Load-dependent strains

Abstract

The present invention provides a microbial production cell for synthesizing a product, further comprising a load-dependent gene circuit, the expression of which confers a selective growth and / or survival advantage to cells synthesizing the product, while limiting the growth of low-producing / non-producing escaper cells.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention provides a microbial production cell for synthesizing a product, further comprising a load-dependent gene circuit, the expression of which confers a selective growth and / or survival advantage to cells synthesizing the product, while limiting the growth of non-producing / low-producing escaper cells. [Background technology]

[0002] Background of the Invention An increasing share of chemical production worldwide relies on microorganisms genetically engineered to function as cell factories and tailored for the biosynthesis of a given molecule. Production processes employing these cell factories typically begin with a starter culture of a small number of production organism cells and undergo phases of cell growth and cell number expansion in large fermentation tanks (up to 1,000,000 L in volume). In some settings, production of a given molecule proceeds through both a growth phase and a subsequent period (batch and fed-batch culture). Alternatively, production may be continuous. Chemostat fermentors grow production organisms in a fermentation broth that is constantly diluted, thus allowing for the product and cells to be flushed out of the culture while fresh nutrient medium is replenished. At an industrial scale, such production processes may continue operating for one to two months before starting a new culture in a clean tank. The fermentation processes and equipment used in this industry are very similar for the production of a wide range of commodity small molecules and therapeutic proteins, and as a result, these processes present similar challenges. In other applications, microbial cells can be engineered to produce non-natural molecules that confer commercial advantages in the environment in which the cells are cultured. Summary of the Invention [Problem to be solved by the invention]

[0003] Cell factories engineered to allocate finite metabolic resources to the biosynthesis of a given molecule are subject to an unnatural stress, typically reflected in slowed growth. This stress exerts selective pressure against cells that cannot synthesize the product molecule (non- or lower-producing cells), a problem that arises particularly during long production runs (e.g., in chemostats). Such non-producing cells in industrial fermentation are highly undesirable because they consume nutrients, oxygen, and space. Because lower-producing and non-producing cells grow faster, they have a strong selective advantage over producing cells. In growing cell cultures, such fitness improvements can lead to significant outcompetition of producing cells over time. This drift from optimal production is the ultimate reason to discard the fermentation broth and replenish the fermentation tank with new producing organisms, expending resources, not to mention nutrients, for cleaning and sterilization. Such non-producing cells originate from genetic mutations that occur in cells of the original producing organism that have undergone many growth divisions.

[0004] Because genetic mutations and non-genetic adaptations inevitably occur within the cells of a production organism, leading to reduced product formation by the cells during production runs, a method is needed to eliminate or slow the growth of non-producing cells in production. Preferably, such a removal method is effective enough to prevent the observed drift from production conditions, thereby extending the duration of industrial fermentation. [Means for solving the problem]

[0005] The present invention addresses the problem of how to prune non-producing members of a cell factory using fitness benefits, such as slowing proliferation among productive members of the cell factory over time, thereby increasing the productivity of the cell factory. [Effects of the Invention]

[0006] A first aspect of the present invention provides a microbial production cell genetically engineered to synthesize a product, the microbial production cell further comprising an essential gene operably linked to a load-sensing promoter, wherein said promoter is heterologous with respect to said essential gene, wherein synthesis of the product imposes a load and / or fitness cost on said cell, and wherein expression of said essential gene is upregulated when said load-sensing promoter is induced due to said load and / or fitness cost relative to a basal level of expression of said essential gene when said load-sensing promoter is not induced.

[0007] A second aspect of the present invention is providing a microbial production cell of the invention; introducing the cells into a culture medium containing a substrate for the production of said product; recovering the product. The present invention provides a method for the biosynthesis of a product comprising:

[0008] A third aspect of the present invention provides the use of an essential gene operably linked to a load-sensing promoter to enhance product yield of a culture population of microbial production cells arising from a single cell, wherein the promoter is heterologous with respect to the essential gene, and production of a product imparts load to the cell, and expression of the essential gene is upregulated when the load-sensing promoter is induced by the load relative to basal level expression of the essential gene when the load-sensing promoter is not induced.

[0009] A fourth aspect of the invention provides the use of a microbial production cell of the invention to produce a biosynthetic product. [Brief explanation of the drawings]

[0010] [Figure 1]Figure 1: Graph showing the growth over time (measured as optical density: OD630nm) of cells of the parental strain E. coli BL21(DE3) (gray line) and cells of the parental strain transformed with the hGH-GFP-producing plasmid pEG34 (black line; pEG34). Growth of four replicate cultures of each strain was measured under conditions inducing hGH-GFP expression; error bars represent the standard error of the mean (n=4). [Figure 2] Figure 2: Graph showing growth over time (measured as optical density: OD630nm) of cells of the parental strain E. coli BL21(DE3) (black line) and cells of the parental-derived, non-productive load-dependent E. coli strains s5.0#3, s7.0#8, and s9.0#8 (as indicated; essential genes folP-glmM are controlled by pyccV, pycjX, and pmutM, respectively). Measured growth rates are based on the average of four replicate cultures (n=4). [Figure 3] Figure 3: Graph showing the growth over time of cells of the load-dependent strain s9.0#8 carrying either the hGH-GFP-producing plasmid pEG34 (black line) or the control plasmid pEG0 (gray line) (measured as optical density: OD630nm). Growth of four replicate cultures of each strain was measured under conditions inducing hGH-GFP expression; error bars represent the standard error of the mean (n=4). [Figure 4] Figure 4: Graph showing the growth over time (measured as optical density: OD630nm) of cells of the parental strain E. coli BL21(DE3) (black line) and of cells of three load-dependent strains (as indicated): s5.0#3, s7.0#8, and s9.0#8 harboring and expressing the hGH-GFP-producing plasmid pEG34. Growth rates measured under conditions inducing hGH-GFP expression are based on the average of four replicate cultures (n=4). [Figure 5]Figure 5: Histogram of GFP / OD values ​​(Y-axis) measured during the first and sixth seedings (seeds 1 and 6) of cultures of hGH-GFP-producing strains: s3.6 (pibpA), s6.6 (pgrpE), s7.6 (pycjX), and s10.6 (pybbN), which contain their respective load-sensing promoters and one of four variant RBS coding sequences driving the expression of the essential genes folP-glmM. The non-load-dependent E. coli strain BEG34 and the parental strain E. coli BL21(DE3) culture BEG0 are included as controls. Measurements are performed on cultures grown under conditions inducing hGH-GFP expression. Error bars represent the standard error of the mean (n = 4). [Figure 6] Figure 6: Histogram of GFP / OD values ​​(Y axis) measured after successive culture transfers (seeds 1–12) of hGH-GFP-producing strains: s3.6#2 (pibpA), s6.6#6 (pgrpE), s7.6#8 (pycjX), and s10.6#7 (pybbN), each containing a load-sensing promoter and one of four variant RBS coding sequences driving expression of the essential genes folP-glmM. Cultures of the non-load-dependent E. coli strain BEG34 and the parental strain E. coli BL21(DE3) are included as controls. Measurements are performed on cultures grown under conditions inducing hGH-GFP expression. Error bars represent the standard error of the mean (n = 3). [Figure 7] Figure 7: Histogram of OD / min as a measure of growth rate of load-dependent hGH-GFP-producing strains s3.6#2, s6.6#6, s7.6#8, and s10.6#7 compared to the non-load-dependent hGH-GFP-producing E. coli strain BEG34 and the parental strain E. coli BL21-DE3 (BEG0). Growth rates for each strain are based on seven replicate cultures measured under hGH-GFP expression-inducing conditions at seed 0; error bars indicate the standard error of the mean (n = 7). [Figure 8]Figure 8: Graph showing GFP / OD values ​​(Y-axis) measured after successive seedings (seeds 1, 4-9) of cultures of hGH-GFP-producing strains, minus the value measured for the empty plasmid control BEG0-empty strain. Strains designated (A) s7.6#8(pycjX), (B) s13.6#2(pfsxA), and (C) s13.6#2evo(pfsxA), each containing the respective load-sensing promoters driving expression of the essential genes folP-glmM (gray line), and the non-load-dependent hGH-GFP-producing E. coli strain BEG34 (black line). Measurements were performed on cultures grown under conditions inducing hGH-GFP expression. Error bars represent the standard error of the mean (n = 5). [Figure 9] Figure 9: Graph showing GFP / OD values ​​(Y-axis) measured after successive seedings (seeds 1, 4-9) of cultures of hGH-GFP-producing strains, minus the value measured for the empty plasmid control BEG0-empty strain. Strains designated (A) s15.6.7(prrnB) and (B) s16.6.6(prrnE), each containing the respective load-sensing promoters driving expression of the essential genes folP-glmM (gray line), and the non-load-dependent hGH-GFP-producing E. coli strain BEG34 (black line). Measurements were performed on cultures grown under conditions inducing hGH-GFP expression. Error bars represent the standard error of the mean (n = 5). [Figure 10] Figure 10: Graph showing relative hGH-GFP production (Y-axis) measured after serial transfers (seeds 2–5 and 7) of cultures of the hGH-GFP-producing strain s29.6#3, subtracted from the value measured for the corresponding empty plasmid control strain BEG0. Strain s29.6#3 contains the osmotic stress-sensing promoter ppoxB (black squares) driving expression of the essential genes folP-glmM compared to the non-load-dependent hGH-GFP-producing E. coli strain BEG34 (gray dots). Serial passage 7 was analyzed after 72 h. Measurements were performed on cultures grown under conditions inducing hGH-GFP expression. Error bars represent the standard error of the mean (n = 5 for s29.6#3 and n = 8 for BEG34). [Figure 11]Figure 11: Histogram showing the rate of S. aureus lysis per minute normalized to the OD of the E. coli culture as a measure of the rate of secreted lysostaphin production in cultures of the lysostaphin-producing strain s6.4#5, which contains the load-sensing promoter pgrpE and one of four variant RBS coding sequences driving expression of the essential genes folP-glmM, compared to the non-load-dependent lysostaphin-producing E. coli strain BENDU5cam. Rates were measured in cultures (seeds 1 and 7) grown under conditions inducing lysostaphin expression after continuous culture induction. Error bars represent the standard error of the mean (n = 3). [Figure 12] Figure 12: Graph showing mevalonate titers (g / L) synthesized by cultures of the load-dependent mevalonate-producing strains s19.1#1 (pcspD) and s9.1.4 (pmutM) measured after continuous culture transfers (seeds 2, 5, and 6) compared to the parental non-load-dependent mevalonate-producing E. coli strain BL21(DE3)-pMevT. The load-sensing promoters pcspD and pmutM are each combined with one of four variant RBS coding sequences that drive expression of the essential genes folP-glmM. Measurements are performed in cultures grown under conditions inducing MevT expression. Error bars represent the standard error of the mean (n=3). [Figure 13] Figure 13: Bar plot showing human serum albumin (hSA) production titers of cultures of Pichia pastoris strain EGS31 containing a genomically integrated copy of a cDNA version of hSA encoding the ALB1 gene under the control of the PDI1 promoter operably linked to the AOX1 promoter and the kanMX selection gene. Cultures were grown under either selective conditions (dependent activated by 750 μg / mL G418) or nonselective conditions (non-activated dependent). The hSA titer of the "non-activated dependent" culture was set to 100%, and the titer of the "activated dependent" culture is shown as a percentage. [Figure 14]Figures 14A and B: Bar plots showing human serum albumin (hSA) production titers from cultures of a load-dependent version of Pichia pastoris strain EGS31, each containing a genomically integrated copy of a cDNA version of the kanMX selection gene operably fused to the load-responsive promoter FPR2, RPL3, or RPL6A, encoding hSA under the control of the AOX1 promoter. Cultures were grown under either nonselective conditions (non-activated dependence) or selective conditions (activated dependence with either 150 or 750 μg / mL G418) for approximately 30 cell divisions ([A] Seed 1) or after an additional 10 cell divisions ([B] Seed 2). The mean hSA titers are shown as a percentage of the titer in the seed 1 "EGS31" culture. Error bars represent the standard error of the mean (n = 4). DETAILED DESCRIPTION OF THE INVENTION

[0011] Abbreviations, Terms and Definitions: Burden refers to general and / or product-specific metabolic toxicity, metabolic burden, and / or metabolic depletion that microbial cells engineered to synthesize a product experience during production of the product under production conditions, and which results in a fitness cost that can be quantified by measuring the percentage decrease in maximum logarithmic growth rate (along the growth curve) of the cells during production of the product under production conditions compared to parental microbial cells incapable of said production when grown under equivalent production conditions.

[0012] A load-sensing promoter refers to a promoter that is induced by the "load" and, when induced, is capable of upregulating expression of an operably linked gene in a microbial producer cell when cultured under production conditions compared to a mutant derivative of the microbial producer cell that synthesizes essentially no, or at least 50% less, product of the producer cell. Such mutant derivatives include less productive escape mutants isolated after prolonged culture of a cell population derived from the producer cell. Non-limiting examples of load-sensing promoters include the promoters of the E. coli genes htpG, ibpA, clpB, yccV, grpE, ycjX, ldhA, mutM, ybbN, prlC, groES, fxsA, htpX, rrnB, rrnE, cspD, katE, xthA, uspE, gadB, ahpC, katG, grxA, oxyS, poxB, trxC (see Table 2 for details) and their homologue versions in other Gram-positive and Gram-negative bacteria; for Saccharomyces cerevisiae, non-limiting examples of load-sensing promoters include the promoters of the genes KAR2, PDI1, SAA1, FPR2, RPL3, RPL6A, RPL28, OGG1, RAD51, RAD54 (see Table 2 for details). Load-sensing promoters may also be hybrid, recombinant or truncated versions of such native promoters, as long as such promoters still maintain their response to load.

[0013] Load-dependent microbial cells refer to microbial cells that have been engineered to contain a genetic circuit comprising the above-described "load-sensing promoter" operably linked to an essential gene of the cell.

[0014] A load-dependent microbial production cell refers to a microbial cell that has been genetically engineered to synthesize a desired product, and the cell has further been genetically engineered to contain a genetic circuit that includes the "load-sensing promoter" operably linked to an "essential gene" of the cell. The desired product can be a protein encoded by a nucleic acid molecule in the engineered microbial cell or the product of a heterologous pathway encoded by one or more nucleic acid molecules expressed by the cell.

[0015] Desired products of the present invention include, but are not limited to, products that, when synthesized by a genetically engineered microbial production cell, impart a fitness cost (burden) to the production cell. Non-limiting examples include organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, amino acids, peptides, enzymes, therapeutic proteins and their precursors, such as human growth hormone, insulin, glucagon-like peptide-1, monoclonal and polyclonal antibodies, and single-chain fragment antibodies.

[0016] An essential gene (or essential gene operon) refers to a gene or genes in a microbial production cell that, when down-regulated, results in a decreased growth rate of the microbial production cell under production conditions. Preferably, essential genes are essential for growth regardless of the nutrient composition of those production conditions, such that sufficient expression of such essential genes to support cell growth is independent of the presence or absence of particular inhibitors or nutrients provided under production conditions. Non-limiting examples of essential genes in standard laboratory conditions include folP-glmM, glmM, murI, asd, thyA, rpoD, nusG, rpsU, accD, degS, fldA, ftsN, hflB, lolA, mraY, mreD, murA, murB, murF, nadD, rplV, and rpsG, and for Bacillus licheniformis or Bacillus subtilis, glmM, ylaN, infA, and dapA. For Saccharomyces cerevisiae, non-limiting examples include URA3, FOL1, MED7, RRP40, NOP8, PGI1, and NEP1. For example, suitable genes encode enzymes involved in synthesizing cell wall components. Furthermore, essential genes used in the context of the present invention preferably do not encode the desired product expressed by the production strain, nor do they encode proteins that facilitate synthesis of the desired product or its intermediates in a heterologous pathway expressed by the production strain.

[0017] Conditionally essential genes allow load-dependent systems in microbial production cells to be "activated" by culturing the cells under essential conditions, such as the addition of antibiotics or the withdrawal of nutrients.

[0018] The basal expression level of an essential gene is the level of transcription of an "essential gene" (as defined above) operably linked to a "load-sensing promoter" in a load-dependent microbial production cell when the "load-sensing promoter" is not induced. The load-sensing promoter to which the essential gene is operably linked is heterologous with respect to the essential gene in the sense that the promoter is not the native promoter of the essential gene (even though the promoter may be present in the same genome), and is therefore not found operably linked to the essential gene in nature. The basal (i.e., uninduced) level of expression of an essential gene is a level sufficient to support growth of cells under production conditions (or during logarithmic growth phase) at a level that is no greater than 10, 20, 50, 90, or 95% of the growth rate of a corresponding cell in which the essential gene is operably linked to its native promoter.

[0019] Fitness cost: quantified by comparing the maximum logarithmic growth rate of a non-load-dependent microbial production cell (i.e., lacking a load-sensing promoter operably linked to an essential gene but containing and expressing one or more genes encoding the product or encoding a metabolic pathway for the biosynthesis of the product) to the maximum logarithmic growth rate of a parent microbial cell from which the microbial production cell is derived that lacks or is unable to express the gene(s) encoding the product or a metabolic pathway for the biosynthesis of the product when grown under equivalent production conditions.

[0020] Production conditions refer to the specific culture conditions used during production and may relate to the medium and / or other culture conditions (e.g., temperature, pH, agitation, aeration, etc.) for production of the desired product by the microbial production cells.

[0021] Ribosome binding site (RBS), translation initiation region, or translation strength element refers to a region in the 5' untranslated region of a gene that controls the translation strength of a particular messenger RNA.

[0022] Detailed description of the invention: I. Load-dependent microbial production cells A first aspect of the present invention provides a microbial production cell genetically engineered to synthesize a product, comprising: a. An essential gene operably linked to a load-sensing promoter wherein the promoter is heterologous with respect to the essential gene; production of a product imparts a load and / or fitness cost to the cell, and expression of the essential gene is upregulated when the load-sensing promoter is induced by the load and / or fitness cost relative to a basal level of expression of the essential gene when the load-sensing promoter is not induced.

[0023] According to one embodiment, the microbial production cells of the present invention comprise: b. one or more genes encoding a product or encoding a metabolic pathway for the synthesis of the product Including, Optionally, the one or more genes are operably linked to a constitutive or inducible promoter.

[0024] According to one embodiment, the burden and / or fitness cost to the microbial production cell of the invention is quantified by comparing the maximum logarithmic growth rate of a microbial production cell that contains one or more genes encoding the product or encoding a metabolic pathway for the synthesis of the product, but lacks a load-sensing promoter operably linked to an essential gene, to the maximum logarithmic growth rate of a parent microbial cell from which the microbial production cell is derived that lacks or cannot express one or more genes encoding a product or encoding a metabolic pathway for the synthesis of the product, and each cell is cultured under essentially identical production conditions. The burden and / or fitness cost to the microbial production cell preferably corresponds to a percentage reduction in the quantified maximum logarithmic growth rate selected from the group consisting of ≥ 5%, ≥ 10%, ≥ 15%, ≥ 20%, ≥ 25%, ≥ 35%, and ≥ 45%.

[0025] Load-dependent microbial production cells according to the present invention can be any prokaryotic or eukaryotic microorganism, such as bacteria, yeast, and filamentous fungi.

[0026] In one embodiment, the microbial production cells of the present invention are prokaryotes. A non-exhaustive list of suitable bacteria includes species belonging to genera selected from the following: Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Bacillus, Acetobacter, Acinetobacter, Pseudomonas; Proprionibacterium, Bacteroides, and Bifidobacterium.

[0027] In another embodiment, the microbial production cells of the present invention are eukaryotic organisms, such as yeast or fungi. In particular embodiments, the eukaryotic organism may be a member of the genera Saccharomyces, Komagataella, or Aspergillus.

[0028] A non-exhaustive list of suitable yeasts includes yeasts belonging to the genus Saccharomyces, such as S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum; yeasts belonging to the genus Kluyveromyces, such as K. lactis, K. marxianus var. marxianus, K. thermotolerans; yeasts belonging to the genus Candida, such as C. utilis, C. tropicalis, C. albicans, C. lipolyticA, C. versatilis; yeasts belonging to the genus Pichia, such as P. stipidis, P. pastoris, P. sorbitophila, or other yeast genera, such as Cryptococcus, Debaryomyces, Hansenula, Yarrowia, Zygosaccharomyces, or Schizosaccharomyces.

[0029] A non-exhaustive list of suitable filamentous fungi includes those belonging to the genera Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma thermomyces, Streptomyces, and Aspergillus. More specifically, filamentous fungi include Fusarium oxysporum, A. niger, A. awamori, A. oryzae, and A. nidulans.

[0030] The product(s) synthesized by the microbial production cells of the invention incur a cellular and fitness cost reflected in a reduced growth rate, and such product(s) include amino acids, organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, peptides, enzymes, therapeutic proteins such as human growth hormone, insulin, glucagon-like peptide-1, monoclonal and polyclonal antibodies, single-chain antibodies, and their precursors. In one embodiment, the product is not a natural product of the microbial production cells of the invention and, therefore, is not produced by the parent cell from which the microbial production cell is derived. In one embodiment, one or more genes encoding the product or encoding a metabolic pathway for synthesizing the product are heterologous with respect to the microbial production cells of the invention, and the one or more genes may be transgenes.

[0031] II. Characteristics of the Microbial Production Cells of the Invention II.i Fitness Cost-Production Load Heterologous expression of a desired product or a pathway leading to a desired product in microbial cells places a burden on the cells. Burden can also result, for example, in overproduction of a natural product in cells of a microbial strain selected after mutagenesis. This burden can be explained as a fitness cost that slows cellular growth (Example 1; Figure 1). This can be due to general and / or product-specific metabolic toxicity, increased use and / or depletion of potentially finite cellular resources and metabolites, which microbial cells experience during biosynthesis of a product of the invention under production conditions. Particularly during industrial-scale fermentation, the combination of fitness cost and growth delay exerts selective pressure on the cell population within the fermenter, which over time leads to a decline in product yield due to the accumulation of non-producing cells due to genetic or non-genetic variations (BEG34 in Example 2, Figure 6).

[0032] In the present invention, the presence and expression of one or more genes encoding a product or encoding a metabolic pathway for the biosynthesis of a product in a microbial production cell confers a fitness cost (burden).

[0033] The fitness cost of producing a product in a microbial production cell is quantified by comparing the growth rate of a non-load-dependent microbial production cell (i.e., lacking a load-sensing promoter operably linked to an essential gene, but containing and expressing one or more genes encoding the product or encoding a metabolic pathway for the biosynthesis of the product) to the growth rate of a parent microbial cell from which the microbial production cell was derived that lacks the gene(s) encoding the product or encoding a metabolic pathway for the synthesis of the product. The load and / or fitness cost to the microbial production cell preferably corresponds to a percentage decrease in quantified growth rate selected from among ≧5%, ≧10%, ≧15%, ≧20%, ≧25%, ≧35%, and ≧45%, more preferably at least 5%.

[0034] Preferably, the determination of relative growth rate is performed for each microbial cell by measuring their maximum logarithmic growth rate when cultured under essentially identical conditions, these conditions being selected to closely mimic those under which large-scale fermentation will ultimately be carried out.

[0035] II.ii Essential genes The microbial production cells of the present invention contain essential genes that encode proteins whose expression is required for cell growth and / or survival.

[0036] Preferably, the essential gene and its expression does not indirectly cause a decrease in the production of the desired product by the production cell. Furthermore, essential genes used in the context of the present invention do not encode or result in the synthesis of a desired product or intermediate of a heterologous pathway expressed in the microbial production cell.

[0037] The essential gene is preferably an unconditionally essential gene, such that expression of the gene is essential for cell growth and / or survival regardless of the composition or conditions of the growth medium in which the cells are cultured.

[0038] In one embodiment of the invention, when the production cell is a prokaryote such as E. coli, the unconditionally essential genes are selected from folP-glmM, glmM, murI, asd, thyA, usA, rpoD, nusG, rpsU, accD, degS, fldA, ftsN, hflB, lolA, mraY, mreD, murA, murB, murF, nadD, rplV and rpsG.

[0039] In a further embodiment of the invention, when the production cell is a prokaryote, such as a Bacillus strain, the non-conditionally essential genes are selected from glmM, ylaN, infA, and dapA.

[0040] In one embodiment of the invention, the essential gene is a conditionally essential gene that leads to the synthesis of a product required for auxotrophic growth or a protein product required for resistance to growth inhibitors such as antibiotics, specific toxins, protoxins, etc.

[0041] In one embodiment of the invention, when the production cell is eukaryotic, the essential genes in the unconditional case are selected from FFOL1, MED7, RRP40, NOP8, PGI1, NEP1; and in the conditional case are selected from URA3, LEU2, TRP1, HIS3.

[0042] If the essential gene is conditionally essential, it will be necessary to adjust the composition of the growth medium / growth conditions of the producer cells, such as by using a growth medium lacking specific nutrients or by supplementing with a growth inhibitor.

[0043] [Table 1] TIFF0007742611000002.tif242162

[0044] The essentiality of the genes can be determined by creating cells in which the respective genes are knocked out, with unconditional gene knockout resulting in loss of cell viability or inability to grow regardless of growth conditions, while conditional gene knockout results in inability to grow or loss of cell viability depending on the media composition / culture conditions.

[0045] The appropriate essential gene is L-arabinose-inducible p BAD (in E. coli), xylose-induced p XYL (in Bacillus subtilis or Bacillus licheniformis) or galactose-inducible p GAL Essential gene assays can be performed by replacing the native promoter of the assayed essential gene with an inducible promoter, such as αβ (in Saccharomyces cerevisiae or Pichia pastoris) or plac (in Escherichia coli or Lactobacillus). In such essential gene assays, an integrative DNA construct containing an inducible promoter is integrated into the chromosome of the host microbial cell using standard methods. In the case of prokaryotes, the integrative DNA preferably possesses an RBS catalog (e.g., Table 3) that directs translation of the essential gene at different rates to account for the different baseline expression levels of different promoters and the required baseline expression levels of the essential gene to support growth at the corresponding wild-type specific growth rate. After targeted integration of the integrative DNA, the integrant cells are plated on plates supplemented with permissive conditions (temperature or inducer concentration: >0.9% L-arabinose, >0.8% xylose, >1% IPTG, >2% galactose). The cells are then tested for their synthetic dependence on the inducer conditions in a standard growth curve assay, and suitable essential genes can be identified as genes for which inducer-dependent growth rates can be determined. Inducer-dependent growth can be observed as a decrease in growth rate of more than 5 percent in the absence of inducing conditions. Suitable essential genes are characterized by a decrease in log-phase specific growth rate of less than 5% under optimal inducer conditions in a production-related culture medium.

[0046] Methods for identifying essential genes are detailed in the January 2015 publication "Gene Essentiality - Methods and Protocols" [DOI 10.1007 / 978-1-4939-2398-4], which allows those skilled in the art to determine whether a gene is essential for microbial cell growth under specific growth conditions. These methods include methods for mapping and identifying essential genes in Campylobacter jejuni, Streptococcus sanguinis, Porphyromonas gingivalis, Escherichia coli, Leptospira, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Candida albicans, primarily by screening transposon-tagged libraries. Furthermore, various "computational tools" described therein allow those skilled in the art to directly predict and identify genes encoding essential proteins and structural features of many known essential genes in microbial genomes, facilitated by the widespread availability of whole-genome microbial sequences. Thus, those skilled in the art are provided with both databases of essential genes and a variety of freely accessible online tools and algorithms for successfully and reproducibly identifying large numbers of essential genes in the genomes of microbial cells without undue burden. Furthermore, it should be noted that 82% of essential genes in Saccharomyces cerevisiae encode essential proteins that are similar to proteins in another organism [Giaever G et al., 2002], demonstrating that most known or identified essential genes in one microorganism (e.g., yeast) have corresponding genes in other microorganisms and can therefore be identified in a selected microorganism by a simple BLAST search.

[0047] II.iii Load-sensing promoter The microbial production cells of the present invention, as described above, comprise a load-sensing promoter operably linked to an essential gene encoding a protein required for cell growth and / or survival. The load-sensing promoter is one that is induced in microbial production cells that experience a load and / or fitness cost from the production or overproduction of the product. Typically, induction of a load-sensing promoter occurs when the production of a product by the microbial cell results in a decrease in the growth rate of the cell, which in turn results from a fitness cost of production. Importantly, the load-sensing promoter according to the present invention is induced by the "load state" within the production cell resulting from the production of the product, rather than by the product itself. The load-dependence imparted by the present invention supports a wide range of uses for microbial production cells, as the use of load-sensing promoters and essential genes to increase the productivity of microbial production cells is largely independent of the product the cells are engineered to make.

[0048] Because the essential gene is operably linked to a load-sensing promoter, its transcription and expression level increases above basal levels only when the load-sensing promoter is induced. A suitable load-sensing promoter is one that allows for basal level expression of the operably linked essential gene that significantly limits or prevents cell growth and / or survival when not induced. Thus, cells containing a load-sensing promoter in the uninduced state grow significantly slower than cells in which the essential gene is operably linked to its native promoter (Example 1, Figure 2). In contrast, microbial production cells containing a load-sensing promoter linked to an essential gene exhibit a significantly increased growth rate when the cells synthesize a desired product compared to cells that do not produce the product (Example 1, Figure 3). Furthermore, despite the inherent burden of producing the product, the microbial production cells of the present invention can surprisingly achieve initial growth rates indistinguishable from those of parental microbial cells from which the microbial production cells are derived that lack the gene(s) encoding the product or the metabolic pathway for the biosynthesis of the product (Example 1, Figure 4). Thus, under production conditions, the microbial production cells of the invention have a selective advantage over escape mutants of the production cells that stop synthesizing the product, thereby reducing the rate of escape.

[0049] In one embodiment, the load-sensing promoter may be a native promoter or a modified native promoter with respect to the microbial production cell, while being heterologous with respect to the essential gene.

[0050] In one embodiment, the promoter contains a natural TF / sigma factor binding site. Suitable load-sensing promoters are promoters of the E. coli σ32 regulon, as they are generally upregulated in response to overexpression of proteins and their intracellular aggregation. A non-exhaustive list of E. coli σ32 promoters is included in Table 2 herein, and further suitable promoters have been identified by Nonaka et al., 2006.

[0051] In one embodiment, the load-sensing promoter is a σ factor-regulated promoter. In a preferred embodiment, the load-sensing promoter is a σ factor-regulated promoter. 32 It is activated via a regulon and can be selected from among the promoters of the E. coli genes htpG, ibpA, clpB, yccV, grpE, ycjX, ldhA, mutM, ybbN, prlC, groES, fxsA, and htpX. For Bacillus, such suitable load-sensing promoters can be selected from among the HrcA-regulated promoters, including, for example, the promoter of groES.

[0052] In one embodiment, the load-sensing promoter is the promoter of the mutM gene, which encodes a functionally conserved DNA-glycosylase involved in initiating repair of one of the most common oxidative stress-induced DNA damages, namely the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG) (Jain et al., 2007).

[0053] Load-sensing promoters also include promoters related to the carbon nutrient status and growth phase of the cell. Thus, in one embodiment, the load-sensing promoter is the promoter of the toxin gene cspD, which is activated by reduced growth rate and carbon starvation (Uppal et al., 2014; Yamanaka and Inouye, 1997), and is therefore predicted to be differentially upregulated in cells subject to load or fitness costs from metabolite production.

[0054] In one embodiment, the load-sensing promoter is a ribosomal RNA promoter, specifically the promoters of the rrnB and rrnE genes, which are nutrient-responsive, induced by the Fis protein, and inhibited by the ppGpp alarmone. rrn promoter activity is induced in cells with high protein production, as seen in their early logarithmic growth phase (Nonaka et al., 2006).

[0055] In one embodiment, the load-sensing promoter is one that responds to oxidative stress associated with metabolic stress and microbial xenogenesis (Dragosits & Mattanovich, 2013). Oxidative stress-responsive promoters include promoters regulated by OxyR, which activates several genes associated with cellular remediation and protection in response to oxidative damage; and rpoS (σ), which is primarily upregulated during protein overexpression during stationary growth. S ) contains promoters of genes belonging to the

[0056] [Table 2] TIFF0007742611000004.tif242162TIFF0007742611000005.tif242162TIFF0007742611000006.tif150162

[0057] The basal expression levels and specific response curves upon induction / activation of different load-sensing promoters (e.g., in Table 2) vary, which can be used to select the load-sensing promoter that best suits (in terms of strength) a specific microbial production cell and its product.

[0058] Suitable load-sensing promoters induced by load and / or fitness costs in microbial production cells during production can be selected from public databases known to those skilled in the art, e.g., https: / / ecocyc.org for E. coli promoters; https: / / bsubcyc.org for B. subtilis promoters (which also work for B. licheniformis promoters) and their respective prokaryotic homologs; https: / / www.yeastgenome.org for S. cerevisiae promoters and their fungal homologs.

[0059] Suitable load-sensing promoters can also be validated in test assays in which a candidate load-sensing promoter is operably linked to a gene encoding a fluorescent protein (e.g., green fluorescent protein) and integrated into microbial producer cells engineered to synthesize the product, isolated after serial dilution culture experiments in production medium for 50-150 cell generations, and integrated into corresponding non-producer cells (controls). The load-sensing promoter induces detectable fluorescent protein expression in producer cells by at least 5%, 7.5%, 10%, 15%, 25%, 60%, 150%, or 300% compared to non-producer cells (controls).

[0060] II.iv Matching load-sensing promoters to microbial production cells Microbial producer cells exhibit a transcriptional state characterized by the expression of specific genes in response to the load or fitness cost of producing a product, expression of which is not induced in the corresponding non-producing parent cells or non-producing escape cells derived from the microbial producer cells during productive fermentation. The physiological nature of the load or fitness cost depends on the product synthesized by a given microbial producer cell and is reflected in the type of gene whose expression is induced. The identity of gene promoters induced in cells producing a given product can be determined by techniques well known in the art (e.g., transcriptomics; see Example 7). Because many of the induced gene promoters are related to or correspond to those listed in Table 2, they provide a starting point for finding a matching load-sensing promoter for a given microbial producer cell. Promoter selection can be expanded by identifying 5 to 15 promoters of genes found to be specifically up-regulated in the production strain of interest. As illustrated in the examples herein, the method used to product-specifically match load-sensing promoters operably linked to essential genes to microbial producer cells is experimentally rapid and can be performed in a simple laboratory setting.

[0061] II.v Translational control elements Regulating the growth of microbial production cells of the invention by expression of an essential gene requires that the response threshold and response curve of the load-sensing promoter and the expression level of the essential gene be matched; basal levels of essential gene expression support limited or no growth, while in highly productive cells, an induced load-sensing promoter promotes sufficient essential gene expression to support a significantly increased growth rate, preferably a growth rate similar to or ≦5% lower than that of a production cell lacking the load-sensing promoter of the invention, when measured in logarithmic growth phase.

[0062] One suitable approach to balancing the load response of a load-sensing promoter against the expression level of an essential gene is to alter the translation strength of the essential gene. In bacteria, translation strength is defined by the Shine-Dalgarno / ribosome binding site (RBS) sequence immediately upstream of the start codon, whereas in eukaryotic cells, translation strength can be altered using a translation initiation region / Kozak element. RBSs in E. coli that confer a wide range of translation strengths are provided in Table 3, and further examples can be found in the literature (e.g., Bonde et al., 2016). Those skilled in the art can balance the translation strength of a regulated essential gene by constructing four ribosome binding site variants for each load-sensing promoter (Rugbjerg et al., 2018, PNAS) and testing which variant allows effective regulation of cell growth rate by the selected essential gene. Exemplary RBSs for use in Bacillus licheniformis or Bacillus subtilis are provided in Table 3.1.

[0063] [Table 3] [Table 4]

[0064] II.vi Engineering load-sensing promoters and translational control elements The selected natural load-sensing promoter generally falls within the -1 to -300 bp region (upstream) of the natural regulatory ORF in prokaryotes and the -1 to -500 bp region in eukaryotes. The sequence boundaries of core promoters and their regulatory sites, e.g., transcription factor binding sites such as σ32, that must be included are common general knowledge (Nonaka et al., 2006). A translational control element / RBS can be added downstream of the promoter to avoid altering the regulatory properties of the selected promoter sequence.

[0065] II.vii Improved Generation Levels A surprising advantage of the microbial production cells of the invention is that, in addition to having an increased initial growth rate compared to non-producing cells, the cells retain significantly improved productivity during large-scale fermentation over many cell divisions compared to microbial production cells lacking engineered load dependence (simulated in Example 2; Figure 6). The surprising advantage conferred by load dependence in the microbial production cells of the invention is obtained regardless of the type of product synthesized, as exemplified by the range of products synthesized by the microbial production cells in Examples 2-5.

[0066] According to one embodiment, the microbial production cells of the invention are characterized by improved product yield after at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 100, 150, 250, or 400 cell divisions from a single cell compared to non-load-dependent production cells after the same number of cell divisions, where product yield is measured as moles or grams of product produced per unit substrate.

[0067] In one embodiment, production levels are increased by at least 10, 25, 50, or 80% after at least 50 cell divisions from a single cell compared to a reference, non-load-dependent producing cell after the same number of cell divisions.

[0068] A common practice in the operation of microbial cell factories is to minimize the burden known to arise from the maintenance and expression of heterologous pathways. The present invention offers a counterintuitive solution for improving cell factory yields because it makes microbial cell growth and survival dependent on cells that produce their product, while subject to a certain burden or fitness cost. Without wishing to be bound by theory, it is speculated that the selective pressure imposed on the microbial producer cells and their progeny of the present invention, where only producer cells can grow / survive under conditions of burden, may serve to progressively select for highly burdened cell subpopulations during long-term culture in a starting population of isogenic cells.

[0069] III. Methods for Preparing and Identifying Cells of Load-Dependent Microbial Production Strains In a preferred method of carrying out the present invention, a load-dependent microbial production strain is prepared and identified by the following steps: To obtain the best functional relationship between the load-sensing promoter and the essential gene, it may be appropriate to prepare and test different candidate load-sensing promoters with different RBSs for regulating the selected essential gene.

[0070] III.i Identification of load-sensing promoter candidate(s) in specific production strains The candidate load-sensing promoters listed in Table 2 are tested. Alternatively, unique, positively differentiating gene transcripts detected in a specific production strain of interest are identified; their respective promoters provide a source of candidate load-sensing promoters. Preferably, such differentiating gene transcripts are identified by comparing the transcript profile of a microbial production strain (in production) with isolated gene or non-gene escape mutant variant(s) from the corresponding production strain, where such escape mutant variants are characterized by a production rate that is up to 50% lower. Positively differentiating gene transcripts can be identified by using transcriptomics, for example, by RNA sequencing of transcribed RNA extracted from highly productive microbial production cell(s) compared to non-producing / low-producing escape mutant variants. The identified promoters may be tested in combination with a given essential gene in microbial production cells.

[0071] A method for identifying one or more load-sensing promoters is exemplified in Example 7, in which the promoter(s) are operably linked to essential genes in the microbial production cells selected for testing.

[0072] III.ii Introduction of load-sensing promoters in growth regulation processes One or more promoter(s) selected from the list of candidate general load-sensing promoters (Table 2) or from the identified specific load-sensing promoter(s) (see IIIi) are tested by operably linking it to an essential gene in the microbial production cell. Different translation strengths of essential genes can be tested simultaneously by providing alternative RBS sequences to the cognate essential gene and testing different essential genes (as described in section IIii). The essential gene can be native or heterologous with respect to the microbial production cell.

[0073] If the essential gene is a native gene, its cognate native promoter may be disrupted (i.e., rendered non-functional), for example, by a mutation or deletion event, and a load-sensing promoter (i.e., heterologous with respect to the essential gene) is then operably linked to the native essential gene by targeted introduction. In another embodiment, the native essential gene promoter is replaced with the load-sensing promoter by targeted introduction.

[0074] Suitable methods for targeted introduction of gene sequences into microorganisms include recombination in bacteria, for example, lambda red recombination in E. coli, while homologous recombination can be used in yeast and filamentous fungi. Both concepts can be used, optionally in combination with CRISPR, to increase the efficiency of gene replacement.

[0075] III.iii Screening for balanced load sensing and growth regulation The microbial production cell clones containing the inserted load-sensing promoter operably linked to an essential gene are then screened to identify clones in which the load-sensing promoter regulates the essential gene in a growth-regulated manner. For example, the growth of a number of such clones (e.g., 8-96 clones) is compared to cells of the corresponding non-load-dependent microbial production strain under conditions in which product production is controlled (e.g., using a microbial production strain in which the production gene is inducible).

[0076] When cell growth is measured under conditions where product production by both strains is low / absent, suitable load-dependent production clones are those that exhibit a growth rate significantly lower, preferably at least 5% lower, than the non-load-dependent production strain (Example 1, Figure 2).

[0077] III.iv Verification of unperturbed central and product metabolism Alterations in transcriptional regulation or expression of essential genes can result in undesirable indirect perturbations of production genes and central carbon or nitrogen metabolism, impairing product formation and thus reducing the load in load-dependent microbial production cells. To exclude such clones, cell growth is measured under conditions in which both strains synthesize the product. Suitable load-dependent production clones are those that exhibit growth rates comparable to or lower than the non-load-dependent production strain (as seen in Example 2, Figure 7).

[0078] III.v Growth rate stability screening Clones that meet the above criteria are tested for growth rate stability under production-mimicking conditions by culturing for at least 20 cell doublings, e.g., by serial passaging and measuring growth rates, or by sampling from various steps of a scale-up production process. Suitable load-dependent microbial production cells maintain a lower growth rate over time than non-load-dependent production strains (e.g., Example 2, Figure 7).

[0079] IV. Methods for Producing Desired Products Using Cells of Load-Dependent Microbial Production Strains A second aspect of the present invention is a. providing a microbial production cell genetically engineered to synthesize a product, said cell further comprising an essential gene operably linked to a load-sensing promoter, said promoter being heterologous with respect to said essential gene; b. introducing the genetically modified microbial cells into a culture medium containing a production substrate for the product; c. recovering the product synthesized by the culture; 1. A method for producing a desired product comprising: wherein synthesis of a product imposes a load and / or fitness cost on the cell, expression of the essential gene is upregulated relative to a basal level of expression of the essential gene when the load-sensing promoter is induced by the load and / or fitness cost, and lack of product synthesis in the load-dependent production strain or its progeny cells reduces the growth rate of the cell.

[0080] IV. USE OF CELLS OF LOAD-DEPENDENT MICROBIAL PRODUCTION STRAINS TO PRODUCE A DESIRED PRODUCT A third aspect of the invention relates to the use of a load-dependent microbial production cell of the invention to produce a desired product, wherein the lack of product synthesis in said load-dependent production strain or its progeny reduces the growth rate of said strain.

[0081] Example Example 1: Engineering a load-dependent E. coli strain that produces recombinant human growth hormone 1.1 Maintenance and expression of heterologous genes in cell factories places an unnatural burden on cells The growth rate of parental E. coli BL21(DE3) cells was compared with that of an engineered derivative transformed with the plasmid pEG34, which contains the gene encoding recombinant human growth hormone (hGH) fused to green fluorescent protein (GFP).

[0082] 1.1.1 Materials and Methods Cells of the host strain E. coli BL21(DE3) (E. coli Genetic Stock Center, Yale University) were made electroporable and transformed with the plasmid pEG34 by standard electroporation methods (1800 V, 25 μF, 200 Ohms, 1 mm cuvette width) and plated on LB agar plates containing chloramphenicol.

[0083] [Table 5]

[0084] Precultures of single-colony transformants were grown in 96-well microtiter plates on 200 μL of 2xYT medium (16 g / L tryptone, 5 g / L NaCl, 10 g / L yeast extract) containing 500 μM IPTG and 30 mg / L chloramphenicol at 37°C with rapid horizontal shaking in an ELx808 plate reader (Biotek) with OD readings every 10 minutes. OD readings were background subtracted using the initial OD reading.

[0085] 1.1.2 Results E. coli cells containing the hGH-GFP expression plasmid pEG34 grew slower than the parental E. coli host cells from which they were derived (Figure 1), illustrating the burden or load imposed on the cells as a result of allocating cellular resources to the maintenance and expression of the synthetic construct.

[0086] 1.2 Operation of load-dependent cell factories A load-dependent strain of the parent E. coli host was engineered to incorporate a genetic circuit designed to confer a selective fitness advantage to the production cells within the cell factory. Specifically, a load-sensing promoter (p mutM , p yccV or p ycjX ) replaced the native promoter of the essential gene operon folP-glmM in the E. coli BL21(DE3) chromosome. The gene circuit also contained a RBS between the promoter and the essential gene, and four different RBSs (Table 3) were tested for regulating the expression level of the essential gene. The growth rate of the strain containing the gene circuit was compared to that of the parent host strain E. coli BL21(DE3).

[0087] 1.2.1 Materials and Methods Load-sensing promoter: promoter p mutM , p yccV or p ycjXThe genes were generated by PCR amplification of a 0.3 kb region immediately upstream of each gene (see Table 3) using the primers identified in Table 5 and genomic DNA from lysed E. coli BL21(DE3) cells as template. PCR mix: 10 μl MQ water, 2 μl forward primer (10 μM), 2 μl reverse primer (10 μM), 1 μl DNA template, 15 μl Phusion U MasterMix (Thermo Scientific). PCR reaction protocol: 95°C for 180 s (1×); 95°C for 20 s, 68–58°C (touchdown) for 30 s, 72°C for 60 s (35×); 72°C for 300 s (1×); and stand at 15°C.

[0088] [Table 6]

[0089] USER cloning was used to generate an integration sequence containing an amplified promoter region fused to a linear 1.5 kb DNA fragment containing the kanR gene for selection of correct recombinant products and a 221-bp target sequence identical to the 221-bp immediately upstream of the folP gene. The amplified promoter region and kanR-folP fragment were fused by mixing equimolar amounts and adding 1 μl of 10× T4 ligation buffer (Thermo Scientific) and 0.75 μl of USER enzyme (New England Biolabs) for a total reaction volume of 10 μl. The USER reaction was incubated at 37°C for 30 minutes. The reaction was then incubated at room temperature for 15 minutes, followed by the addition of 0.75 μl of T4 DNA ligase (Thermo Scientific) and incubation at room temperature for 30 minutes. An example of such an integration sequence containing the promoter, KanR resistance gene, and folP target sequence can be found in SEQ ID NO: 140 (s9_pmutM_folP) in the Sequence Listing. The ligated product was then amplified to approximately 250 ng / μl using primers "rev" (specific promoter-specific, Table 5) and P493 (Table 5) using the following PCR reaction protocol: 98°C for 180 s (1x); 85°C for 20 s, 72-68°C (touchdown) for 30 s, 72°C for 60 s (35x); 72°C for 5 min (1x); and stand at 15°C. The primer overhang provided a 50-bp folP identity target sequence to direct recombination into the folP locus.

[0090] Chromosomal integration of the load-dependent promoter: The promoter was integrated upstream of the folP gene into the genome of E. coli BL21(DE3) cells as follows: 100 ml of 2xYT medium containing tetracycline was inoculated with 600 μl of an overnight culture of BL21(DE3) previously transformed with pSIM5-tet (Koskiniemi et al., 2011). The cells were grown at 30°C, and when they reached an OD600 of 0.20, the culture was transferred to a shaking chamber at 42°C for 15 minutes to allow expression of the recombinant enzyme located in pSIM5-tet. The culture was then transferred to 2 x cold 50 ml centrifuge tubes and centrifuged at 4000 g for 10 minutes. The supernatant was discarded, and the remaining cell pellet was washed with 20 ml of ice-cold 10% glycerol. The partially resuspended cells were then centrifuged at 4000 g for 6 minutes. The supernatant was discarded, and the cell pellet was washed with 20 ml of ice-cold 10% glycerol. The partially resuspended cells were centrifuged at 4000 g for 6 minutes. Each cell pellet was carefully resuspended in 495 μl of ice-cold 10% glycerol and pooled. 90 μl of the resuspended cells were added to electroporation cuvettes, each containing 1 μl (>250 ng) of one load-sensing promoter integration sequence. The cells were electroporated at the following settings: 1800 V, 25 μF, 200 Ohms, 1 mm cuvette width. 900 ml of 2xYT medium was added to the cuvette immediately after electroporation, and the cells were allowed to recover for 1.5 hours at 37°C in a 1.5 ml Eppendorf tube. Electroporated cells were incubated overnight at room temperature to allow time for recombination; then, they were plated onto LB agar plates containing 50 mg / L kanamycin and grown overnight at 37°C to ensure the integrity of pSIM5-tet. Correct targeting to the essential locus was verified using primers P525 and P526. Generally, a single kanamycin R Colonies were selected that had an average size or a size smaller than the population of colonies.

[0091] Load-dependent promoter integration in the genomes of kanR-selected colonies was verified by colony PCR using Taq DNA polymerase and primers targeting the folP promoter region. Furthermore, the identity of the RBS in selected colonies was determined by Sanger sequencing.

[0092] The growth rate of strains containing the gene circuits was then compared to the parent host strain E. coli BL21(DE3) by culturing them in 200 μl of 2×YT supplemented with 500 μM IPTG at 37°C with horizontal shaking.

[0093] 1.2.2 Results The growth rates of the selected load-dependent E. coli strains were slowed to various degrees compared to the parental E. coli host from which they were derived (Figure 2; growth of load-dependent strains s5.0#3, s7.0#8, and s9.0#8 is shown, and the essential genes folP-glmM are expressed by the load-sensing promoter p yccV , p ycjX , and p mutM Because cell growth depends on the expression levels of the essential folP-glmM genes, it can be concluded that the levels of essential gene expression driven by the respective load-sensing promoters in the non-productive load-dependent strain are insufficient to support growth at the level of the wild-type parent strain.

[0094] The observed reduction in growth rate in these load-dependent strains provides a measure of the penalty that can be imposed on non-producing cells that spontaneously evolve during culture of a cell factory population containing a load-dependent gene circuit. The degree of the penalty is determined by the choice of the load-sensing promoter in combination with the strength of the selected RBS. The greater the penalty, the wider the window of "negative selection" for low-producing or non-producing variants (e.g., due to mutations in producing genes) that spontaneously arise during culture.

[0095] 1.3 Generation increases the growth rate of load-dependent cell factories A load-dependent E. coli strain that confers a growth penalty for nonproduction (Fig. 2) was used as a host strain to demonstrate a selective fitness advantage for highly productive load-dependent cells.

[0096] 1.3.1 Materials and Methods The slowest-growing clones of the resulting load-dependent E. coli strains (s9.0#8; s5.0#3; and s7.0#8) were electroporated and transformed with pEG34 or pEG0 (Table 4), respectively, using standard electroporation techniques (1800 V, 25 μF, 200 Ω, 1 mm cuvette width) and plated onto LB agar plates containing chloramphenicol and kanamycin. Single-colony transformants were grown overnight in 2xYT medium containing chloramphenicol and then used to inoculate 96-well microtiter plates containing 200 μL of 2xYT medium (500 μM IPTG, chloramphenicol) and incubated at 37°C with rapid horizontal shaking in an ELx808 plate reader (Biotek), with OD630 readings every 10 minutes. Background subtraction was performed using the initial OD630 reading.

[0097] The E. coli strains were transformed with the plasmids pEG34 or pEG0 (Table 4), respectively, and their growth rate characteristics were measured when the cells were induced to synthesize the recombinant protein hGH fused to GFP.

[0098] 1.3.2 Results The load-sensing promoter p controls the expression of the folP-glmM essential genes mutM The load-dependent strain, E. coli strain s9.0#8, containing the β-glucanase (β-glucanase), showed the slowest growth of the strains tested when compared to the parent E. coli BL21(DE3) strain (Figure 2). This decrease in logarithmic growth rate was reversed in cells of E. coli strain s9.0#8 transformed with the pEG34 plasmid and induced to express the recombinant protein hGH-GFP, in contrast to cells of E. coli strain s9.0#8 transformed with the empty plasmid BEG0 (Figure 3). This was due to the load-sensing promoter p mutMis induced by the cellular burden or load brought about by the synthesis of the recombinant protein hGH-GFP, which in turn leads to upregulated expression of the folP-glmM genes and enhanced logarithmic growth.

[0099] Synthesis of hGH-GFP in each of the load-dependent E. coli strains s5.0#3, s7.0#8, and s9.0#8 harboring the pEG34 plasmid not only resulted in a reversal of the decline in logarithmic growth rate, but furthermore, the growth rates of these strains were indistinguishable from that of the parental E. coli BL21(DE3) (Fig. 4 ).

[0100] In summary, this example demonstrates that load-dependent gene circuits can be used to confer a selective fitness advantage to those cells in a cell factory whose protein or metabolite synthesis is high enough to constitute a burden or load, and that this burden or load can be detected by a load-sensing promoter operably linked to an essential gene within the cells. In contrast, non-producing variant cells (e.g., resulting from a producing gene mutation) that spontaneously emerge during culture of a load-dependent cell factory are subjected to negative selection pressure as their growth slows to a rate supported by basal expression of essential genes. The reduced growth rate is itself sufficient to slow the increase in frequency of such variant cells in the cell factory, thereby slowing the decline in cell factory productivity over time.

[0101] Example 2: Load-dependent E. coli strains producing human growth hormone exhibit enhanced long-term production stability Load-sensing promoters have been shown to be promoters that can sense and activate the load-induced state within cells resulting from cellular synthesis of the recombinant protein hGH-GFP. Once activated, load-sensing promoters have been shown to elevate expression of the essential genes folP-glmM to levels sufficient to confer a selective growth advantage to cells compared to non-producing cells. To maximize the dynamic range of essential gene expression in response to their cognate load-sensing promoters, load-sensing promoters are randomly combined with variant RBS coding sequences (Table 3) that confer different translational strengths.

[0102] Promoters with load-sensing properties suitable for use in load-dependent gene circuits are shown to include heat shock promoters, DNA damage responsive promoters, oxidative stress responsive promoters, and rRNA promoters, as exemplified by the following engineered production strains cultured under simulated large-scale production conditions:

[0103] First, a number of clones carrying random variant RBS coding sequences were selected for each of the load-sensing promoters, and their hGH-GFP synthesis was followed over many cell divisions to demonstrate their relative ability to increase / preserve recombinant hGH-GFP synthesis over time.

[0104] 2.1 Materials and Methods Load-sensing promoter: promoter p yccV , p ycjX , p ibpA , p grpE , p ldhA and p ybbN , and their respective integration sequences were generated by PCR and USER cloning as described in Example 1.2.1 using the primers specified in Table 6, while the ligation products were amplified using primers "rev" (with specific promoters in Table 6) and P493 (Table 5).

[0105] [Table 7] TIFF0007742611000012.tif121162

[0106] Chromosomal integration of load-dependent promoters: Each promoter was integrated upstream of the folP gene in the genome of E. coli strain BEG34 (corresponding to E. coli BL21(DE3) harboring plasmid pEG34) as follows. Cells of E. coli strain BEG34, previously transformed with recombinant plasmid pSIM5-tet, were prepared and transformed by electroporation with each of the promoter integration sequences as described in Example 1.2.1. After the described pSIM5-tet recombination and curing steps, eight colonies corresponding to eight clones with the same promoter integration but with random variant RBS sequences were picked from each plate (see Table 6). Each clone was then transferred to a well of a 96-well plate containing 200 μl of 2xYT supplemented with chloramphenicol for maintenance of plasmid pEG34. 2 μl of each culture clone was used to verify promoter integration via colony PCR as described in Example 1.2.1 before freezing the 96-well plates.

[0107] Short-term hGH-GFP production screening assay: Frozen 96-well plates were thawed, and cells were transferred using a pin replicator to a new 96-well plate containing 200 μL of 2xYT supplemented with chloramphenicol. The plate was sealed with a Breathe-Easy sealing membrane (Sigma-Aldrich) and placed in a Synergy H1 plate reader (Biotek) at 37°C and 754 rpm for 20 hours with linear shaking overnight. The OD (600 nm) and GFP fluorescence (ex / em 485 nm / 528 nm) of each well were measured every 10 minutes over the 20 hours. The plate was then placed on a conventional benchtop plate shaker at room temperature for 4 hours, after which 2 μL of culture from each well was transferred to a new 96-well plate containing 200 μL of 2xYT supplemented with chloramphenicol and 0.5 mM IPTG. A new 96-well plate was similarly sealed with Breathe-Easy sealing membrane (Sigma-Aldrich) and placed in a Synergy H1 plate reader at 37°C with a linear shake of 754 rpm for 20 hours overnight. The OD and GFP fluorescence of each well were measured at 600 nm and ex / em 485 nm / 528 nm every 10 minutes for 20 hours. The following day, 2 μL of the culture was transferred to a new 96-well plate containing 200 μL of 2xYT supplemented with chloramphenicol and 0.5 mM IPTG. This process was repeated daily for a total of 6 days.

[0108] Long-term hGH-GFP production assay (Figure 6): A single colony selected from each load-dependent hGH-GFP-producing strain was used to inoculate a 15 mL Greiner culture tube containing 4 mL of 2xYT supplemented with chloramphenicol and 0.5 mM IPTG. The cultures were grown at 37°C on a shaking table at 250 rpm for 23 hours. 2 μL of each culture was then inoculated into a new Greiner culture tube under the same conditions at a 2000-fold dilution (corresponding to approximately 11 cell divisions), and these process steps were repeated for a total of 12 seeds. After each passage, a 200 μL sample of the culture was taken to determine hGH-GFP synthesis by measuring OD600 and GFP fluorescence (ex / em 485 nm / 528 nm) on a Syngergy H1 plate reader (Biotek).

[0109] Long-term hGH-GFP production assay (Figure 8): A single colony selected from each load-dependent hGH-GFP-producing strain was used to inoculate 24-deep-well plates containing 1.8 mL of 2xYT supplemented with chloramphenicol and 0.5 mM IPTG. The cultures were grown at 30°C on a shaking table at 200 rpm for 23 hours. 2 μL of each culture was then inoculated into a new deep-well plate under the same conditions at a 1:1000 dilution (corresponding to approximately 10 cell divisions), and these process steps were repeated for a total of 9 seedings. After each passage, a 200 μL sample of the culture was taken to determine hGH-GFP synthesis by measuring OD600 and GFP fluorescence (ex / em 485 nm / 528 nm) on a Syngergy H1 plate reader (Biotek).

[0110] Growth rate measurement of selected strains: The selected high-hGH-producing strains, s3.6#2, s6.6#6, s7.6#8, and s10.6#7, were streaked onto LB agar plates containing chloramphenicol and kanamycin. Strains BEG34 and BEG0 were streaked onto LB agar plates containing chloramphenicol. Seven colonies from each plate were used to inoculate 96-well plates containing 200 μl of 2xYT supplemented with chloramphenicol, which were then sealed with Breathe-Easy sealing membranes (Sigma-Aldrich). Growth was measured overnight at 37°C and 754 rpm with a linear shaker in a Syngergy H1 plate reader (Biotek). A 2 μl sample from each well was transferred to a new 96-well plate containing 200 μl of 2xYT supplemented with chloramphenicol and 0.5 mM IPTG. The 96-well plates were sealed with Breathe-Easy film and incubated at 37°C with a linear shake of 754 rpm for 20 hours, and growth (OD600nm) was measured every 10 minutes using a Syngergy H1 plate reader.

[0111] Strain catalog: BEG34 = E. coli BL21(DE3) harboring pEG34; BEG0 = E. coli BL21(DE3) harboring the empty plasmid pEG0 sX.Z#Y = E. coli BL21(DE3) carrying pEG34 with the wt folP promoter replaced with the load-dependent promoter X, where X refers to the promoter ID in Table 2. The number Y after the # indicates a clone selected from a pool of four RBS variants introduced by degenerate primers (Table 7). Z refers to the genes encoding hGH-GFP (0 ​​or 6), mevalonate (1), and lysostaphin (4), respectively.

[0112] [Table 8]

[0113] 2.2 Results 2.2.1 Short-term hGH-GFP productivity screening: Load-sensing promoters (p) selected from the group of heat shock promoters ibpA , p grpE , p ycjX , and p ybbN The productivity of four hGH-GFP producing strains containing the RBS (p3.6#1) and carrying one of four RBS coding sequence variants was tested under simulated large-scale production conditions as follows. These strains were serially passaged by 100-fold inverse dilution daily for six consecutive days, which corresponds to approximately six generations per seed. By day 6 (seed 6), several strains showed elevated hGH-GFP synthesis compared to the non-load-dependent producing strain BEG34, both between seed 1 and seed 6. A strain with higher short-term hGH-GFP productivity compared to the BEG34 strain was s3.6#2 (p ibpA ), s6.6#6(p grpE ), s7.6#8(p ycjX ), and s10.6#7(p ybbN )—see Figure 5.

[0114] 2.2.2 Improved Long-Term hGH-GFP Productivity: The hGH-GFP productivity of the load-dependent strains s3.6#2, s6.6#6, s7.6#8, and s10.6#7, as well as the non-load-dependent strains BEG34 and non-producing strain BEG0, was monitored under simulated large-scale fermentation with continuous transfer. After seed 2, the load-dependent strains s3.6#2, s6.6#6, s7.6#8, and s10.6#7 and the non-load-dependent control strain BEG34 performed similarly well in terms of hGH-GFP synthesis (Figure 6). However, after approximately 20 further generations (seed 4), hGH-GFP productivity by the control strain BEG34 declined significantly. By seed 6, the strain had essentially ceased production. Surprisingly, the load-dependent strain s7.6#8 (p controlling folP-glmM) showed no significant hGH-GFP production. ycjX The load-dependent strain s3.6#2 (containing the folP-glmM-controlling p ibpA s10.6#7 (having folP-glmM controlling p ybbN s6.6#6 (having folP-glmM controlling pgrpE ) also showed significantly better long-term productivity compared to the non-load-dependent strain BEG34.

[0115] 2.2.3 Growth rates of load-dependent strains: The growth rates of the hGH-GFP producing load-dependent strains s3.6#2, s6.6#6, s7.6#8, and s10.6#7 were not higher than the non-load-dependent hGH-GFP producing strain BEG34, and therefore their improved hGH-GFP production levels over time were not simply due to a lower initial production level and therefore an inherently lower load (Figure 7).

[0116] 2.2.4 Dependence of the load-dependent gene circuit on selected essential genes: the heat shock promoter p controlling the essential genes folP-glmM fxsA We compared the load-dependent hGH-GFP-producing strain (s13.6#2(fxsA)) containing glmM to a mutant derivative strain, s13.6#2evo(fxsA), containing a frameshifted folP gene that abolishes folP expression, whose growth was solely dependent on glmM expression. Although loss of folP expression resulted in a lower growth rate in complex 2xYT medium (data not shown), the essential gene glmM alone was shown to be sufficient to confer improved long-term hGH-GFP production stability in the load-dependent strain s13.6#2evo(fxsA), comparable to that of strain s7.6#8(pycjX), when compared to the non-load-dependent hGH-GFP-producing strain BEG34 (Figure 8).

[0117] 2.2.5 Use of ribosomal RNA promoters in load-dependent gene circuits: In addition to heat shock promoters, p rrnB and p rrnE It has been shown that ribosomal RNA promoters such as ribosomal RNA promoters can sense and respond to load-dependent conditions within cells to control essential gene expression, such as ribosomal RNA promoters, enhancing long-term productivity in hGH-GFP producing strains. As can be seen in Figure 9, strain s15.6.7 (p rrnB ) and s16.6.6(p rrnEThe long-term stability of hGH-GFP production in E. coli strain BEG34 was significantly improved under simulated large-scale production, equivalent to approximately 110 cell divisions, when compared to the non-load-dependent hGH-GFP-producing E. coli strain BEG34.

[0118] 2.2.6 Use of oxidative stress-sensing promoters in load-dependent gene circuits:p poxB It has been shown that oxidative stress-sensing promoters such as s29.6#3(p) can sense stress-dependent conditions within cells and respond to them to control essential gene expression, such as enhancing long-term productivity in hGH-GFP-producing strains. As can be seen in Figure 10, s29.6#3(p) poxB Both the production level and long-term stability of hGH-GFP production in the ) strain were significantly increased when compared to the non-load-dependent hGH-GFP-producing E. coli strain BEG34 under simulated large-scale production and extended cultivation of the final seed for seven serial passages, equivalent to approximately 90 generations. As can be seen in Figure 10, p poxB Generation using a load-dependent genetic circuit based on surprisingly results in increased production over time, indicating enrichment of non-genic high-performing variants in the population.

[0119] In summary, this example demonstrates that load dependence enhances long-term stability and production in an E. coli strain engineered to synthesize human growth hormone fused to GFP by linking the essential folP-glmM transcripts to one of the E. coli heat shock promoter, oxidative stress-responsive promoter, DNA damage-responsive promoter, and rRNA promoter.

[0120] Example 3: Load-dependent E. coli strains producing lysostaphin exhibit enhanced long-term production stability Lysostaphin is a 27 kDa endopeptidase that cleaves the cross-linked pentaglycine bridge in the cell wall peptidoglycan of Staphylococcus aureus, leading to cell lysis. This antibacterial agent can be recombinantly synthesized in Escherichia coli. Load-dependent gene circuits containing promoters with load-sensing properties have been shown to enhance the production stability of such E. coli strains engineered to synthesize lysostaphin, as demonstrated by the following load-dependent E. coli strains that produce lysostaphin under simulated large-scale production conditions. Productivity of load-dependent production strains has been further optimized by selecting strains in which the load-sensing promoter is combined with an RBS sequence (Table 6) to confer optimized translation strength of the cognate essential gene. These advantages are reflected in the development of the load-dependent strain s6.4#5 (p grpE ) is exemplified by

[0121] 3.1 Materials and Methods Load-sensing promoter: Promoter p grpE The promoter integration sequence containing the p grpE PCR was performed using specific primers. grpE The promoter was amplified as described in Example 1.2.1.

[0122] Chromosomal integration of a load-dependent promoter: The grpE promoter was integrated upstream of the folP-glmM genes in the genome of the E. coli strain BENDU5cam (corresponding to E. coli BL21(DE3) harboring the lysostaphin-producing plasmid pENDU5cam) as follows: Cells of the E. coli strain BENDU5cam, previously transformed with the recombinant plasmid pSIM5-tet, were prepared as described in Example 1.2.1, and p grpEThe plasmid pENDU5cam was transformed by electroporation with the promoter integration sequence. After the described steps of recombination and curing of pSIM5-tet, five colonies were selected, corresponding to clones with the same promoter integration but with random variant RBS sequences (see Table 6). Each clone was then transferred and cultured in a 15 mL Greiner culture tube containing 4 mL of 2xYT supplemented with chloramphenicol for maintenance of the plasmid pENDU5cam. 2 μl of each cultured clone was used to verify promoter integration as described in Example 1.2.1 before freezing.

[0123] Lysostaphin production screening: Escherichia coli strain s6.4#5(p) with the highest lysostaphin productivity grpE ) compared with the non-load-dependent production strain BENDU5cam, and the RBS and p grpE Three other potential combinations of the strains were identified using a screening assay in which strains were cultured in 2xYT medium supplemented with chloramphenicol in a 96-well format over six serial passages (equivalent to 60 generations) at 1:1000 dilutions. Strains were grown in 3 mL of 2xYT medium containing 30 mg / L chloramphenicol in 15 mL culture tubes for 21 hours at 37°C. 150 μL of the culture was mixed with 50 μL of 50% glycerol and stored in a 96-well plate at -80°C. An additional 30 μL of the culture was transferred to 3 mL of fresh 2xYT medium supplemented with chloramphenicol and grown under the same conditions for an additional 21 hours. This transfer was repeated five times, with frozen stocks being made from the overnight cultures at each transfer.

[0124] Lysostaphin Expression and Detection Assay: A 96-well plate containing all frozen stocks from five transfers was thawed on ice. 20 μL from each well was transferred to a 96-well plate containing 180 μL of 2xYT supplemented with chloramphenicol, and the cultures were grown at 37°C in a plate reader until most wells reached an OD of 0.35. Then, 10 μL of 2xYT supplemented with 30 mg / L chloramphenicol and 20 mM IPTG was added to each well to a final concentration of 1 mM IPTG, sufficient to induce lysostaphin gene expression. The induced culture was grown at 37°C for 4 hours, and the plate was then centrifuged at 4000 RPM for 10 minutes. 100 μL of the supernatant was transferred to a new 96-well plate containing 100 μL of an overnight culture of S. aureus. Lysis of S. aureus in each well was monitored at OD630 every 10 minutes for 2 hours at 37° C. in a plate reader. The rate of S. aureus lysis was quantified by dividing the change in OD of S. aureus by the 60 minute time frame in which the change occurred. The equation can be seen below:

number

[0125] Specific lysostaphin synthesis rates were determined by normalizing the measured rates to the final OD630 reading of each production E. coli culture.

[0126] 3.2 Results p grpE A load-dependent lysostaphin-producing strain harboring one of four RBS coding sequence variants controlling the expression of the promoter and essential genes folP-glmM was cultivated under simulated large-scale production conditions achieved by serial passaging. As can be seen in Figure 11, strain s6.4#5 (p grpE ) maintained significantly higher levels of lysostaphin production compared to the sharp decline in production seen in the non-load-dependent lysostaphin-producing BENDU5cam strain. grpE) and BENDU5cam strains had the same initial lysostaphin production rate. grpE The smaller decrease in the production rate of ) is not due to the inherently lower initial load from lysostaphin production.

[0127] Summary: Load-dependent expression of the E. coli heat shock promoter p grpE We demonstrate that linking the transcription of the essential genes folP-glmM to the lysostaphin gene enhances long-term stability and production in E. coli engineered to synthesize secreted lysostaphin.

[0128] Example 4: Load-dependent E. coli strains producing mevalonate exhibit enhanced long-term production stability E. coli BL21(DE3) cells were engineered to synthesize mevalonate by introducing the plasmid pMevT, which expresses a heterologous three-step enzymatic pathway (Martin et al., 2003) that converts glucose to mevalonate via the acetyl-CoA pool. Load-dependent gene circuits containing promoters with load-sensing properties and controlling transcription of essential genes have been shown to enhance the production stability of such mevalonate-producing E. coli strains under simulated large-scale production conditions. Productivity of load-dependent production strains has been shown to be further optimized by combining load-sensing promoters with RBS coding sequences (Table 5) and selecting strains that confer optimized translation strength of the cognate essential genes. Specifically, the load-dependent promoter p, which controls folP-glmM transcription, cspD (oxidative stress and glucose starvation sensing promoter), or p mutM Both mevalonate-producing strains containing (heat shock / DNA damage sensing promoters) have been shown to be more resilient to production loss than non-load-dependent strains due to their dependence on transcriptional signals resulting from mevalonate production.

[0129] 4.1 Materials and Methods Load-sensing promoter: Promoter p mutM and p cspDThe promoter integration sequence containing the p mutM and p cspD PCR was performed using specific primers. mutM and p cspD It was generated by amplifying the promoter.

[0130] [Table 9]

[0131] Chromosomal integration of load-dependent promoters:p mutM and p cspD were individually integrated upstream of the folP-glmM genes in the genome of E. coli BL21(DE3)pMevT cells harboring the lysostaphin-producing plasmid pMevT, as follows. Cells of the E. coli strain BL21(DE3)pMevT, previously transformed with the recombinant plasmid pSIM5-tet, were prepared and transformed by electroporation with each promoter integration sequence, as described in Example 1.2.1. After the described steps of pSIM5-tet recombination and curing, five colonies were selected, corresponding to clones with the same promoter integration but with random variant RBS sequences (see Table 8). Each clone was then transferred to a well of a 96-well plate containing 200 μl of 2xYT supplemented with chloramphenicol for maintenance of the pMevT plasmid, and cultured. 2 μl of each cultured clone was used to verify promoter integration as described in Example 1.2.1 before freezing the 96-well plate.

[0132] Mevalonate production screening: Escherichia coli strain s19.1.1(p) with the highest mevalonate productivity cspD ) and s9.1.4(p mutM) was identified using a screening assay in which strains containing four other potential combinations of RBS and promoter were cultured in parallel with the non-load-dependent production E. coli strain BL21(DE3)pMevT in 96-well plates over six serial passages (equivalent to 60 generations) of 1000-fold dilutions, as follows: These strains were grown in 200 μL of 2xYT medium containing 30 mg / L chloramphenicol and 0.5 mM IPTG in microtiter plates sealed with breathe-easy seals for 21 hours at 37°C with horizontal shaking. 150 μL of the culture was mixed with 50 μL of 50% glycerol and stored in the 96-well plates at -80°C. Additionally, 2 μL of the 10-fold diluted culture was transferred to 200 μL of fresh 2xYT medium supplemented with chloramphenicol and 0.5 mM IPTG and grown for an additional 21 hours under the same conditions. In total, five transfers identical to the one above were performed and frozen stocks were made from overnight cultures at each transfer.

[0133] Mevalonate synthesis and detection assay: 96-well plates containing frozen stocks from the second, fifth, and sixth transfers were thawed on ice and used to inoculate 10 mL of 2xYT with 0.5 mM IPTG and 30 mg / L chloramphenicol, which were then grown for 54 h at 37°C with horizontal shaking (250 rpm). A 300 μL aliquot from each culture was treated with 23 μL of 20% sulfuric acid, shaken vigorously, and spun down at 13,000 × g for 2 min. Supernatant (medium) samples were injected onto an Ultimate 3000 high-performance liquid chromatograph running a 5 mM sulfuric acid mobile phase (0.6 mL / min) on an Aminex HPX-87H ion-exclusion column (300 mm × 7.8 mm, Bio-Rad Laboratories) at 50°C. A refractive index detector was used for detection. A standard curve for mevalonate was constructed using mevalonolactone (Sigma-Aldrich) dissolved in 2xYT medium supernatant of a non-producing E. coli strain incubated under the same conditions.

[0134] 4.2 Results p cspD or p mutMLoad-dependent mevalonate-producing strains harboring one of four RBS coding sequence variants controlling the promoter and expression of the essential genes folP-glmM were cultivated under simulated large-scale production conditions achieved by serial passaging. As can be seen in Figure 12, the selected load-dependent strain s19.1.1(p cspD ) and s9.1.4(p mutM ) maintained significantly higher levels of mevalonate production compared to the sharp decline in production seen in the non-load-dependent mevalonate-producing strain BL21(DE3)pMevT. The initial mevalonate production rate of each load-dependent strain was the same as the control BL21(DE3)pMevT strain at seed 0, so strain s19.1.1(p cspD ) and s9.1.4(p mutM The smaller decrease in the production rate of ) is not due to the inherently lower initial load from mevalonate production.

[0135] Summary: Load-dependent expression of the E. coli oxidative stress- and glucose starvation-sensing promoter p cspD , and heat shock / DNA damage sensing promoter p mutM We demonstrate that linking the transcription of the essential genes folP-glmM to enhances long-term stability and production in E. coli engineered to synthesize mevalonate.

[0136] Example 5: Evaluation of promoters as load sensors to make yeast cells dependent on the load of engineered recombinant protein production Promoters that can sense the cellular burden or load imposed by recombinant expression of a protein or biosynthetic pathway and then drive expression of essential genes can be used to create load-dependent gene circuits tailored for use in yeast. Methods for evaluating candidate promoters are exemplified in yeast cells genetically engineered to synthesize recombinant human serum albumin (hSA) or insulin precursor (IP), optionally translationally fused to green fluorescent protein (GFP).

[0137] To induce growth responsive to the activity of a candidate load-sensing promoter, the native promoter of an essential gene in the yeast Saccharomyces cerevisiae is replaced with the candidate promoter using homologous recombination of a linear DNA construct transformed into yeast cells with a standard selectable marker. For example, candidate promoters can be selected from upregulated promoters of ribosomal RNA genes, such as those transcribed by RNA polymerase I (Laferte et al., 2006), DNA damage-sensing promoters (e.g., pOGG1), and unfolded protein-responsive promoters upregulated by the HAC1 transcription factor (Kimata et al., 2006). It may be necessary to fine-tune the expression level of the essential gene to ensure that the load-sensing promoter, once activated, confers a selective growth advantage to cells compared with a non-sensing promoter. Varying translation strengths can be engineered by varying the translation initiation region introduced along with the load-sensing promoter.

[0138] Clones harboring potentially different combinations of candidate load-sensing promoters are selected and evaluated for sustained protein production over 30-100 cell divisions.

[0139] 5.1 Materials and Methods Growth media: YPD medium contains 1% yeast extract, 2% peptone, and 2% glucose. SC medium contains 6.7 g / L of yeast nitrogen base with ammonium sulfate without amino acids, but lacks uracil.

[0140] Chromosomal integration and verification of promoter constructs: Constructs for chromosomal integration contain 300-600 bp upstream of the naturally regulated gene. Chromosomal integration of the promoter construct is achieved by transformation using standard electroporation procedures in Saccharomyces cerevisiae and homologous recombination. Correct chromosomal integration of the promoter was verified using colony PCR.

[0141] Long-term cultivation and production: A single colony from each strain is transferred to a 24-deep-well plate and cultured in 1.8 mL of YPD medium at 250 rpm and 30°C under conditions conducive to recombinant protein production. After 48 hours of cultivation, the cells are passaged into a new deep-well plate under the same conditions by 1000-fold reverse dilution. Samples are analyzed for production using a recombinant protein-specific assay (e.g., GFP detection), and cell density is monitored by OD600.

[0142] Growth rate measurement of selected strains: The growth rate of each strain is compared to a non-load-dependent production strain. The 96-well plates are sealed with breathe-easy film and growth is measured in a Syngergy H1 plate reader at 37°C and 754 rpm linear shaking for 20 hours. OD600 is measured every 10 minutes.

[0143] 5.2 Results 5.2.1 HAC1 upregulated promoters The HAC1 upregulated promoter, which contains an unfolded protein response (UPR) element, such as KAR2 (SEQ ID NO: 91), PDI1 (SEQ ID NO: 92), SSA1 (SEQ ID NO: 93), or FPR2 (SEQ ID NO: 94), has been demonstrated to be useful for regulating growth when introduced before a native growth regulator gene (e.g., URA3, a conditionally essential gene encoding orotidine 5'-phosphate decarboxylase essential for pyrimidine biosynthesis) in recombinant protein-producing Saccharomyces strains producing human insulin precursor or human serum albumin, optionally linked to GFP. The stability of the production was monitored under long-term production conditions, simulated by serial passaging corresponding to 60-80 cell divisions. In Saccharomyces strains containing the HAC1 upregulated promoter controlling transcription of the growth regulator gene (URA3), production is expected to be more stable than in the corresponding parent recombinant protein-producing Saccharomyces strain.

[0144] 5.3.2 RNA polymerase I upregulated promoters RNA polymerase I transcribes ribosomal RNA genes in yeast. Gene promoters: RNA polymerase I upregulated promoters such as RPL3 (SEQ ID NO: 95), RPL6A (SEQ ID NO: 96), and RPL28 (SEQ ID NO: 97) are useful for regulating the growth of essential yeast genes. Such upregulated promoters are introduced in front of native growth-regulating genes (e.g., the conditionally essential gene URA3) in recombinant protein-overproducing strains producing human insulin precursor or human serum albumin, possibly linked to GFP. The stability of production is monitored by experimentally simulating long-term production through serial passaging corresponding to 60-80 cell divisions. In Saccharomyces strains containing the RNA polymerase I upregulated promoter URA3, which regulates transcription of the conditionally essential gene, production is more stable than in the corresponding parent recombinant protein-producing Saccharomyces strain.

[0145] 5.3.3 DNA damage responsive promoters The DNA damage response in yeast resulting from heterologous expression broadly induces transcription of DNA repair systems, including OGG1 (SEQ ID NO:98), RAD51 (SEQ ID NO:99), and RAD54 (SEQ ID NO:100). Promoters of genes encoding OGG1, RAD51, or RAD54, when operably linked to essential genes, are useful for regulating growth of yeast production cells of the invention. Such promoters are introduced in front of a native essential gene (e.g., a growth regulatory gene encoding URA3) in cells of a yeast protein production strain that produces human insulin precursor or human serum albumin, optionally fused to GFP.

[0146] The stability of the production is followed by long-term production simulated by serial passages corresponding to 60-80 cell divisions. RAD51 and / or p RAD54 In strains with essential genes upregulated, production is more stable.

[0147] Summary: Load dependence enhances long-term stability and production in Saccharomyces cerevisiae cells engineered to produce human serum albumin or synthesize insulin precursors by linking essential gene transcription to load-sensing promoters selected from promoters activated during the load of recombinant protein production or promoters associated with ribosomal RNA promoters (Table 2).

[0148] Example 6: Examples of load-dependent yeast strains producing human serum albumin that exhibit enhanced long-term production stability Load-dependent systems for use in yeast strains include promoters derived from genes encoding 1) protein isomerase PDI1, which encodes a chaperone belonging to the unfolded protein response in yeasts such as Saccharomyces cerevisiae and Pichia pastoris and whose abundance is frequently upregulated in response to recombinant protein overexpression; 2) ribosomal subunits encoded by the RPL6A and RPL3 genes; and 3) FPR2, which encodes a peptidyl-prolyl cis-trans isomerase known to be activated during DNA replication stress. Load-dependent systems based on these promoters were introduced into Pichia pastoris strains engineered to express and secrete human serum albumin (hSA) to determine their effects on long-term human serum albumin (hSA) production stability.

[0149] 6.1 Materials and Methods: The Pichia pastoris (Komagataella phaffii) strain EGS31 is a derivative of strain CBS7435 (NRRL-Y11430 or ATCC 76273) that has been engineered to secrete hSA by genomic integration of a cDNA version of hSA encoding the ALB1 gene under the control of the AOX1 promoter.

[0150] Strain Construction: A load-dependent version of strain EGS31 was generated by genetic integration using constructs containing the kanMX conditionally selectable G418 resistance gene operably linked to one of the load-responsive promoters: pPDI1, pFPR2, pRPL3, and pRPL6A. These load-dependent constructs were integrated into the KU70 genomic locus of Pichia pastoris strain EGS31 by transforming EGS31 cells with linear integrating DNA flanked by >750 bp homology arms (sequences N1–N3, respectively). Transformation was performed using standard electroporation procedures on logarithmically growing cells pretreated with lithium acetate and dithiothreitol (Wu & Letchworth, 2004).

[0151] Table: Recombinant genes and load-dependent constructs introduced into Pichia pastoris [Table 10]

[0152] Growth media: BMGY and BMMY liquid media (1 L) were prepared as follows: 10 g of yeast extract and 20 g of peptone were added to 700 mL of HO, mixed using a magnetic stirrer, and then autoclaved. After cooling to room temperature, the following was added to the solution: 100 mL of 1 M potassium phosphate buffer (pH 6.0); 100 mL of 13.4% (w / v) yeast nitrogen base without amino acids and containing ammonium sulfate; 2 mL of 0.02% (w / v) biotin; and, in the case of BMGY, 100 mL of 10% (v / v) glycerol. In the case of BMMY, 100 mL of 5% (v / v) methanol was added.

[0153] Culture: EGS31 and load-dependent EGS31 strains were streaked onto YPD (1% yeast extract, 2% peptone, 2% D-glucose) agar plates and incubated overnight at 30°C. Single colonies were selected and precultured in 2 mL of BMGY. Cultures of load-dependent strains were supplemented with 50 μg / mL of G418 and grown overnight at 30°C with horizontal shaking at 300 rpm. Expression cultures were inoculated with 1 μL of preculture into 500 μL of BMMY medium containing different concentrations of G418 (0 μg / mL, 750 μg / mL) in a 96-well deep-well plate with a vented lid to generate "Seed 1."

[0154] The cultures were incubated at 30°C for 72 hours (300 rpm horizontal shaking). To generate the next seed, the expanded cultures were serially passaged (500-fold dilution) into fresh 500 μL of BMMY medium containing different concentrations of G418 (0 μg / mL, 750 μg / mL) in a 96-well deep-well plate with a vented lid four more times. At each serial passage, glycerol stocks (20% glycerol) were stored at -80°C from the expanded cultures.

[0155] To quantify the production of secreted hSA, quantitative cultures were regrown from glycerol stocks in 500 μL of BMMY medium containing different concentrations of G418 (0 μg / mL and 750 μg / mL) in 96-well deep-well plates with vented lids for 72 h.

[0156] Cultures were centrifuged at 3000 g for 15 minutes and the concentration in 50 μL of supernatant was quantified using an hSA-specific ELISA kit (Abcam catalogue number: ab179887: Human Albumin SimpleStep ELISA® Kit) according to the manufacturer's instructions.

[0157] 6.2 Results Strains with the genomically integrated load-dependent promoter PDI1 operably linked to the selectable kanMX gene showed higher production of secreted hSA when the strain's load-dependent system was activated by the addition of G418 (Figure 13). Furthermore, improved hSA production was seen with higher selection (750 μg / mL G418) for each of the other load-dependent promoters tested after approximately 30 cell divisions (Figure 14A—Seed 1). When further cultured for approximately 10 cell divisions (Figure 14B—Seed 2), these strains showed enhanced production levels of hSA when load-dependent activation was activated (150–750 μg / mL G418) compared to the non-load-dependent control and the strain without load-dependent activation (0 μg / mL G418).

[0158] In conclusion, culturing yeast cells containing the exemplary load-dependent systems of the present invention, under conditions that activate their respective load-dependent systems, is believed to enrich for high-producing yeast variants within the culture population. While increased hSA production can be detectable after a relatively short culture period (30 cell divisions), enrichment for high-producing yeast variants is maintained and even enhanced over longer culture periods, when traditional cultures generally exhibit a significant decrease in productivity.

[0159] Example 7 Identification of suitable load-sensing promoter candidates The different engineered production genes and pathways elicit different transcriptional responses indicative of the production process. To identify suitable promoter candidates for use as loading sensors, the following experiments were performed.

[0160] Methods: Exemplary genetic escaper cells were isolated from long-term cultures using genetically engineered microbial production cells of interest in the intended fermentation medium. Suitable genetic escaper cells are characterized by having a logarithmic phase growth rate at least 5% higher and a production rate or yield at least 30% lower than the original genetically engineered production cells.

[0161] Producer cells and corresponding escaper cells were cultured under intended fermentation conditions, scaled-down conditions mimicking intended fermentation conditions, or shake flask conditions. At the time point corresponding to the highest production rate in the producer cells, samples were taken for RNA sequencing. Total RNA was purified using the Purelink RNA Mini Kit (Thermo Fischer) according to the kit manufacturer's instructions and prepared using the TruSeq Stranded mRNA Kit (Illumina). Reads were mapped to the reference genome of the strain and analyzed, and then analyzed for differential expression between the producer cells and the corresponding escaper cells.

[0162] Results: Candidate suitable promoters were identified as those driving the expression of genes that showed differential expression of more than three-fold higher in the production organism relative to at least one isolated gene escape strain. References [Table 11] TIFF0007742611000018.tif129162

Claims

1. A microbial production cell genetically engineered to synthesize a product, comprising: a. An essential gene operably linked to a load-sensing promoter and a synthetic ribosome binding site (RBS). Including, the sequence of said synthetic RBS is selected to alter the translation strength of said essential gene and to enable said essential gene to regulate the growth rate of a cell; the essential gene is either a gene whose expression is essential for cell growth regardless of the nutritional composition of the production conditions of the microbial production cell, independent of the presence or absence of a specific inhibitor or nutrient, or a gene whose expression is conditional upon the removal of a nutrient from the production conditions of the microbial production cell, and whose knockout results in a loss of cell viability; the load-sensing promoter is heterologous with respect to the essential gene, and synthesis of the product confers a load on the microbial production cell; the burden imparted by synthesis of the product has a fitness cost measured as a percent reduction in log-phase growth rate of the microbial producer cell selected from among ≧5%, ≧10%, ≧15%, ≧20%, ≧25%, ≧35%, and ≧45%, relative to a corresponding non-producer microbial cell, when the essential gene in the microbial producer cell and the non-producer microbial cell is operably linked to its native promoter; expression of the essential gene is upregulated when the load-sensing promoter is induced by the load, relative to a basal level of expression of the essential gene when the load-sensing promoter is not induced; and the load-sensing promoter is induced by the load and upregulates the essential gene in the microbial production cell when cultured under production conditions compared to a derivative of the microbial production cell that produces the product at least 50% less; the load-sensing promoter is not induced by the product itself; Microbial producing cells.

2. The microbial production cells b. one or more genes encoding said product or encoding a metabolic pathway for the synthesis of said product; 2. The microbial production cell of claim 1, further comprising:

3. The microbial production cells i. a bacterium belonging to a genus selected from the genera Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Bacillus, Acetobacter, Acinetobacter, Pseudomonas, Proprionibacterium, Bacteroides, and Bifidobacterium; or ii. A yeast belonging to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Komagataella, Cryptococcus, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, and Schizosaccharomyces; or iii. Filamentous fungi selected from the genera Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma, Thermomyces, Streptomyces, and Aspergillus.

3. The microbial production cell of claim 1 or 2,

4. The microbial production cell of any one of claims 1 to 3, wherein the essential gene is an unconditionally essential gene.

5. 5. The microbial production cell of any one of claims 1 to 4, wherein the microbial production cell is a bacterium and the essential gene is an E. coli gene or operon selected from among folP-glmM, glmM, murI, asd, thyA, usA, rpoD, nusG, rpsU, accD, degS, fldA, ftsN, hflB, lolA, mraY, mreD, murA, murB, murF, nadD, rplV and rpsG or homologs thereof.

6. 6. The microbial production cell of any one of claims 1 to 5, wherein the essential gene is operably linked to a synthetic RBS whose sequence is selected to modify the translation strength of the essential gene independently of induction of the load-sensing promoter.

7. The load-sensing promoter i. σ ３２ , σ Ｂ and σ Ｓ a σ factor-regulated promoter selected from among σ factor-regulated promoters; ii. ribosomal RNA promoter, iii. A HAC1 upregulated promoter containing a UPR element, and iv. DNA damage-sensing promoters The microbial production cell according to any one of claims 1 to 6, selected from:

8. A microbial production cell according to any one of claims 1 to 7, wherein the load-sensing promoter is a promoter selected from among htpG, ibpA, clpB, yccV, grpE, ycjX, ldhA, mutM, ybbN, prlC, groES, fxsA, htpX, rrnB, rrnE, cspD, katE, xthA, uspE, gadB, ahpC, katG, grxA, oxyS, poxB, trxC, KAR2, PDI1, SAA1, FPR2, RPL3, RPL6A, RPL28, OGG1, RAD51, and RAD54.

9. 9. The microbial production cell of any one of claims 1 to 8, wherein the microbial production cell is characterized by increased product yield after at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 cell divisions from a single cell compared to a parent microbial production cell lacking the essential gene operably linked to a load-sensing promoter.

10. 10. The microbial production cell of any one of claims 1 to 9, wherein the microbial production cell is characterized by an increased product yield of at least 10, 25, 50, or 80% after at least 50 cell divisions from a single cell compared to a parental microbial production cell lacking the essential gene operably linked to a load-sensing promoter.

11. i. Providing a microbial production cell according to any one of claims 1 to 10; ii. introducing the microbial production cells into a culture medium containing a substrate for production of the product; iii. Recovering the product A method for product biosynthesis comprising:

12. 12. The method for product biosynthesis of claim 11, wherein the product is selected from amino acids, organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, peptides, enzymes, therapeutic proteins and their precursors, human growth hormone, insulin, glucagon-like peptide-1, monoclonal and polyclonal antibodies, and single chain fragment antibodies.

13. 1. Use of an essential gene operably linked to a load-sensing promoter and a synthetic RBS to increase product yield of a culture population of microbial production cells arising from a single cell, comprising: the essential gene is either a gene whose expression is essential for cell growth regardless of the nutritional composition of the production conditions of the microbial production cell, independent of the presence or absence of a specific inhibitor or nutrient, or a gene whose expression is conditional upon the removal of a nutrient from the production conditions of the microbial production cell, and whose knockout results in a loss of cell viability; the load-sensing promoter is heterologous with respect to the essential gene; the sequence of said synthetic RBS is selected to alter the translation strength of said essential gene and to enable said essential gene to regulate the growth rate of a cell; production of the product stresses the microbial production cells; the burden imparted by synthesis of the product has a fitness cost measured as a percent reduction in log-phase growth rate of the microbial producer cell selected from among ≧5%, ≧10%, ≧15%, ≧20%, ≧25%, ≧35%, and ≧45%, relative to a corresponding non-producer microbial cell, when the essential gene in the microbial producer cell and the non-producer microbial cell is operably linked to its native promoter; wherein the expression of the essential gene is upregulated when the load-sensing promoter is induced by the load, relative to a basal level of expression of the essential gene when the load-sensing promoter is not induced; the load-sensing promoter is induced by the load and upregulates the essential gene in the microbial production cell when cultured under production conditions compared to a derivative of the microbial production cell that produces the product at least 50% less; the load-sensing promoter is not induced by the product itself; Use of essential genes.

14. 11. Use of a microbial production cell according to any one of claims 1 to 10 to produce a biosynthetic product.

15. 15. Use of microbial production cells according to claim 14, wherein the product is selected from among amino acids, organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, peptides, enzymes, therapeutic proteins and their precursors, human growth hormone, insulin, glucagon-like peptide-1, monoclonal and polyclonal antibodies, and single chain fragment antibodies.

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