to food or foodstuff. As used herein, a "feedstock" is a type of culture medium. Thus, as used herein, "culture medium" includes, but is not limited to, raw materials. Thus, whenever reference is made herein to "medium", the feedstock is also explicitly contemplated.
Prior to culturing the transformed microbial cells, it may be useful to determine the effect (if any) or preferably conditions that allow survival and optionally growth of a host that has received the self-replicating extrachromosomal nucleic acid molecule.
In some embodiments, a microbial cell or microbial host cell or synthetic microbial host cell comprising a self-replicating extra-chromosomal nucleic acid molecule is provided comprising a first nucleic acid sequence conferring a advantage to the microbial host in genetic activity and optionally a second nucleic acid sequence directly or indirectly involved in the production of a product of interest, wherein the genetic activity of the first nucleic acid sequence is controlled.
In some embodiments, there is provided a self-replicating extra-chromosomal nucleic acid molecule comprising a first nucleic acid sequence conferring a advantage to a microbial host in genetic activity and optionally a second nucleic acid sequence directly or indirectly involved in production of a product of interest, wherein the genetic activity of the first nucleic acid sequence is controlled.
Various features of the microbial host and the self-replicating extrachromosomal nucleic acid molecule have been described herein.
General description
The terms used herein have their ordinary and customary meaning as understood by those skilled in the art upon reading this disclosure and may include the following general description.
Microorganisms
As used herein, "microorganism (microbe)", "microbe (microbe)", "microbial cell" or "microbial host" and variations of these root terms (e.g., plurals, etc.) have their customary and ordinary meaning as understood by those skilled in the art in view of this disclosure, including any naturally occurring species or synthetic or fully synthetic prokaryotic or eukaryotic unicellular organisms as well as archaea species. Thus, the expression may refer to bacterial species, fungal species and algal cells. Exemplary microorganisms that may be used in accordance with embodiments herein include, but are not limited to, bacteria, yeasts, filamentous fungi, and algae, such as photosynthetic microalgae. In addition, a fully synthetic microbial Genome can be Synthesized and transplanted into a single microbial Cell to produce a synthetic microorganism capable of continuous self-replication (see Gibson et al (2010), "Creation of bacterial Cell Controlled by a chemical Synthesized Genome," Science 329:52-56, incorporated herein by reference in its entirety). Thus, in some embodiments, the microorganism is fully synthetic. Desired combinations of genetic elements, including elements that regulate gene expression, and elements that encode gene products (e.g., immunomodulators, toxic agents, antidotes, and industrially useful molecules, also referred to as target products), can be assembled into partially or fully synthetic microorganisms on desired substrates. A description of genetically engineered microorganisms 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 the genetic element will be described later herein.
According to embodiments herein, a variety of bacterial species and strains may be used, and genetically engineered variants or synthetic bacteria based on known species of "chassis" may be provided. Exemplary bacteria having industrially applicable properties that can be used according to embodiments herein include, but are not limited to, bacillus species (e.g., bacillus coagulans, bacillus subtilis, and bacillus licheniformis), paenibacillus species, streptomyces species, microsphericium species, corynebacterium species, acetobacter species, cyanobacterium species, salmonella species, rhodococcus species, pseudomonas species, lactobacillus species, enterococcus species, alcaligenes species, klebsiella species, paenibacillus species, arthrobacterium species, corynebacterium species, brevibacterium species, thermus aquaticus, pseudomonas stutzeri, clostridium thermocellum, and escherichia coli.
According to embodiments herein, a variety of yeast species and strains may be used, and genetically engineered variants or synthetic yeasts based on the "chassis" of known species may be provided. Exemplary yeasts having industrially useful properties that may be used according to embodiments herein include, but are not limited to, Saccharomyces species (e.g., Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces boulardii), Candida species (e.g., Candida utilis, Candida krusei), Schizosaccharomyces species (e.g., Schizosaccharomyces pombe, Schizosaccharomyces japonicus), Pichia or Hansenula species (e.g., Pichia pastoris or Hansenula polymorpha), and Brettanomyces species (e.g., Brettanomyces clausii).
Various algal species and strains can be used according to embodiments herein, and genetically engineered variants or synthetic algae based on known species "underpinnes" can be created. In some embodiments, the algae comprises, consists essentially of, or consists of photosynthetic microalgae. Exemplary algae species that can be used for biofuels and that can be used in accordance with embodiments herein include botryococcus braunii, chlorella sp, dunaliella salina, gracilaria sp. In addition, many algae can be used in food, fertilizer products, waste neutralization, environmental remediation, and carbohydrate production (e.g., biofuels).
A variety of filamentous fungal species and strains may be used according to embodiments herein, and genetically engineered variants or synthetic filamentous fungi based on the known species "chassis" may be provided. Exemplary filamentous fungi with industrially useful properties that may be used according to embodiments herein include, but are not limited to, Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Staphylococcus, Ceriporiopsis, Chaetomium, Chrysosporium, Claviceps, Courospora, Coprinus, Coptotermes, Cladosporium, Pleurospora, Cryptococcus, Chromospora, Auricularia, Filibasidium, Fusarium, Gibberella, Trichophyton, Humicola, lrpeyrex, Lentinus, Leptospaa, Leptosporium, Asperulina, Melanocarpus nigra, Maitake (Melanocarpus), Mucor, myceliophthora, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Rumezia, Poitaria, Pseudoperonospora, Pseudomyces (Pyrenophora), Cephalosporium, Talaromyceliophytium, Thielavia, Tolymus, Talaromycelia, Talaromyceliophthora, Talaromycelitis, Talaro, Trichoderma, Colletotrichum, Verticillium, Endocarpium or Xylaria.
Species include Acremonium cellulosum, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fogeri, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium faecalis (Chrysosporium merdarium), Chrysosporium pannicum (Chrysosporium pannicola), Chrysosporium queenslandicum (Chrysosporium queenslandicum), Chrysosporium tropicalis, Chrysosporium zonatum (Chrysosporium zonatum), Fusarium sporogenes, Fusarium oxysporum, Fusarium, Fusarium crookwellense, Fusarium flavum, Fusarium graminearum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium polyspora, Fusarium roseum, Fusarium oxysporum, Fusarium venenatum, Fusarium, myceliophthora thermophila, neurospora crassa, penicillium funiculosum, penicillium purpurogenum, phanerochaete chrysosporium, fuschonia leucotrichum and (Thielavia achromatis), haloxylobium cambium (Thielavia albomyces), haloxylobium bailii (Thielavia albolosa), haloxylobium australis (Thielavia australis), haloxylobium faecalis (Thielavia fimeti), haloxylobium parvum, haloxylobium ovani (Thielavia oviispora), haloxylobium pernici (Thielavia perviana), haloxylobium trichoderma (Thielavia setosum), haloxylobium oncophora (Thielavia spodonium), haloxylonium thermonatum (Thielavia subthermophila), trichoderma viride, trichoderma harzianum, trichoderma longibrachiatum or trichoderma longibrachiatum.
Antibiotic
"antibiotics" and variations of this term have their customary and ordinary meaning as understood by those skilled in the art in view of this disclosure, and include intermediates that can kill or prevent at least one microbial cell growth metabolite or metabolic pathway. Some antibiotics can be produced by microbial cells (e.g., bacteria). Some antibiotics can be chemically synthesized. It is understood that bacteriocins differ from antibiotics at least in that bacteriocins refer to gene products (which in some embodiments undergo additional post-translational processing) or synthetic analogs thereof, while antibiotics refer to intermediates or products of metabolic pathways or synthetic analogs thereof.
Sequence identity and similarity
Sequence identity has its plain and ordinary meaning as understood by those of skill in the art in view of this disclosure, including the 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. Typically, sequence identity or similarity is compared over the entire length of the sequences being compared. In the art, "identity" may 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 of one polypeptide and its conservative amino acid substitutions 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 full length of the sequences identified herein.
Some methods of determining identity are designed to provide the largest match between the tested sequences. Methods for determining identity and similarity have been codified in publicly available computer programs. Computer program methods for determining identity and similarity between two sequences include, for example, BestFit, BLASTP, BLASTN, and FASTA (Altschul, S.F.et al., J.mol.biol.215: 403-.
Alternatively, so-called "conservative" amino acid substitutions may also be considered by those skilled in the art in determining the degree of amino acid similarity, as will be clear to those skilled in the art. 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 with 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 sulfur-containing side chains is cysteine and methionine. Suitable conservative amino acid substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substituted variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue inserted in its place. In some embodiments, the amino acid changes are conservative. Suitable conservative substitutions for each naturally occurring amino acid 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.
Homology
The term "homologous" has its customary and ordinary meaning as understood by those skilled in the art in view of this disclosure, including when used to indicate a relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, it is understood to mean that the nucleic acid or polypeptide molecule is produced in nature by a host cell or organism (optionally the same species or strain) of the same species. If homologous to a host cell, the nucleic acid sequence encoding the polypeptide will typically be operably linked to another promoter sequence that is different from its native environment. The term "homologous" when used to indicate the relatedness of two nucleic acid sequences has its customary and ordinary meaning as understood by those skilled in the art in view of this disclosure, and may refer to a single-stranded nucleic acid sequence that is hybridizable 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 sequences and hybridization conditions, such as the temperatures and salt concentrations mentioned previously. The identity region may be greater than about 5bp, and the identity region may be greater than 10 bp. In some embodiments, two nucleic acid or polypeptide sequences are said to be homologous when they are greater than 80% identical.
From a source of foreign origin
The term "heterologous" has its customary and ordinary meaning as understood by those 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 referred to as a polypeptide or enzyme) that does not naturally occur as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or is found at a different location or locations in a cell or genome or DNA or RNA sequence than the location or locations in the cell or genome or DNA or RNA sequence in which the nucleic acid or protein is found in nature. A heterologous nucleic acid or protein is not endogenous to the cell into which it is introduced, but is obtained from another cell or produced synthetically or recombinantly. Typically, although not necessarily, the protein encoded by such a nucleic acid is not normally a protein normally produced by a cell that transcribes or expresses the DNA. Similarly, the exogenous RNA encodes a protein that is not normally expressed in the cell in which it is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. In this context, the term "heterologous nucleic acid or protein" encompasses any nucleic acid or protein that would be recognized by one of skill in the art as being heterologous or foreign to the cell in which it is expressed. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations in which at least two of the combined sequences are foreign relative to each other.
Is operably connected to
As used herein, the term "operably linked" has its customary and ordinary meaning as understood by those skilled in the art upon consideration of this disclosure, and can refer to the linkage of polynucleotide elements (or coding sequences or nucleic acid molecules) 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 example, 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 generally contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
Promoters
As used herein, the term "promoter" has its usual and ordinary meaning as understood by those of skill in the art in view of this disclosure, and can refer to a nucleic acid fragment that is used to control transcription of one or more nucleic acid molecules, is located upstream relative to the direction of transcription of the transcription initiation site of the nucleic acid molecule, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, transcription initiation site, and any other DNA sequences (including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known by those of skill in the art to directly or indirectly regulate/control the amount of transcription from a 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 a non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Furthermore, the verb "consist of …", which may be replaced by "consist essentially of …", means that the self-replicating extra-chromosomal nucleic acid molecule, microbial host (or method), as defined herein, may comprise additional components (or additional steps) in addition to the explicitly specified composition (or step), which do not alter the unique characteristics 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. Thus, the indefinite article "a" or "an" generally means "at least one".
All patents and references cited in this specification are incorporated herein by reference in their entirety. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
Sequence Listing (DNA, unless otherwise specified)
1 enterococcus faecalis peptide SEQ ID NO
Motif characteristic of bacteriocin of SEQ ID NO 2
3 hybrid bacteriocin, Ent35-MccV, SEQ ID NO
SEQ ID NO 4-698, 720-759: sequences in tables 1-3 (half DNA/half protein, as shown in the tables)
SEQ ID NO:699McbG
SEQ ID NO:700MccE
C-terminal part of SEQ ID NO 701MccE
SEQ ID NO:702pSyn2-McbG
SEQ ID NO:703pSyn2-McbE/F
SEQ ID NO:704pMcbG1.0
SEQ ID NO:705pMcbG.1.1.
C-terminal portion (amino acid) of SEQ ID NO 706MccE
707P24 promoter of SEQ ID NO
708proC promoter of SEQ ID NO
SEQ ID NO:709Cvi
710proC-McbG-CterMccE (proC) (Cter may be replaced by C-terminal)
711proC-Cvi-Cter-MccE (proC) (Cter may be replaced by C-terminal)
SEQ ID NO:712pBACT5.0
SEQ ID NO:713pBACT2.0
SEQ ID NO:714pBACT5.0-mcherry
SEQ ID NO:715McbG-MccE
716McbG-Cter part MccE (Cter can be replaced by C-terminal)
SEQ ID NO:717Cvi-MccE
SEQ ID NO 718Cvi-Cter portion McE (Cter may be replaced by C-terminal)
719 SEQ ID NO vector pUC-ColV
Drawings
FIG. 1: construct (a): pSyn 2-McbE/F: genes McbE and F are contained under Ptac.
FIG. 2: construct (a): pSyn 2-McbG: the gene McbG is contained under Ptac.
FIG. 3: construct (a): pMcbG 1.1: the gene McbG is contained in P24.
FIG. 4: construct (a): pMcbG 1.0: the gene McbG is contained under P24 LacO.
FIG. 5: pBACT5.0 vector
FIG. 6: pBACT2.0 vector
FIG. 7: pBACT5.0-mcherry vector
FIG. 8: the promoter is tuned. In the absence of a sensor (upper part), a repressor (repressor) may bind to the operator and prevent expression of the selection gene. In the presence of the sensor (lower part), the repressor does not bind to the operator that allows expression of the selected gene.
FIG. 9: comparison of protein X overexpression in E.coli with KanR (pKan-pLac) and with 2 immunizations against microcin C7 and ColV (pBACT6.0-pLac). 5mg of total extract was analyzed in SDS-PAGE.
FIG. 10: comparison of iota-Carrageenase protein overexpression in E.coli with KanR (pKan-T7prom) and with 2 immunizations against microcin C7 and ColV (pBACT5.0-T7 prom). 5mg of total extract was analyzed in SDS-PAGE.
FIG. 11: comparison of lambda-carrageenase protein overexpression in E.coli with KanR (pKan-T7prom) and with 2 immunizations against microcin C7 and ColV (pBACT5.0-T7 prom). 5mg of total extract was analyzed in SDS-PAGE.
Detailed Description
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