Genetically engineered bacteria and genetically stable methods for producing metabolites, peptides, and / or recombinant proteins

Abstract

The present invention relates to genetically engineered bacteria for producing at least one metabolite, peptide, or recombinant protein, comprising a gene encoding a recombinant protein of interest or at least one gene encoding an enzyme involved in the production of a peptide or metabolite of interest. The present invention is further directed to methods of using the bacteria to produce at least one metabolite, peptide, or recombinant protein.

Description

Detailed Description of the Invention

[0001] The present invention relates to genetically engineered bacteria capable of producing at least one metabolite, peptide or recombinant protein, and to methods for the bioproduction of at least one metabolite, peptide or recombinant protein.

[0002] In a more general aspect, the present invention relates to the application of synthetic biology in metabolic engineering for the production of metabolites, peptides and heterologous proteins of biotechnological or medical interest.

[0003] Many microorganisms are capable of synthesizing compounds such as biofuels, bulk fine chemicals, or medicinal molecules. Modifying such microorganisms to optimize or maximize production of compounds of interest is one of the major challenges in the field of biotechnology.

[0004] Genetic engineering allows for the redesign of metabolic fluxes in microorganisms, or cells more generally. In particular, engineered microbial cells can ideally be switched from growth (i.e., biomass production) to product synthesis (i.e., production of a compound of interest). Therefore, industrial processes generally involve two major steps: a first step in which a cell population is grown to a desired size and the compound of interest is produced in parallel; and a second step in which cell growth is stopped to produce the compound of interest without using the resources required for cell growth. In the first step, most of the available energy and resources are used for biomass formation. As a result, the yield of the compound of interest is low in the first step. However, inhibiting cell growth decouples the production of the compound of interest from biomass production. In other words, once cell growth is stopped, microbial cells can focus on producing the compound of interest. Reference: Chotani et al. (2000) Biochim. Biophys. Acta 1543:434-455 and Sonderegger M, Schuperli M, Sauer U. 2005. Selection of quiescent Escherichia coli with high metabolic activity. Metab Eng 7:4-9.

[0005] Generally, growth arrest occurs naturally when cell density reaches a certain limit. However, if the cell density is too high, it is generally accompanied by a high level of cellular pathology and a severe decline in intracellular metabolic activity. This significantly reduces the productivity of the target compound, adversely affecting industrial processes.

[0006] Therefore, controlling bacterial growth is necessary in biotechnological processes, especially on an industrial scale. The ability to externally control bacterial growth is essential for many metabolic engineering applications, which aim to simultaneously optimize titer, productivity, and yield. See: Stephanopoulos G. 2012, Synthetic biology and metabolic engineering, ACS Synth Biol 1:514-525 and Van Dien S. 2013, From the first drop to the first truckload: Commercialization of microbial processes for renewable chemicals, Curr Opin Biotechnol 24:1061-1068. Classical approaches for growth control are based on bacterial responses to temperature, antibiotics, and nutrient limitation. Sonderegger M, Schuperli M, Sauer U. 2005, Selection of quiescent Escherichia coli with high metabolic activity, Metab Eng 7:4-9; Tunner JR, Robertson CR, Schippa S, Matin A. 1992, Use of glucose starvation to limit growth and induce protein production in Escherichia coli, Biotechnol Bioeng 40:271-279; Li S, Jendresen CB, Nielsen AT. 2016, Increasing production yield of tyrosine and mevalonate through inhibition of biomass formation, Process Biochem 51:1992-2000; Rowe DC, Summers DK.1999, The quiescent-cell expression system for protein synthesis in Escherichia coli, Appl Environ Microbiol 65:2710-2715; Harder BJ, Bettenbrock K, Klamt S. 2018, Temperature-dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli, Biotechnol Bioeng 115:156-164; and Schramm T, Lempp M, Beuter D, Sierra SG, Glatter T, Link H. 2020, High-throughput enrichment of temperature-sensitive argininosuccinate synthetase for two-stage citrulline production in E. coli, Metab Eng 16:14-24。.

[0007] Over the past decade, synthetic biology has enriched the available technologies for bacterial growth control. Emerging technologies include genome and plasmid manipulation (see Kosuri S, Church G. 2014, Large-scale de novo DNA synthesis: technologies and applications, Nat Methods 11:499-507, and Csorgo B, Nyerges A, Posfai G, Feher T. 2016, System-level genome editing in microbes, Curr Opin Microbiol 33:113-122), computer-aided design and construction of genetic circuits (see Nielsen AAK, Der BS, Shin J, Vaidyanathan P, Paralanov V, Strychalski EA, Ross D, Densmore D, Voigt CA. 2016, Genetic circuit design automation, Science (80- ) 352:aac7341), and optogenetic control of gene expression (see Olson EJ, Tabor JJ. 2014, Optogenetic characterization methods overcome key challenges in synthetic and systems biology, Nat Chem Biol 10:502-511), as well as fluorescent reporters for live monitoring of gene expression (see: Zaslaver A, Bren A, Ronen M, Itzkovitz S, Kikoin I, Shavit S, Liebermeister W, Surette MG, Alon U. 2006, A comprehensive library of fluorescent transcriptional reporters for Escherichia coli, Nat Methods 3:623-628), allowing feedback control of cellular processes.Indeed, synthetic biology approaches to growth control have explored feedback regulation of genes responsible for amino acid synthesis (see Milias-Argeitis A, Rullan M, Aoki SK, Buchmann P, Khammash M. 2016, Automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth, Nat Commun 7:12546), competition between host RNA polymerase and phage T7 RNA polymerase (see Stargardt P, Feuchtenhofer L, Cserjan-Puschmann M, Striedner G, Mairhofer J. 2020, Bacteriophage inspired growth-decoupled recombinant protein production in Escherichia coli, ACS Synth Biol 9:1336-1348), and feedback regulation of mRNA decay (see Venturelli OS, Tei M, Bauer S, Chan LJG, Petzold CJ, Arkin AP. 2017, Programming mRNA decay to modulate synthetic circuit resources). allocation, Nat Commun 8:15128), and a toggle switch to decouple growth and lactate production (Reference: Venayak N, Raj K, Jaydeep R, Mahadevan R. 2018, An optimized bistable metabolic switch to decouple phenotypic states during anaerobic fermentation, ACS Synth Biol 7:2854-2866).

[0008] The present applicant has previously developed a fundamentally different approach to bacterial growth control based on a so-called synthetic growth switch. Details of the technology involved in the synthetic growth switch are disclosed in EP 3 047 031 and further in Izard J, Gomez Balderas C, Ropers D, Lacour S, Song X, Yang Y, Lindner AB, Geiselmann J, De Jong H. 2015, A synthetic growth switch based on controlled expression of RNA polymerase, Mol Syst Biol 11:840. In summary, the synthetic growth switch allows for the control of cell growth by controlling the expression of a gene encoding RNA polymerase with a specific chemical compound. This technology thus involves growth-related genes that can be selectively expressed or repressed by supplying the microorganism with the chemical compound. In other words, the expression or repression of the mutant gene depends on the presence or absence of the chemical compound.

[0009] However, the synthetic proliferation switch is inherently prone to spontaneous breakdown, primarily due to the tendency of cells to accumulate mutations in the system responsible for inhibiting RNA polymerase, thus promoting proliferation. As a result, the synthetic proliferation switch system is often rendered inoperable by spontaneous mutations.

[0010] There is a need to provide genetically modified bacteria that have improved genetic stability while maintaining metabolic production capacity.

[0011] To that end, an object of the present invention is a genetically engineered bacterium for producing at least one metabolite, peptide or recombinant protein, comprising a gene encoding a recombinant protein of interest or at least one gene encoding an enzyme involved in the production of a peptide or metabolite of interest, and further comprising a gene encoding the ββ' subunit of RNA polymerase operably linked to an inducible promoter, wherein the native promoter of said gene has been replaced by said inducible promoter, and wherein said genetically engineered bacterium has the following characteristics: The following features (according to the first embodiment): - the gene is operably linked to a second inducible promoter, and the native promoter of the gene is replaced by the second inducible promoter; - the gene is inserted by homologous recombination into a locus that is not essential for bacterial growth (in other words, a locus that does not contain genes involved in bacterial growth) or into a non-coding sequence, and the locus does not undergo spontaneous homologous recombination; - the gene encoding the α subunit of RNA polymerase is located at a distance of up to 500,000 nucleotides from the origin of replication; and - the gene is oriented in the opposite direction to the upstream gene; A gene encoding an α subunit of an RNA polymerase having the formula: the native gene encoding the α subunit of the RNA polymerase has been deleted from its native location, or Alternatively (according to a second embodiment), the vector further comprises at least one gene encoding at least one small regulatory RNA (srRNA) that inhibits translation of messenger RNA from a gene encoding the ββ' subunit of RNA polymerase so as to inhibit synthesis of the ββ' subunit of RNA polymerase, wherein the gene encoding the small regulatory RNA is operably linked to a second inducible promoter.

[0012] As used herein, "a locus that does not undergo spontaneous homologous recombination" means a gene sequence that has a probability of being subject to spontaneous homologous recombination of less than 30%, preferably less than 20%, and more preferably less than 10%.

[0013] These dual mechanisms for controlling gene expression allow for increased stability of bacterial growth alterations, and indeed, bacterial growth continues to be controlled by these mechanisms even if a mutation occurs in one of the inducible promoter regions.

[0014] In particular, in the case of the first embodiment, the stability of growth control is increased by additionally controlling the transcription of the α subunit.

[0015] It should be noted that genetically stable bacteria cannot be obtained if the subunit-encoding genes are not replaced. This is because the gene encoding the α subunit of RNA polymerase is located in the middle of an operon containing ribosomal genes. Failure to replace the gene encoding the α subunit of RNA polymerase may disrupt operon control and inhibit the expression of ribosomal genes involved in the translation process.

[0016] The same problem occurs if the above insertion criteria are not taken into account.

[0017] Advantageously, when the first and second promoters are of the same type, the expression of the α and ββ' subunits of RNA polymerase can be controlled simultaneously.

[0018] In particular, in the second embodiment, the stability of growth control can be enhanced by additional and indirect control of the translation of the ββ' subunit mRNA by snRNA. This structure is particularly interesting in that it allows growth control not only at the translational level but also at the transcriptional level.

[0019] In this second embodiment, there can be more than one srRNA that targets the messenger RNA of the β or β' subunit. If only one srRNA is produced, it is preferentially designed to inhibit translation of the RNA encoding the β subunit.

[0020] Advantageously, in these embodiments, when the first and second promoters are distinct, precise and simultaneous control of subunit and srRNA expression can be achieved, allowing precise control of proliferation.

[0021] According to a variation of the first embodiment, the coding sequence for the alpha subunit of RNA polymerase operably linked to a second inducible promoter is inserted by homologous recombination into any of the insG, yjhD, insO-insl1-insM, insA-insB1, ryjB, yjhR, envZ-ompR, arcZ, or sokA-insKJ-hokA loci.

[0022] According to variations of these embodiments, the first inducible promoter and the second inducible promoter are two different types of inducible promoters.

[0023] According to a variation of these embodiments, at least one of the inducible promoters depends on two different external inducer molecules.

[0024] In variations of these embodiments, at least one of the inducible promoters is selected from the following promoters: an IPTG-dependent promoter, an arabinose-dependent promoter (such as AraC), a rhamnose-dependent promoter.

[0025] According to a modification of the first embodiment, the gene encoding the ββ' subunit of RNA polymerase is the rpoBC gene of the rpoBC operon, and the gene encoding the α subunit of RNA polymerase is the rpoA gene of the rpsM operon.

[0026] According to a variation of these embodiments, the bacterium is Escherichia coli.

[0027] According to a variation of these embodiments, the original rpoA locus is replaced by a selectable marker, preferentially one that confers chloramphenicol resistance (cmR), by homologous recombination.

[0028] According to a variation of these embodiments, the bacterium contains at least three copies of the lacI gene.

[0029] According to a variant of the first embodiment, the bacterium of the invention comprises a nucleotide sequence having at least 80%, preferentially 90%, more preferentially 100% identity to at least one nucleotide sequence from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0030] According to a variant of the first embodiment, the bacterium of the invention comprises a nucleotide sequence having at least 80%, preferentially 90%, more preferentially 100% identity to SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0031] Another object of the present invention is a method (of the first embodiment) for producing at least one metabolite, peptide or recombinant protein of interest, comprising the steps of: -a. Cultivating a bacterium comprising: (i) a gene encoding the recombinant protein or at least one gene encoding an enzyme involved in the production of the peptide or metabolite; (ii) a gene encoding the ββ' subunit of bacterial RNA polymerase operably linked to a first inducible promoter, wherein the native promoter of the gene is replaced by the first inducible promoter; and (iii) a gene encoding the α subunit of bacterial RNA polymerase, in a first culture medium that induces expression of a gene encoding the ββ' subunit and further induces expression of a gene encoding the α subunit, thereby inducing bacterial growth; the α subunit of RNA polymerase has the following characteristics: said gene is operably linked to a second inducible promoter, such that the native promoter of said gene is replaced by said second inducible promoter; said gene is inserted by homologous recombination into a locus comprised of genes that are not essential for bacterial growth or into a non-coding sequence, said locus not undergoing spontaneous homologous recombination; said gene encoding the α subunit of RNA polymerase is located at a distance of up to 500,000 nucleotides from the origin of replication, and said gene is oriented in the opposite direction to an upstream gene, and the native gene encoding the α subunit of RNA polymerase has been deleted from its native location; -b. culturing the bacterium in a second medium that inhibits expression of the gene encoding the ββ' subunit and that inhibits expression of the gene encoding the α subunit, thereby producing the metabolite, peptide, or recombinant protein while inhibiting bacterial growth; -c. Optionally, sequentially repeating steps a. and b.; and -d. Optionally, recovering said metabolite, peptide or recombinant protein produced by said bacterium.

[0032] According to a variant of the first embodiment, each inducible promoter is an IPTG-dependent promoter or a lactose-dependent promoter.

[0033] According to a variant of the first embodiment, the first medium in step a. contains IPTG or lactose, and the second medium in step b. does not contain IPTG or lactose.

[0034] Another object of the present invention is a method (of the second embodiment) for producing at least one metabolite, peptide or recombinant protein of interest, comprising the steps of: -a. culturing a bacterium comprising: (i) a gene encoding the recombinant protein or at least one gene encoding an enzyme involved in the production of the peptide or metabolite, (ii) a gene encoding the ββ' subunit of bacterial RNA polymerase operably linked to a first inducible promoter, wherein the native promoter of the gene has been replaced by the first inducible promoter, and (iii) at least one gene encoding at least one small regulatory RNA that inhibits translation of messenger RNA of the gene encoding the ββ' subunit of RNA polymerase, wherein the gene encoding the small regulatory RNA is operably linked to a second inducible promoter, the bacterium being cultured in a first medium that induces expression of the gene encoding the ββ' subunit of RNA polymerase and inhibits expression of the gene encoding the small regulatory RNA, thereby inducing bacterial growth; -b. culturing the bacterium in a second medium that inhibits expression of the gene encoding the ββ' subunit and further induces expression of the gene encoding a small regulatory RNA, thereby producing the metabolite, peptide, or recombinant protein while inhibiting bacterial growth; -c. Optionally, sequentially repeating steps a. and b.; and -d. Optionally, recovering said metabolite, peptide or recombinant protein produced by said bacterium.

[0035] According to variations of these embodiments, the gene encoding the recombinant protein or the at least one gene encoding an enzyme involved in the production of the peptide or metabolite is transcribed by a second RNA polymerase having a catalytic subunit or catalytic subunits different from the ββ' and α subunits of the RNA polymerase operably linked to an inducible promoter.

[0036] According to a variation of these embodiments, the second RNA polymerase is bacteriophage T7 polymerase.

[0037] In a variation of these embodiments, the bacterium is Escherichia coli.

[0038] Other features and advantages of embodiments according to the invention will stand out and / or become apparent on reading the following description and drawings, which include specific examples given in an illustrative and non-limiting manner:

[0039] [Figure 1] shows a conceptual diagram of an embodiment according to the present invention;

[0040] [Figure 2] shows the gene construction for IPTG-dependent control of the transcription of rpoBC and rpoA in the mutant according to the present invention;

[0041] [Figure 3] shows the arrangement according to the variant of the present invention, in which rpoBC is under the control of an IPTG-dependent promoter;

[0042] [Figure 4] shows the first part of the sequence according to the modification of the present invention, in which rpoA is replaced by Cm in the operon of ribosomal proteins starting from rpsM;

[0043] [Figure 5] shows the second part of the sequence in Figure 4;

[0044] [Figure 6] shows the first part of the sequence according to the variant of the invention, in which rpoA is under the control of an IPTG-dependent promoter;

[0045] [Figure 7] shows the second part of the sequence in Figure 6;

[0046] [Figure 8] shows the third part of the sequence in Figure 6;

[0047] [Figure 9] shows the fourth part of the sequence in Figure 6;

[0048] [Figure 10] shows the improvement of the genetic stability of bacterial strains due to the modifications of the present invention;

[0049] [Figure 11] shows the growth, glucose consumption, and glycerol production of the wild-type strain and strains with single and double control of RNA polymerase expression;

[0050] [Figure 12A] shows the genomic region predicted after integration of the sequence carrying the inducible rpoA gene into the yhim-yhiN locus;

[0051] [Figure 12B] shows the results of agarose gel migration of DNA fragments obtained under different PCR conditions; and

[0052] FIG. 13 shows a conceptual diagram of another embodiment according to the present invention.

[0053] The drawings and description herein contain elements that are, for the most part, distinct in nature, and therefore the description and drawings are used not only to better understand the embodiments of the invention, but also, where appropriate, to contribute to its definition.

[0054] The present applicant previously engineered a bacterial strain of Escherichia coli in which transcription of RNA polymerase, a key component of the gene expression apparatus, is under the control of an inducible promoter. See EP 3 047 031. By varying the inducer concentration in the medium, it is possible to adjust the RNA polymerase concentration, thereby switching bacterial growth between zero and the maximum growth rate supported by the medium. The present applicant conferred the ability to produce glycerol when grown on glucose to both wild-type E. coli strains and modified strains of the present invention. Growth-arrested cells remain metabolically active and produce glycerol at a yield twofold higher than cells in which RNA polymerase expression is naturally regulated. The resulting yield was shown to be close to the theoretical maximum. Reference: Izard J, Gomez Balderas C, Ropers D, Lacour S, Song X, Yang Y, Lindner AB, Geiselmann J, De Jong H. 2015, A synthetic growth switch based on controlled expression of RNA polymerase, Mol Syst Biol 11:840. The synthetic switch thus decouples growth from target compound production, an approach that is optimal from a control-theoretic perspective (see Yegorov I, Mairet F, de Jong H, Gouze JL. 2019, Optimal control of bacterial growth for the maximization of metabolite production, J Math Biol 78:985-1032) and is commonly adopted in the art.References: Lo TM, Chng SH, Teo WS, Cho HS, Chang MW. 2016, A two-layer gene circuit for decoupling cell growth from metabolite production, Cell Syst 3:133-143; Venayak N, Anesiadis N, Cluett WR, Mahadevan R. 2015, Engineering metabolism through dynamic control, Curr Opin Biotechnol 34:142-152; and Lalwani MA, Zhao EM, Avalos JL. 2018, Current and future modalities of dynamic control in metabolic engineering, Curr Opin Biotechnol 52:56-65.

[0055] As mentioned above, most synthetic circuits are vulnerable from an evolutionary perspective, in the sense that host strains harboring the circuit are counterselected by the environment in which it must function (Renda BA, Hammerling MJ, Barrick JE. 2014, Engineering reduced evolutionary potential for synthetic biology, Mol Biosyst 10:1668-1678). This is particularly true in the case of growth switches, where derepression of the gene encoding the RNA polymerase ββ' subunit would allow growing mutant bacteria to quickly overtake growth-arrested populations. The design of growth switches includes safeguards, particularly redundancy of the gene encoding the repressor LacI (see EP 3 047 031). However, when moving to high-density cultures in large-scale bioreactors, where deleterious mutations are expected to arise at higher frequencies, these safeguards may not be sufficient.

[0056] Although several strategies to improve the genetic reliability of synthetic circuits have been proposed in the literature, there is room for improvement.

[0057] Embodiments of the present invention dramatically improve synthetic circuits to enhance the genetic stability of production strains. To this end, the present invention provides a genetic architecture in which a single mutation does not affect its function (also known as mutational robustness). In particular, the genetic structure of the microorganisms of the present invention incorporates redundancy in the regulation of RNA polymerase expression.

[0058] More precisely, according to a first embodiment, the applicant placed an additional subunit of RNA polymerase, the α-subunit, under the control of the same or (preferably) a different inducible system used to control the expression of the ββ'-subunit. In other words, the gene encoding the ββ'-subunit is under the control of a first inducible promoter, while the gene encoding the α-subunit is under the control of a second inducible promoter. As a result, only simultaneous mutations in both promoter regions upstream of the α- and ββ'-encoding genes result in loss of control of RNA polymerase expression, and therefore proliferation. In practice, this is an extremely unlikely event, since the theoretical probability that a mutation will disable the modified proliferation switch is the square of the mutation probability of the original genetic circuit. The proliferation switch that redundantly controls RNA polymerase expression is thus genetically stable.

[0059] In order to obtain a genetically stable bacterium according to the first embodiment, a number of criteria must be met for the replacement of the gene encoding the α subunit.

[0060] First, the gene encoding the α subunit must be operably linked to a second inducible promoter (replacing the gene's native promoter). Furthermore, the gene encoding the α subunit must be inserted by homologous recombination into a locus not essential for bacterial growth or into a non-coding sequence, located up to 500,000 nucleotides away from the origin of replication. Additionally, the gene sequence selected for insertion of the α subunit-encoding gene operably linked to the second inducible promoter must not be susceptible to spontaneous homologous recombination. The α subunit-encoding gene must also be oriented in the opposite direction to the upstream gene (i.e., the gene upstream of the promoter of the α subunit-encoding gene). Finally, the α subunit-encoding gene is deleted from its native site. More precisely, the native α subunit-encoding gene may simply be deleted or may correspond to the α subunit-encoding gene replaced according to the above conditions, but the bacterium must not contain a copy of this α subunit-encoding gene in its native site. It has been observed that if one or more of these criteria are omitted, the resulting genetically modified bacteria are not sufficiently stable.

[0061] For example, the gene encoding the α subunit can be inserted by homologous recombination into one of the following insertion sites: insG, yjhD, insO-insl1-insM, insA-insB1, ryjB, yjhR, envZ-ompR, or arcZ, whose functions are not essential for bacterial growth under typical biotechnological production conditions, but are listed in the table below:

[0062] [Table 1]

[0063] Thus, the method of this first embodiment includes a bacterial growth step in which expression of the genes encoding the ββ' and α subunits, i.e., rpoB, rpoC, and rpoA, is induced. This results in intracellular production of RNA polymerase. Bacterial growth occurs during this step, resulting in the production of natural amounts of metabolites, peptides, or recombinant proteins. The method then includes a further step of inhibiting expression of the genes encoding the ββ' and α subunits. Without the synthesis of new subunits, the intracellular concentration of RNA polymerase falls below a level that can no longer support bacterial growth. However, because the enzymes of the relevant metabolic pathway are sufficiently expressed and stable, they remain present even after growth has stopped, and metabolite production continues. Furthermore, peptide or recombinant protein expression can continue even in the presence of low concentrations of RNA polymerase.

[0064] The above method is enhanced when the expression of a metabolic pathway enzyme, or of a peptide or homologous protein, is placed under the transcriptional control of a strong promoter. In fact, the amount of RNA polymerase remaining in the cell preferentially transcribes this strong promoter. A strong promoter is one that is at least 10% as strong as a so-called rrn promoter. See Liang et al. (1999), J. Mol. Biol. 292:19-37.

[0065] According to a second embodiment, the applicant has placed one or more genes encoding one or more small regulatory RNAs that inhibit messenger RNA translation from the gene encoding the ββ' subunit of RNA polymerase under the control of a separate induction system. This latter induction system is different from the induction system used to control the expression of the ββ' subunit. The small regulatory RNAs used in this embodiment are intended to inhibit the synthesis of the ββ' subunit of RNA polymerase. Therefore, only simultaneous mutations in both the promoter region upstream of the ββ'-encoding gene and the small regulatory RNAs result in loss of control of RNA polymerase expression, and therefore growth control.

[0066] In this second embodiment, there can be more than one srRNA targeting the β or β' subunit. For example, it is possible to produce a modified bacterium with an srRNA for the β subunit and an srRNA for the β' subunit. If only one type of srRNA is produced, the srRNA designed to inhibit translation of messenger RNA from the gene encoding the β subunit will be preferentially selected.

[0067] Thus, the method of this second embodiment of the present invention includes a bacterial growth control step in which the expression of genes encoding the ββ' subunit is induced, i.e., the expression of rpoB and rpoC is induced, while the expression of genes encoding small regulatory RNAs is inhibited. This results in the production of RNA polymerase within the cells. During this step, bacterial growth occurs, resulting in the production of natural amounts of metabolites, peptides, or recombinant proteins. The method then includes a further step in which the expression of genes encoding the ββ' subunit is inhibited, while the expression of genes encoding small regulatory RNAs is induced to inhibit the translation of potential mRNAs for the ββ' subunit. Thus, repression of the expression of the gene encoding the ββ' subunit is strengthened. Without the synthesis of new subunits, the intracellular concentration of RNA polymerase falls below a level that can no longer support bacterial growth. However, because the enzymes of the relevant metabolic pathway are sufficiently expressed and stable, they remain present even after growth has stopped, allowing the production of metabolites to continue. Furthermore, peptide or recombinant protein expression can continue even in the presence of low concentrations of RNA polymerase.

[0068] Expression of genes encoding metabolite-producing enzymes or of target peptides or recombinant proteins can be further improved by adding a second polymerase with a catalytic subunit different from the ββ' and / or α subunits of the host RNA polymerase operably linked to an inducible promoter. This second RNA polymerase can specifically increase transcription of genes encoding target metabolite-producing enzymes or of peptides or recombinant proteins. However, the presence of such a second RNA polymerase is not essential. In fact, the applicant integrated a fluorescent reporter gene (mCherry under the control of the rrnBp2 promoter) into the genome of E. coli IJ40 strain. Results showed that the average fluorescence per cell increased from 80 to 120 relative fluorescence units after growth arrest. This indicates that growth-arrested cells are synthesizing proteins and are metabolically active.

[0069] To implement the method of the first embodiment of the present invention, the applicant designed a bacterium in which an inducible promoter is used to control the expression of the rpoBC operon and the rpoA operon, which encode the ββ'- and α-subunits of RNA polymerase, respectively. In contrast, in the second embodiment, the applicant designed a bacterium in which an inducible promoter is used to control the expression of an operon encoding a small regulatory RNA that inhibits the translation of messenger RNA from the rpoBC operon and the gene encoding the ββ'-subunit. In this bacterium (or bacterial strain), the intracellular concentration of RNA polymerase can be externally set to a desired level. As a result, bacterial gene expression can be controlled.

[0070] The inducible promoters may be of the same type or of different types, depending on the embodiment and / or the desired control of gene expression.

[0071] In particular, in a first embodiment, two identical promoters are preferably used to simultaneously control the expression of the genes encoding the ββ' and α subunits of RNA polymerase.

[0072] Conversely, for the second embodiment, two different promoters are used to differentially control the expression of the gene encoding the ββ' subunit of RNA polymerase and the small regulatory RNA that inhibits translation of messenger RNA from the gene encoding the ββ' subunit.

[0073] In these embodiments, the inducible promoter can be, for example, a lactose- or β-D-1-thiogalactopyranoside (IPTG)-dependent promoter, a variant of the endogenous lac promoter. Removal of IPTG (or lactose) from the culture medium can switch off cell growth, or at least dramatically reduce it from the wild-type bacterial growth rate to a very low growth rate. Remaining cell growth in the absence of IPTG in the medium is due to RNA polymerase molecules that remained in the bacteria before the IPTG concentration was reduced. Increasing the IPTG (or lactose) concentration in the medium can switch cell growth back on to restore or further increase the bacterial population size.

[0074] Chemically regulated promoters, such as the tet promoter or the araBAD promoter, or physically regulated promoters, such as light- or temperature-dependent promoters, can also be used in solution. Such promoters are well known to those skilled in the art. See Terpe (2006), Appl. Microbiol Biotechnol, 72:211-222.

[0075] As mentioned above, the solution of the present invention is an improvement based on the method and engineered bacteria described in EP 3 047 031 (originally filed as PCT / EP2014 / 069719 and published in WO2015 / 036622 based on priority EP13306266).Therefore, the terms used herein are well known to those skilled in the art.Further details regarding the specific definitions used herein are described in EP 3 047 031, and the reader is referred to therein.

[0076] Figure 1 is a schematic diagram of an embodiment according to the present invention. The molecular components on the right side of Figure 1, related to IPTG-dependent regulation of rpoBC, were already present in the previous version of the growth switch disclosed in EP 3 047 031. The components on the left side dramatically improve upon known growth switches, particularly improving the robustness and stability of the genetic system. More precisely, the components on the left side are related to the regulation of rpoA by the same IPTG-dependent regulatory mechanism as that for rpoBC regulation. The mutational robustness of this system is achieved by the fact that mutations in only one of the two promoter regions (rpoBC or rpoA) do not lead to loss of growth control, while simultaneous mutations in both promoter regions are highly unlikely. This measure of robustness is in addition to the safeguard already included in the original design, i.e., the presence of three lacI sites on the genome, which protects against a single mutation that abolishes LacI binding to the promoter.

[0077] More precisely, Figure 1 shows the structure of a synthetic circuit for controlling RNA polymerase expression according to a preferred embodiment. The genes rpoBC encoding the ββ'-subunit and rpoA encoding the α-subunit are placed under the control of the LacI repressor by replacing their native promoters with synthetic promoters. Addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the growth medium relieves LacI repression, allowing growth; however, in the absence of IPTG, the rpoA and rpoBC genes are not expressed, and therefore no RNA polymerase is produced.

[0078] There are two α subunits per RNA polymerase molecule: α I and α II , also called α2. Each α subunit contains two domains: the αNTD (N-terminal domain) and the αCTD (C-terminal domain). The αNTD contains determinants for RNA polymerase assembly, and the αCTD contains determinants for interaction with promoter DNA.

[0079] The α subunit is overproduced during cell growth. Given the abundance of the α subunit of RNA polymerase, the results of embodiments according to the present invention are quite surprising in that a control switch located in the rpoA region can radically enhance the genetic stability of known control switches while maintaining their metabolic production capacity. The combination of genetic stability and the high-throughput metabolic production capacity of the present invention is a dramatic improvement compared to state-of-the-art systems.

[0080] FIG. 2 shows the gene construct for IPTG-dependent regulation of the transcription of rpoBC and rpoA in the mutant according to the present invention.

[0081] Figure 2A shows the control of rpoBC expression via the IPTG-inducible T5 promoter (disclosed and discussed in EP 3 047 031). More specifically, the rpoBC upstream sequence was modified by inserting an IPTG-inducible T5 promoter (T5p) with two lacO binding sites and a selectable marker carrying a spectinomycin resistance gene and its promoter, spcRp. Inserting this sequence via homologous recombination allowed rpoBC expression to be controlled by adjusting the IPTG concentration in the medium (see Lutz R, Bujard H. 1997, Independent and tight regulation of transcriptional units in Escherichia coli via the LacR / O, the TetR / O, and AraC / I1-I2 regulatory elements, Nucleic Acids Res 25:2003-20). In the absence of an inducer, the LacI protein binds to the lacO site and inhibits RNA polymerase binding.

[0082] Figure 2B shows the control of rpoA expression via the same regulatory system. This demonstrates the realization of genetic control of rpoA at the sequence level in an embodiment of the present invention. The RNA subunit α is encoded by the rpoA gene. The original rpoA locus on the E. coli genome was modified by homologous recombination (Sharan, SK, Thomason, LC, Kuznetsov, SG, and Court, DL (2009). Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4, 206-223.), replacing the rpoA coding region with that of a selectable marker (cmR) that confers chloramphenicol resistance. The introduction of a gene encoding chloramphenicol resistance provides ideal selection for successful recombinants. According to one aspect of the present invention, the rpoA gene containing an inducible promoter was moved from its original location on the chromosome to a different location, the sokA-insKJ-hokA sequence. This sequence was chosen because it is approximately the same distance from the rpoBC operon and the origin of replication. The sokA-insKJ-hokA genes are not essential for bacterial growth and were therefore removed by the cloning procedure. According to a variant of the present invention, the expression of both rpoBC and rpoA can be controlled by adjusting the IPTG concentration in the medium.

[0083] Figure 2C shows the location of genes on the E. coli chromosome and summarizes the changes that occurred on the E. coli chromosome due to the above modifications.

[0084] Figure 3 shows a variant of the sequence of the invention in which rpoBC is under the control of an IPTG-dependent promoter. All promoter elements and genes are shown.

[0085] Figures 4 and 5 show the sequence of a modification of the present invention in which rpoA is replaced by Cm in the ribosomal protein operon starting with rpsM. This modification was made to avoid expression of rpoA under natural conditions and to confer chloramphenicol resistance (cmR). All relevant genes are annotated in the figures according to standard nomenclature.

[0086] Figures 6, 7, 8 and 9 show variants of the present invention in which rpoA under the control of an IPTG-dependent promoter has been inserted in place of and in place of the sokA-insKJ-hokA sequence. All relevant genes are annotated in the figures according to standard nomenclature.

[0087] Thus, variants of the bacterium (or bacterial strain) of the invention comprise a nucleotide sequence having at least 80%, preferentially 90%, more preferentially 100% identity to the sequence of Figure 3 (SEQ ID NO: 1).

[0088] Another variant of the bacterium of the invention comprises a nucleotide sequence having at least 80%, preferentially 90%, more preferentially 100% identity to the sequence of Figures 4 and 5 (SEQ ID NO: 2).

[0089] Another variant of the bacterium of the invention comprises a nucleotide sequence having at least 80%, preferentially 90%, more preferentially 100% identity to the sequence of Figures 6, 7, 8 and 9 (SEQ ID NO: 3).

[0090] Preferentially, another variant of the bacterium of the invention comprises a nucleotide sequence having at least 80%, preferentially 90%, more preferentially 100% identity to the sequences of Figures 3 to 9 (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3).

[0091] The procedure for increasing production yield in bioreactors using the new strain is the same as for the previous strain; see EP 3 047 031. Testing showed that the new strain retained the ability to switch growth on and off by supplying and removing IPTG from the medium, respectively. It also showed that the glycerol production yield from glucose was maintained after growth was stopped. Furthermore, the genetic stability of the improved strain was shown to be higher than that of the original strain.

[0092] FIG. 10 shows the improved genetic stability of the strain with regulated expression of the ββ' and α subunits compared to regulated expression of the ββ' subunit alone.

[0093] In Figure 10A, a strain in which expression of the rpoBC gene is under the control of an IPTG-inducible promoter was precultured in 40 biological replicates in M9 minimal medium containing 0.2% glucose and supplemented with IPTG (250 μM). Each replicate culture was then washed, diluted, and resuspended in the same medium without IPTG to block synthesis of the new ββ' subunit of RNA polymerase. As a control, three replicates were washed, diluted, and resuspended in the same medium with IPTG. Starting from an optical density as low as 0.005, growth was monitored 24 and 96 hours after the initiation of cultivation. None of the cultures escaped after 24 hours, but 33 of 40 escaped after 96 hours.

[0094] Figure 10B shows the same for a strain in which expression of both the rpoBC and rpoA genes is under the control of IPTG-inducible promoters, demonstrating the dramatically increased genetic stability of strains with dual expression control according to embodiments of the present invention.

[0095] FIG. 11 shows the growth, glucose consumption, and glycerol production of the wild-type strain and both the single- and double-regulated strains of RNA polymerase expression.

[0096] In Figure 11A, wild-type strains transformed with plasmids enabling overexpression of enzymes involved in glycerol synthesis were grown in triplicate in M9 minimal medium containing 0.2% glucose. Optical density (OD600) measurements were performed at different time points during the experiment (blue dots, i.e., crossed dots). The same measurements were performed under the same conditions for a strain in which expression of the rpoBC gene is under the control of an IPTG-inducible promoter (red dots, i.e., crossed dots), and a strain in which expression of both the rpoBC and rpoA genes is under the control of IPTG-inducible promoters (yellow dots, i.e., crossed dots). Contrary to the wild-type strain, the latter two strains cease growth after inoculation due to the absence of IPTG in the medium.

[0097] Figure 11B shows the results of measuring the glucose concentration in the medium. The strain in which RNA polymerase expression was regulated in an IPTG-dependent manner continued to consume glucose even after growth stopped.

[0098] Figure 11C shows the glycerol concentration in the medium. While the wild-type strain produces only small amounts of glycerol, which is consumed after glucose depletion, the growth-arrested strain converts glucose to glycerol at a much higher level. This supports the claim that inhibiting RNA polymerase expression can arrest population growth and redirect metabolic flux toward the biosynthesis of a target metabolite (glycerol in this case) at high yields.

[0099] The applicant has pointed out that if the insertion parameters of the first embodiment (described above) are not respected, genetically stable bacteria cannot be obtained. In other words, merely transferring the gene encoding the α subunit of RNA polymerase is not sufficient to obtain genetically modified bacteria with improved stability. This was demonstrated by homologous recombination of an inducible gene encoding the α subunit into a site that does not meet the insertion criteria of the first embodiment.

[0100] For testing purposes, we engineered bacteria containing the inducible rpoA gene of RNA polymerase inserted into the insertion site yhiM-yhiN by homologous recombination, while deleting the native rpoA gene, as shown in the scheme in Figure 12A. The corresponding modified bacteria have a gene encoding the ββ' subunit of RNA polymerase operably linked to a separate inducible promoter, as described above. Furthermore, the chosen site is less than 500,000 nt from the replication origin. However, the yhiM-yhiN region is susceptible to spontaneous homologous recombination. Therefore, it does not meet all of the predefined insertion criteria.

[0101] Figure 12B shows the agarose gel electrophoresis results (30 minutes, 75 V) of fragments obtained under various PCR (polymerase chain reaction) conditions using the primers shown in Figure 12A. Specifically, PCR was performed before and after recombination. Primer 1 and Primer 2 were used to verify whether the genomic region was present before and after recombination under eight different PCR conditions.

[0102] Before recombination, a region flanked by the primers and containing the cloning cassette is present (positive control in the gel, around 2500 bp). However, after recombination, the region expected to contain the inducible rpoA sequence does not appear (there is no spot around 1400 bp). The applicant believes that this attempt was unsuccessful because all the criteria for insertion were not met. In particular, the selected region is prone to spontaneous recombination or transposon insertion.

[0103] These results demonstrate that the construct shown in Figure 12A does not provide genetic stability for the engineered bacteria, and therefore highlight the complexity of obtaining engineered bacteria with improved stability.

[0104] Figure 13 is a schematic diagram of a second embodiment of the present invention. The molecular components on the left side of Figure 13 are related to IPTG-dependent regulation of rpoBC, as described above. The components on the right side are related to regulation of srRNA (inhibition of rpoBC mRNA translation) by an aTc-dependent regulator (a tetracycline derivative). The mutational robustness of this system is achieved by the fact that mutations in only one of the two promoter regions (upstream of rpoBC or srRNA) do not lead to loss of growth control, while simultaneous mutations in both promoter regions are highly unlikely.

[0105] More precisely, Figure 13 shows the architecture of the synthetic circuit for controlling RNA polymerase expression according to the second embodiment. The genes encoding the ββ'-subunit, rpoBC, and srRNA, are placed under the control of the LacI and TetR repressors, respectively, by replacing their native promoters with synthetic promoters. Addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the growth medium relieves LacI repression, allowing the production of mRNA and, therefore, the β'-subunit of RNA polymerase. In the absence of IPTG, the rpoBC gene is not expressed, and therefore no RNA polymerase is produced.

[0106] Conversely, the addition of aTc to the medium relieves repression of TetR and prevents translation of the ββ'-subunit mRNA of RNA polymerase, thus preventing growth, whereas in the absence of aTc in the medium, the ββ'-subunit mRNA is translated, thus producing RNA polymerase.

[0107] Thus, dual regulation of these two induction circuits allows dual regulation of RNA polymerase production and therefore bacterial growth. In particular, additional regulation of the translation of the ββ'-subunit mRNA can repress RNA polymerase synthesis. Therefore, only the highly unlikely double mutation of the two promoters leads to RNA polymerase production and bacterial growth.

[0108] As known to those skilled in the art, in bacteria, the core RNA polymerase enzyme generally consists of five subunits: β, β', α', α'' and ω, while the final subunit, ω, can often be removed without side effects. Furthermore, those skilled in the art know that the structure of bacterial RNA polymerases is universal. This is discussed, for example, in the review by Borukhov and Nudler, "RNA polymerase: the vehicle of transcription," Trends in Microbiol., 2008, 16, 126-134. This review states that "the catalytically competent core (subunit composition α, β', β, and ω) has been evolutionarily conserved in terms of its primary sequence, ternary structure, and function" (see p. 126 of the review). This means that the transcription behavior, which directly depends on the catalytically competent core, is also conserved across bacterial species. As a result, the genetically engineered bacteria and methods of the present embodiments function in E. coli or other bacteria, such as Bacillus subtilis, Bacillus thuringiensis, Lactobacillus fermentum, Synechocystis sp. PCC6803, Corynebacterium glutanicum, Deinococcus radiodurans.

[0109] Further variations of the invention include bacteria or methods according to embodiments of the invention: wherein said gene encoding the ββ' subunit of RNA polymerase is the rpoBC gene of the rpoBC operon; - the gene encoding the alpha subunit of RNA polymerase is the rpoA gene of the rpoA operon.

[0110] The method of the present invention may be modified in particular as follows: - In step a., the bacteria were incubated at 0.1–100 OD 600 Cultivate until a population density of 1000 is reached; a variant comprising, between step a. and step b., a substep a1. consisting of harvesting the bacteria cultivated in step a., preferentially washing them and transferring them to a second culture medium in step b.; - a variant including measuring the metabolic activity of the cultured bacteria during step b.; - a variant in which step c. is carried out when the metabolic activity measured during step b. is decreased; - a variant in which the first medium in step a. comprises M9 minimal medium supplemented with 0.4% glucose and 0.5 mM IPTG (or another inducer such as lactose), and the second medium in step b. comprises M9 minimal medium supplemented with 0.4% glucose and no IPTG; - a variant in which the first medium of step a. comprises M9 minimal medium supplemented with 0.2% glucose and 0.25 mM IPTG (2.5 μL of IPTG 100 mM in 1 mL of M9), and the second medium of step b. comprises M9 minimal medium supplemented with 0.2% glucose and without IPTG; - The method may be configured with or without a variation that includes, prior to step a., providing a bacterium containing (1) a gene encoding the recombinant protein or at least one gene encoding an enzyme involved in the production of the peptide or metabolite, and (2) genes encoding the ββ' and α subunits of RNA polymerase operably linked to an inducible promoter. Certain inducible promoters may also include chemicals other than IPTG or different frequencies of light (optogenetics). [Brief explanation of the drawings]

[0111] [Figure 1] 1 shows a conceptual diagram of an embodiment according to the present invention; [Figure 2] 1 shows the gene construction for IPTG-dependent regulation of the transcription of rpoBC and rpoA in the mutants according to the present invention. [Figure 3] 1 shows a modified sequence of the invention in which rpoBC is under the control of an IPTG-dependent promoter. [Figure 4] 1 shows the first part of the sequence according to the variant of the invention, in which rpoA is replaced by Cm in the operon of ribosomal proteins starting with rpsM. [Figure 5] This shows the second part of the sequence in FIG. [Figure 6] The first part of the sequence according to the variant of the invention is shown, in which rpoA is under the control of an IPTG-dependent promoter. [Figure 7] This shows the second part of the array in FIG. [Figure 8] This shows the third part of the sequence of FIG. [Figure 9] This shows the fourth part of the array in FIG. [Figure 10] 1 shows the improvement of the genetic stability of bacterial strains due to the modifications of the present invention. [Figure 11] Growth, glucose consumption, and glycerol production of the wild-type strain and strains with single and double control of RNA polymerase expression are shown. [Figure 12A] The genomic region predicted after integration of the sequence carrying the inducible rpoA gene into the yhim-yhiN locus is shown. [Figure 12B] Agarose gel migration results of DNA fragments obtained under different PCR conditions are shown. [Figure 13] 10 shows a conceptual diagram of another embodiment according to the present invention.

Claims

1. 1. A genetically engineered bacterium for producing at least one metabolite, peptide, or recombinant protein, comprising: a gene encoding a recombinant protein of interest, or at least one gene encoding an enzyme involved in the production of a peptide or metabolite of interest, and further comprising a gene encoding the ββ' subunit of RNA polymerase operably linked to a first inducible promoter, wherein the native promoter of the gene is replaced by said first inducible promoter, comprising: A gene encoding the α subunit of RNA polymerase, having the following characteristics: - said gene is operably linked to a second inducible promoter, said second inducible promoter replacing the native promoter of said gene; - the gene is inserted by homologous recombination into a locus consisting of genes that are not essential for bacterial growth or into a non-coding sequence, the locus being a genetic sequence that has a probability of being subject to spontaneous homologous recombination of less than 30%, preferably less than 20%, and even more preferably less than 10%; - the gene encoding the α subunit of RNA polymerase is located at a distance of up to 500,000 nucleotides from the origin of replication, and - the gene is oriented in the opposite direction to the gene upstream of the promoter of the gene encoding the α subunit; and further comprising a gene having the formula: the native gene encoding the α subunit of the RNA polymerase is deleted from its native location; Genetically engineered bacteria.

2. 2. The genetically engineered bacterium of claim 1, wherein the first inducible promoter and the second inducible promoter are two different types of inducible promoters.

3. 3. The genetically engineered bacterium of claim 1, wherein at least one of the inducible promoters depends on two different external inducer molecules.

4. 4. The genetically engineered bacterium of claim 1, wherein at least one of the inducible promoters is selected from the following promoters: an IPTG-dependent promoter, an arabinose-dependent promoter, a rhamnose-dependent promoter.

5. The genetically engineered bacterium of any one of claims 1 to 4, wherein the gene encoding the ββ' subunit of RNA polymerase is the rpoBC gene of the rpoBC operon, and the gene encoding the α subunit of RNA polymerase is the rpoA gene of the rpsM operon.

6. The genetically engineered bacterium of any one of claims 1 to 5, wherein the bacterium is of the species Escherichia coli.

7. 7. The genetically engineered bacterium of claim 5 or 6, wherein the original rpoA locus has been replaced by a selectable marker, preferentially one that confers chloramphenicol resistance (cmR), by homologous recombination.

8. 8. The genetically engineered bacterium of any one of claims 1 to 7, wherein the bacterium comprises at least three copies of the lacI gene.

9. 2. The genetically engineered bacterium of claim 1, comprising a nucleotide sequence having at least 80%, preferentially 90%, more preferentially 100% identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

10. 2. The genetically engineered bacterium of claim 1, comprising a nucleotide sequence having at least 80%, preferentially 90%, more preferentially 100% identity to SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

11. 1. A method for producing at least one metabolite, peptide or recombinant protein of interest, comprising the steps of: a. Bacteria, including: (i) a gene encoding said recombinant protein or at least one gene encoding an enzyme involved in the production of said peptide or metabolite; (ii) a gene encoding the ββ' subunit of bacterial RNA polymerase operably linked to a first inducible promoter, wherein the native promoter of the gene is replaced by the first inducible promoter; and (iii) a gene encoding the α subunit of bacterial RNA polymerase, having the following characteristics: - said gene is operably linked to a second inducible promoter, said second inducible promoter replacing the native promoter of said gene; - the gene is inserted by homologous recombination into a locus consisting of genes that are not essential for bacterial growth or into a non-coding sequence, the locus being a genetic sequence that has a probability of being subject to spontaneous homologous recombination of less than 30%, preferably less than 20%, and even more preferably less than 10%; - the gene encoding the α subunit of RNA polymerase is located at a distance of up to 500,000 nucleotides from the origin of replication, and - the gene is oriented in the opposite direction to the gene upstream of the promoter of the gene encoding the α subunit; a gene having Cultivating the the native gene encoding the α subunit of the RNA polymerase has been deleted from its native location; culturing in a first medium that induces expression of the gene encoding the ββ' subunit and also induces expression of the gene encoding the α subunit, thereby inducing bacterial growth; b) culturing the bacterium in a second medium that inhibits expression of the gene encoding the ββ' subunit and also inhibits expression of the gene encoding the α subunit, thereby producing the metabolite, peptide, or recombinant protein while inhibiting bacterial growth; c. Optionally, sequentially repeating steps a. and b.; and d. Optionally, recovering the metabolite, peptide or recombinant protein produced by the bacterium.

12. 12. The method of claim 11, wherein each inducible promoter is an IPTG-dependent promoter or a lactose-dependent promoter.

13. 13. The method of claim 11 or 12, wherein the first medium in step a. contains IPTG or lactose, and the second medium in step b. does not contain IPTG or lactose.

14. The method of claim 12 or 13, wherein the gene encoding the recombinant protein or the at least one gene encoding an enzyme involved in the production of the peptide or metabolite is transcribed by a second RNA polymerase having one or more catalytic subunits different from the ββ' subunit and α subunit of the RNA polymerase operably linked to the inducible promoter.

15. The method of any one of claims 11 to 14, wherein the second RNA polymerase is bacteriophage T7 polymerase.

16. The method of any one of claims 11 to 15, wherein the bacterium is an Escherichia coli bacterium.

Images