Load-dependent producers

Abstract

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

Description

[Technical field]

[0001] The present invention provides a productive microbial cell for the synthesis of a product, which further comprises a load-dependent genetic circuit, the expression of which confers a selective growth and / or survival advantage to the cell synthesizing the product, while limiting the growth of non-producing / low-producing escape cells. [Background technology]

[0002] A growing share of chemical production in the world relies on microorganisms genetically engineered to act as cell factories and tailored specifically for the biosynthesis of a given molecule. Production processes using these cell factories typically begin with a starter culture of a small number of cells of the producer organism, which undergo a period of growth and cell number expansion in large fermentation tanks (up to 1,000,000 L in volume). In some setups, production of a given molecule proceeds in two stages, with a growth phase and a subsequent period (batch and fed-batch culture); alternatively, production may be continuous. Chemostat fermenters grow the producer organism in a constant dilution of fermentation broth, so that product and cells are flushed out of the culture as fresh nutrient medium is replenished. At an industrial scale, such production processes may continue to operate for one to two months before starting a new culture in a clean tank. The fermentation processes and equipment used in the industry are very similar for both the production of a wide range of commodity small molecules and therapeutic proteins, and these processes are prone to similar problems. In other applications, microbial cells may be engineered to produce commercially advantageous non-natural molecules in the environment in which the cells are cultured.

[0003] Cell factories engineered to allocate limited metabolic resources to the biosynthesis of a given molecule are subject to an unnatural stress that is usually reflected by slower growth. This stress is especially problematic when production is long term (e.g., in a chemostat) since it creates a selective pressure for cells that are unable to synthesize the product molecule (non- or low-producing). Such non-producing cells consume nutrients and oxygen and take up space, making them highly undesirable in industrial fermentation. Low- and non-producing cells grow faster and therefore have a significant selection advantage over producing cells. In growing cell cultures, such fitness improvements can lead to the substantial displacement of producing cells over time. This deviation from optimal production results in the disposal of fermentation broth and the waste of resources in cleaning, sterilization, not to mention nutrients to replenish the fermentation tank with new producing organisms. Such non-producing cells result from genetic mutations that occur during the multiple proliferation and division of the original producing organism cells.

[0004] Because the occurrence of genetic mutations and non-genetic adaptations in producing biological cells that reduce the cells' product production during production runs is inevitable, methods are needed to eliminate or slow the growth of non-producing cells in production. Preferably, such elimination methods are effective enough to prevent deviations from the observed productive state, thereby extending the useful life of industrial fermentation.

[0005] The present invention aims to delay the proliferation of non-productive cells present in cells that make up a productive cell factory over time, thereby improving the productivity of the cell factory, and addresses the problem of how to eliminate non-productive cells that have an adaptive advantage in a cell factory. Summary of the Invention

[0006] In a first aspect of the present invention, there is provided a producing microbial cell genetically engineered to synthesize a product, wherein the cell further comprises: a first essential gene operably linked to a first load-sensitive promoter; and a second essential gene operably linked to a second load-sensitive promoter; wherein said first load-sensitive promoter is heterologous to said first essential gene; and said second load-sensitive promoter is heterologous to said second essential gene; wherein synthesis of said product imposes a burden and / or fitness cost on said cell; and wherein expression of the first essential gene is upregulated when the first load-sensing promoter is induced by the load and / or fitness cost compared to basal level expression of the essential gene when the first load-sensing promoter is not induced, and expression of the second essential gene is upregulated when the second load-sensing promoter is induced by the load and / or fitness cost compared to basal level expression of the second essential gene when the second load-sensing promoter is not induced.

[0007] In a second aspect of the present invention, there is provided a method for product biosynthesis comprising the steps of: providing at least one producer microbial cell of the invention; introducing at least one cell into a substrate-containing culture medium for the production of said product; recovering the product.

[0008] In a third aspect of the present invention there is provided the use of first and second essential genes operably linked to first and second load-sensing promoters, respectively, for enhancing product yield in a cultured cell population of a producing microbial cell having arisen from a single cell for at least 50 generations or more; wherein said first load-sensitive promoter is heterologous to said first essential gene; and said second load-sensitive promoter is heterologous to said second essential gene; wherein production of the product imparts a load to the cell; and wherein expression of the first essential gene is upregulated when the first load-sensing promoter is induced by the load, compared to basal level expression of the first essential gene when the first load-sensing promoter is not induced, and expression of the second essential gene is upregulated when the second load-sensing promoter is induced by the load, compared to basal level expression of the second essential gene when the second load-sensing promoter is not induced.

[0009] In a fourth aspect of the invention, there is provided a use of a producing microbial cell of the invention for producing a biosynthetic product.

[0010] In a further aspect of the present invention, there is provided a producing microbial cell genetically engineered to synthesize a product, wherein the cell further comprises: · An essential gene operably linked to a load-sensitive promoter; wherein said load-sensitive promoter is heterologous to said essential gene; wherein synthesis of said product imposes a burden and / or fitness cost on said cell; wherein expression of the essential gene is upregulated when the load-sensing promoter is induced by the load and / or fitness cost compared to basal level expression of the essential gene when the load-sensing promoter is not induced; and wherein the producing microbial cell belongs to a genus selected from the genera Bacillus, Corynebacterium, and Aspergillus. [Brief description of the drawings]

[0011] [Figure 1] Graph showing growth (measured at optical density OD630 nm) over time for parental E. coli BL21(DE3) cells (gray line) and cells transformed with the hGH-GFP producing plasmid pEG34 (black line; pEG34). Each strain was cultured in quadruplicate and growth was measured under conditions inducing hGH-GFP expression; error bars indicate standard error of the mean (n=4). [Diagram 2] Graph showing growth (measured at optical density OD630nm) over time of cells of the parental strain E. coli BL21(DE3) (black line) and (as indicated) of the non-productive load-dependent E. coli strains s5.0#3, s7.0#8, and s9.0#8 derived from the parental strain, where the essential genes folP-glmM are controlled by pyccV, pycjX, and pmutM, respectively. Measured growth rates are based on the average of cultures grown in quadruplicate (n=4). [Diagram 3] Graph showing cell growth (measured by optical density OD630nm) over time of the load-dependent strain s9.0#8 containing either the hGH-GFP producing plasmid pEG34 (black line) or the control plasmid pEG0 (grey line). Each strain was cultured in quadruplicate and growth was measured under conditions inducing hGH-GFP expression; error bars indicate standard error of the mean (n=4). [Figure 4] Graph showing the growth over time (measured at optical density OD630 nm) of cells of the parental strain E. coli BL21(DE3) (black line) and of cells of three load-dependent strains s5.0#3, s7.0#8, and s9.0#8 containing and expressing the hGH-GFP producing plasmid pEG34 (as indicated in the figure). Growth rates measured under conditions inducing hGH-GFP expression are based on the average of quadruplicate cultures (n=4). [Diagram 5] Figure 1 shows histograms of GFP / OD values ​​(Y-axis) measured in the first and sixth culture passages (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 each of the load-sensing promoters directing the expression of the essential genes folP-glmM and one of the four mutant RBS coding sequences. As controls, cultures of the non-load-dependent E. coli strain BEG34 and the parental strain E. coli BL21(DE3), BEG0, are included. Measurements are performed on cultures grown under conditions inducing hGH-GFP expression. Error bars indicate standard error of the mean (n=4). [Figure 6]Histograms of GFP / OD values ​​(Y-axis) measured after sequential culture passages (seeds 1-12) of cultures of hGH-GFP producing strains s3.6#2 (pibpA), s6.6#6 (pgrpE), s7.6#8 (pycjX), and s10.6#7 (pybbN), which contain each of the load-sensing promoters directing expression of the essential genes folP-glmM and one of the four mutated RBS coding sequences. As controls, cultures of the non-load-dependent E. coli strain BEG34 and the parental strain E. coli BL21(DE3) are included. Measurements are performed on cultures grown under conditions inducing hGH-GFP expression. Error bars indicate standard error of the mean (n=3). [Figure 7] 1 is a 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 standard error of the mean (n=7). [Figure 8] Graph showing GFP / OD values ​​(Y-axis) measured after successive passaging (seed 1, 4-9) of cultures of hGH-GFP producing strains minus that of the empty plasmid control, BEG0-empty strain. The strains shown in (A)-(C): (A) s7.6#8(pycjX), (B) s13.6#2(pfsxA), and (C) s13.6#2evo(pfsxA), each contain the respective load-sensing promoters directing the expression of the essential genes folP-glmM (gray line), and the non-load-dependent hGH-GFP producing E. coli strain BEG34 (black line). Measurements are performed on cultures grown under inducing conditions for hGH-GFP expression. Error bars indicate standard error of the mean (n=5). [Figure 9]Graph showing GFP / OD values ​​(Y-axis) measured after successive passaging (seed 1, 4-9) of cultures of hGH-GFP producing strains minus that of the empty plasmid control, BEG0-empty strain. The strains shown in (A) and (B): (A) s15.6.7(prrnB) and (B) s16.6.6(prrnE), each contain the respective load-sensing promoters directing the expression of the essential genes folP-glmM (gray line), and the non-load-dependent hGH-GFP producing E. coli strain BEG34 (black line). Measurements are performed on cultures grown under conditions inducing hGH-GFP expression. Error bars indicate standard error of the mean (n=5). [Figure 10] Figure 1 shows the relative hGH-GFP production values ​​(Y-axis) measured after sequential passaging (seeds 2-5, and 7) of cultures of the hGH-GFP producing strain s29.6#3 and subtracted from the measurements of the corresponding empty plasmid control, strain BEG0. Strain s29.6#3 contains the osmotic stress-sensing promoter ppoxB directing the expression of the essential genes folP-glmM (black squares) compared to the non-load-dependent hGH-GFP producing E. coli strain BEG34 (grey dots). The seventh passage of the sequential passaging was analyzed after 72 h. Measurements are performed on cultures grown under conditions inducing hGH-GFP expression. Error bars indicate standard error of the mean (n=5 for s29.6#3 and n=8 for BEG34). [Figure 11] 1 is a histogram showing the lysis rate of S. aureus per minute normalized to the OD of E. coli cultures as a measure of the secreted lysostaphin production rate of cultures of lysostaphin-producing strain s6.4#5, which contains the load-sensing promoter pgrpE driving the expression of the essential genes folP-glmM and one of four mutated RBS coding sequences, compared to the non-load-dependent lysostaphin-producing E. coli strain BENDU5cam. The rates were measured for cultures grown under lysostaphin expression-inducing conditions after successive passages (seeds 1 and 7). Error bars indicate the standard error of the mean (n=3). [Figure 12]Graph showing mevalonate titers (g / L) synthesized by cultures of load-dependent mevalonate-producing strains s19.1#1(pcspD) and s9.14(pmutM) measured after successive passages (seeds 2, 5 and 6) compared to the non-load-dependent mevalonate-producing parent E. coli strain BL21(DE3)-pMevT. The load-sensing promoters pcspD and pmutM are each combined with one of four mutated RBS coding sequences directing the expression of the essential genes folP-glmM. Measurements are performed on cultures grown under inducing conditions for MevT expression. Error bars indicate standard error of the mean (n=3). [Figure 13] 1 is a bar plot showing human serum albumin (hSA) production titers of Pichia pastoris strain EGS31 cultures containing a genomically integrated copy of a cDNA corresponding to the ALB1 gene encoding hSA under the control of the AOX1 promoter, as well as the PDI1 promoter operably linked to the kanMX selection gene. Cultures were grown under either selective (dependence activated with 750 mg / mL G418) or nonselective (dependence unactivated) conditions. The hSA titer of the "dependence unactivated" culture is taken as 100%, and the titer of the "dependence activated" culture is shown as a percentage. [Figure 14] 14A and B. Bar plots showing human serum albumin (hSA) production titers in cultures of a load-dependent version of the Pichia pastoris strain EGS31, which contains a genomically integrated copy of a cDNA corresponding to the ALB1 gene encoding hSA under the control of the AOX1 promoter and the kanMX selection gene operably fused to the load-responsive promoter FPR2, or RPL3 or RPL6A, respectively. Cultures were grown under nonselective conditions (without activation of dependency) or selective conditions (with activation of dependency with either 150 or 750 mg / mL G418) for a period of approximately 30 cell divisions ([A] seed 1), or after an additional 10 cell divisions ([B] seed 2). The average hSA titers represent the percentage of titers in the seed 1 "EGS31" cultures. Error bars indicate the standard error of the mean (n=4). [Figure 15] Comparison of load-dependent IgA-GFP production of EGS340 and parental EGS84 in long-term culture. Demonstration of load-dependence of sustained production in B. subtilis using the IgA-producing load-sensing perR promoter controlling transcription of the essential iscU gene operon (sufC-sufD-sufS-sufU-sufB). Comparison of EGS340 and EGS84 in an experiment using serial passaging at 30°C with 500-fold diluted seeds transferred to fresh medium every 24 hours. Points are means and error bars indicate standard error (n=7). [Figure 16] Comparison of EGS343 and EGS84 in long-term culture. Demonstration of load-dependence of sustained production in B. subtilis using the perR promoter to control transcription of the essential accC gene operon (accB-accC-yhqY). Comparison of IgA-GFP productivity of EGS343 and EGS84 by serial passage at 30°C with 500-fold diluted seeds transferred to fresh medium every 24 hours. Points are means and error bars indicate standard error (n=7). [Figure 17] Use of multiple loading sensors to simultaneously regulate different essential genes and thereby sustain heterologous protein production in B. subtilis. Comparison of IgA-GFP producing strains after 5 successive passages (seeds of 500-fold dilution), corresponding to approximately 75 generations. The single loading sensor strain EGS340 contains only the pperR-based loading sensor that controls transcription of the iscU essential gene operon (EGS340). EGS340 was compared to derivatives in which transcription of the accC essential gene operon is also controlled by the pctsR-based loading sensor (EGS460), the pdnaK-based loading sensor (EGS462), and the phrcA-based loading sensor (EGS466), respectively. Bars indicate the mean and error bars indicate the standard error (n=4). [Figure 18]Comparison of relative IgA-GFP levels measured by an IgA-specific ELISA after approximately 75 generations of cell culture of the non-load-dependent control strain EGS084 and the doubly load-dependent derivative EGS466 (based on pperR controlling the iscU essential gene operon and phrcA controlling the accC essential gene operon). [Figure 19] Use of TIS variations in essential genes to control growth rate. Duplicate experiments on the growth of the parent strain (EGS621) and strains with variations in the TIS sequences of the essential gene CIA1 (EGS1100, EGS1101, EGS1102, EGS1104) grown in YPD. These Pichia strains showed growth that correlated with the relative intensities of the four TIS sequences. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Abbreviations, Terms and Definitions Burden refers to the cellular state of a microbial cell (such as a cell genetically engineered to synthesize a product) producing a product under production conditions, which causes a fitness cost due to the synthesis of the product, especially in cells that exhibit a high level of efficient synthesis of the product. The fitness cost is a quantifiable measure of the burden, which can be quantified by measuring the percentage decrease in the maximum log phase growth rate (measured according to a growth curve) of the cell during the synthesis of the product under production conditions, when grown under similar production conditions, compared to a parent microbial cell that cannot synthesize the product.

[0013] A load-sensing promoter refers to a promoter that is induced by the "load" and is capable of upregulating the expression of a gene operably linked to the promoter in a producing microbial cell cultured under production conditions, compared to a mutant derivative of the producing microbial cell that essentially does not synthesize any product of interest or synthesizes at least 50% less of the product than the producing cell when the load-sensing promoter is induced. Such mutant derivatives include non-productive escape mutants isolated after prolonged culture of a population of cells derived from the producing cell. Non-limiting examples of load-sensing promoters (see Table 2 for details) include: These 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, and homologs of the above in other Gram-positive and Gram-negative bacteria; Non-limiting examples of load-sensitive promoters in Saccharomyces cerevisiae include the promoters of genes KAR2, PDI1, SAA1, FPR2, RPL3, RPL6A, RPL28, OGG1, RAD51, RAD54; Non-limiting examples of load-sensing promoters in Bacillus include the promoters of the genes groES, ctsR, dnaK, perR, hrcA, spx, sigB, yflT and their homologous genes; Non-limiting examples of Corynebacterium load-sensing promoters include the promoters of the genes groES, kata, cplX, mutM and their homologous genes; Non-limiting examples of Aspergillus load-sensing promoters include the promoters of the genes bipA, clxA and their homologues. The load-sensing promoter may be a hybrid, scrambled or truncated version of such a natural promoter, so long as it maintains a response to load.

[0014] Load-dependent microbial cells refer to microbial cells that have been engineered to contain a genetic circuit that includes at least one "load-sensitive promoter" that is "operably linked" to an essential gene of the cell.Preferably, the cell has a genetic circuit that includes two essential genes, namely, a first essential gene that is operably linked to a first load-sensitive promoter, and a second essential gene that is operably linked to a second load-sensitive promoter, where the first load-sensitive promoter is heterologous to the first essential gene; and the second load-sensitive promoter is heterologous to the second essential gene.

[0015] A load-dependent productive microbial cell refers to a microbial cell genetically engineered to synthesize a desired product, wherein the cell is further genetically engineered to contain a genetic circuit comprising said at least one, preferably two "load-sensing promoters" "operably linked" to an "essential gene" of the cell, as defined above. The desired product may be a protein encoded by a nucleic acid molecule in the engineered microbial cell, or a product of a heterologous pathway encoded by one or more nucleic acid molecules expressed by the cell.

[0016] A non-load-dependent productive microbial cell is considered to be the "parent" cell of said load-dependent productive microbial cell. It is a productive microbial cell that contains and expresses one or more genes encoding a product or encoding a biosynthetic metabolic pathway of said product, but lacks a genetic circuit comprising said at least one, preferably two, load-sensing promoters operably linked to essential genes as disclosed in the present invention. The at least one, preferably two, essential genes in this non-load-dependent productive microbial cell, each operably linked to its native promoter.

[0017] The desired products of the present invention include, but are not limited to, products that impose a fitness cost (burden) on the producing cells when synthesized by genetically engineered producing microbial cells. Non-limiting examples of such products 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 or polyclonal antibodies, single fragment antibodies, and nanobodies). Examples further include proteins naturally occurring in eggs (such as ovalbumin) or naturally occurring in milk (such as casein, lactadherin, α-lactoferrin), immunoglobulin A and immunoglobulin G secreted as secretory components. Examples of enzymes include amylase, lipase, protease, barnase, β-galactosidase, crystal protein, cutinase, pectase, and laccase, as well as enzymes that act on carbohydrates (such as xylanases, licheninases, cellulases, lytic polysaccharide monooxygenases, and pectases).

[0018] An essential gene (or essential gene operon) refers to a gene in a producing microbial cell that, when down-regulated, causes the producing microbial cell to undergo a reduced growth rate under the production conditions. Preferably, the essential gene is essential for growth, regardless of the nutrient composition of these production conditions, and sufficient expression of such an essential gene to support cell growth does not depend on the presence or absence of specific inhibitors or nutrients provided under the production conditions. Non-limiting examples of essential genes in E. coli under 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, rpsG, and their homologs. Non-limiting examples for Bacillus subtilis include the iscU operon (sufC-sufD-sufS-sufU-sufB), the accC operon (accB-accC-yhqY), glmM, ylaN, infA, dapA and homologs thereof. Non-limiting examples for Saccharomyces include FOL1, MED7, RRP40, NOP8, PGI1, NEP1, URA3, LEU2, TRP1, HIS3 and homologs thereof. Non-limiting examples for Aspergillus include ARG3, adeA and homologs thereof. Non-limiting examples for Corynebacterium include alr, glmM and homologs thereof (e.g., suitable genes encode enzymes that synthesize cell wall components). Furthermore, the essential genes used in the context of the present invention preferably do not encode a desired product expressed by the production strain, nor do they encode a protein that facilitates synthesis of the desired product or its intermediate in a heterologous pathway expressed by the production strain.

[0019] Conditionally essential genes are those that allow the load-dependent systems of a producing microbial cell to be "activated" by culturing said cells under essential conditions, such as the addition of an antibiotic or the withdrawal of a nutrient.

[0020] The basal expression level of an essential gene is the transcription level of each "essential gene" (as defined above) operably linked to a "load-sensing promoter" in a load-dependent producing microbial cell when the "load-sensing promoter" is not induced. The load-sensing promoter operably linked to the essential gene is heterologous with respect to the essential gene in the sense that the promoter is not the native promoter of the essential gene (even if the promoter is present in the same genome) and is therefore not operably linked to the essential gene in nature. The basal (i.e., non-induced) level of expression of an essential gene is equal to or less than 10%, 20%, 50%, 90% or 95% of the growth rate of the corresponding cell in which each essential gene is operably linked to its native promoter, and is sufficient to support the growth of the cell under production conditions (or in logarithmic growth phase). In those cells in which the load-sensing promoter is inactive, the reduced growth rate due to basal level expression of the essential gene constitutes a selective disadvantage for non-productive cells (e.g., non-productive mutants that arise during product production).

[0021] The fitness cost is quantified by comparing the maximum logarithmic phase growth rate (measured according to a growth curve) of the non-load-dependent producing microbial cell or cell culture thereof (i.e., lacking a load-sensing promoter operably linked to at least one, preferably two essential genes, 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 phase growth rate of the parental microbial cell (or cell culture thereof) from which the producing microbial cell is derived, which either lacks or is unable to express the gene encoding the product or a metabolic pathway for the biosynthesis of the product (or (ii) the maximum logarithmic phase growth rate of escape cells derived from the non-load-dependent producing microbial cell, which produce less than 50% of the product compared to the non-load-dependent producing microbial cell), when grown under similar production conditions. The maximum logarithmic phase growth rate of each cell or cell culture thereof is measured at the start of production after each cell or cell culture thereof is introduced into a substrate-containing culture medium to produce the product.

[0022] Production conditions refer to the particular culture conditions used for production and may relate to the medium and / or other culture conditions (e.g., temperature, pH, agitation, aeration, etc.) for the production of a desired product by the producing microbial cells.

[0023] Ribosome binding site (RBS), translation initiation region, or translation strength element refers to a region in the 5' untranslated region of a gene that regulates the translation strength of a particular messenger RNA.

[0024] [I] Load-dependent productive microbial cells In a first aspect of the present invention, there is provided a producing microbial cell genetically engineered to synthesize a product, wherein the cell further comprises: (a) An essential gene operably linked to a load-sensitive promoter; wherein said promoter is heterologous with respect to said essential gene; wherein production of said product imposes a burden and / or fitness cost on said cell; and wherein expression of the essential gene is upregulated when the load-sensitive promoter is induced due to the load and / or fitness cost, compared to basal level expression of the essential gene when the load-sensitive promoter is not induced. In a further aspect of the invention, a producing microbial cell genetically engineered to synthesize a product comprises: (a) and (b) (a) a first essential gene operably linked to a first load-sensitive promoter; and (b) a second essential gene operably linked to a second load-sensitive promoter; wherein said first load-sensing promoter is heterologous with respect to said first essential gene and said second load-sensing promoter is heterologous with respect to said second essential gene; wherein synthesis of said product imposes a burden on said cell; and wherein expression of the first essential gene is upregulated when the first load-sensitive promoter is induced by the load, compared to a basal level expression of the first essential gene when the first load-sensitive promoter is not induced; and Expression of the second essential gene is upregulated when the second load-sensing promoter is induced by the load, compared to basal level expression of the second essential gene when the second load-sensing promoter is not induced.

[0025] In one embodiment, the first and second essential genes are different.In another embodiment, the first and second essential genes are the same.In a particular embodiment, the first and second essential genes are the same, but the first and second load-sensitive promoters are different.

[0026] In one embodiment, the first and second load-sensitive promoters are different.In another embodiment, the first and second load-sensitive promoters are the same.In a particular embodiment, the first and second load-sensitive promoters are the same, but the first and second essential genes are different.

[0027] In one embodiment, the producing microbial cell comprises three or more essential genes each operably linked to a load-sensing promoter. In one embodiment, the invention provides a producing microbial cell engineered to synthesize a product, the microbial cell comprising two, three, four, five, six or more load-sensing promoters controlling essential genes in the cell.

[0028] In one embodiment, the microbial cell comprises three essential genes each operably linked to a load-sensing promoter. In one embodiment, the microbial cell comprises four essential genes each operably linked to a load-sensing promoter. In one embodiment, all the load-sensing promoters are different. In another embodiment, parts of the load-sensing promoters are identical, but they are linked to different essential genes. In one embodiment, all the essential genes are different. In another embodiment, parts of the essential genes are identical, but they are linked to different load-sensing promoters.

[0029] A producing microbial cell genetically engineered to synthesize a product is a cell that contains one or more genes that encode the product or that encode a metabolic pathway for the synthesis of the product, optionally wherein the one or more genes are operably linked to a constitutive or inducible promoter. In one embodiment, the one or more genes that encode the product or that encode a metabolic pathway for the synthesis of the product may be recombinant.

[0030] In one embodiment, the burden and / or fitness cost imposed on the producing microbial cells of the present invention is quantified by comparing the maximum logarithmic growth rate of a producing microbial cell that lacks or is unable to express the gene encoding the product or encoding a metabolic pathway for the synthesis of the product, and 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 at least one, preferably two essential genes), to the maximum logarithmic growth rate of the parental microbial cell from which the producing microbial cell is derived, when the respective cells are cultured under essentially the same production conditions. The burden and / or fitness cost imposed on the producing microbial cell preferably corresponds to a percentage decrease in the quantified maximum logarithmic growth rate selected from ≧5%, ≧10%, ≧15%, ≧20%, ≧25%, ≧35%, and ≧45%.

[0031] The load-dependent producing microbial cells of the present invention may be any prokaryotic or eukaryotic microorganism, such as bacteria, yeast, and filamentous fungi.

[0032] In one embodiment, the producer microbial cells of the invention are prokaryotes. A non-exhaustive list of suitable bacteria includes: A biological species belonging to a genus selected from the genera Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Bacillus, Acetobacter, Acinetobacter, Pseudomonas; Propionibacterium, Bacteroides, and Bifidobacterium.

[0033] In a preferred embodiment, the present invention provides a producer microbial cell genetically engineered to synthesize a product, wherein the cell comprises an essential gene operably linked to a load-sensing promoter; wherein the producer microbial cell belongs to the genus Bacillus. In another embodiment, the producer microbial cell of the present invention is a eukaryotic organism, such as a yeast or a fungus. In a particular embodiment, the eukaryotic organism can be an organism belonging to the genera Saccharomyces, Komagataella, or Aspergillus.

[0034] 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, and 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, for example yeasts belonging to the genera Cryptococcus, Debaryomyces, Hansenula, Yarrowia, Zygosaccharomyces or Schizosaccharomyces.

[0035] A non-exhaustive list of suitable filamentous fungi includes: A filamentous fungus belonging to the genera Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma, Thermomyces, Streptomyces, and Aspergillus. More specifically, the filamentous fungus may be selected from Fusarium oxysporum, A. niger, A. awamori, A. oryzae, and A. nidulans.

[0036] The products synthesized by the producer microbial cells of the invention incur a burden and fitness cost for the cells reflected in a reduced growth rate, such products include amino acids, organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, peptides, enzymes, therapeutic proteins and their precursors, such as human growth hormone, insulin, glucagon-like peptide 1, monoclonal and polyclonal antibodies, single fragment antibodies and nanobodies. Further examples include proteins naturally occurring in eggs, such as ovalbumin, or in milk, such as casein, lactadherins, α-lactalbumin, β-lactoglobulin, osteopontin, lactoferrin, immunoglobulin A and immunoglobulin G secreted as secretory components. Further examples of enzymes include amylase, lipase, protease, barnase, β-galactosidase, crystal protein, cutinase, petase, and laccase, as well as enzymes acting on carbohydrates, such as xylanases, licheninases, cellulases, lytic polysaccharide monooxygenases, and pectases. In one embodiment, the product is not a natural product for the producing microbial cell of the invention, and thus the parent cell from which the producing microbial cell is derived does not produce the product. In one embodiment, the one or more genes encoding the product or encoding a metabolic pathway for the synthesis of the product are heterologous with respect to the producing microbial cell of the invention, where the one or more genes may be transgenes.

[0037] [II] Characteristics of the Producing Microbial Cells of the Present Invention (II.i) Fitness costs – production burden Heterologous expression of a desired product or a pathway leading to a desired product in a microbial cell places a burden on the cell. A burden can also occur, for example, by overproduction of a natural product in the cells of a microbial strain selected after mutagenesis. The burden can also be explained as a fitness cost that makes the cells grow slower (Example 1; Figure 1). This can be due to general and / or product-specific metabolic toxicity, increased utilization and / or depletion of potentially finite cellular resources and metabolites, which the microbial cells experience during the biosynthesis of the product of the invention under production conditions. Particularly in industrial-scale fermentation, the combination of fitness cost and slower growth exerts a selective pressure on the cell population in the fermenter, causing a decrease in product yield over time due to the accumulation of non-producing cells resulting from genetic mutations or non-genetic changes (Example 2, BEG34 in Figure 6).

[0038] In the present invention, the presence and expression in a producing microbial cell of one or more genes that encode a product or that encode a metabolic pathway for the biosynthesis of a product imposes a fitness cost (burden).

[0039] The fitness cost of product production in a producing microbial cell is quantified by comparing the growth rate of a non-load-dependent producing microbial cell (i.e. lacking at least one, and preferably two, load-sensing promoters operably linked to said essential genes, but containing and expressing one or more genes encoding said product or encoding a metabolic pathway for the biosynthesis of said product) with the growth rate of a cell of (i) or (ii): (i) a parental microbial cell from which a non-load-dependent producing microbial cell is derived, wherein the parental microbial cell lacks a gene encoding the product or a gene encoding a metabolic pathway for the biosynthesis of the product; or (ii) escape cells derived from non-load-dependent productive microbial cells, wherein the escape cells produce less than 50% of the product compared to the non-load-dependent productive microbial cells, wherein the load and / or fitness cost in the productive microbial cells corresponds to a quantified percentage reduction in growth rate, preferably selected from ≥ 5%, ≥ 10%, ≥ 15%, ≥ 20%, ≥ 25%, ≥ 35%, and ≥ 45%, more preferably at least 5%.

[0040] Preferably, the determination of relative growth rates is performed by measuring the maximum log phase growth rate for each microbial cell cultured under essentially identical conditions, which are selected to closely mimic the conditions of the large-scale fermentation that will ultimately be performed. Any use of the term "growth rate" refers to the term specific growth rate.

[0041] (II.ii) Essential genes A producer microbial cell of the invention contains at least one, and preferably two, essential genes, each encoding at least one, and preferably two proteins, the expression of which is necessary for the growth and / or survival of the cell.

[0042] In one embodiment, the producer microbial cell comprises first and second essential genes encoding first and second proteins (wherein the first and second proteins may be the same or different), wherein expression of both the first and second proteins, respectively, is required for cell growth and / or survival.

[0043] Preferably, said at least one, preferably two, essential genes, and the expression thereof, does not indirectly cause a reduced production of said desired product by the producing cell.Furthermore, said essential genes as used in the context of the present invention do not code for or cause the synthesis of a desired product or intermediate of a heterologous pathway expressed in the producing microbial cell.

[0044] The at least one, preferably two, essential genes are preferably non-conditionally essential genes, the expression of which is essential for cell growth and / or survival regardless of the composition of the growth medium or growth conditions in which the cells are cultured.

[0045] In one embodiment of the invention, when the production cell is a prokaryote such as E. coli, the at least one, preferably two, non-conditionally 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 and homologues thereof.

[0046] In a further embodiment of the invention, when the production cell is a prokaryote, such as a Bacillus subtilis strain, the at least one, preferably two, non-conditionally essential genes are selected from the iscU operon, the accC operon, glmM, ylaN, infA, and dapA and homologs thereof.

[0047] In one embodiment of the invention, when the production cell is a prokaryote, such as a Corynebacterium strain, the at least one, preferably two, non-conditionally essential genes are selected from alr, glmM and homologues thereof.

[0048] In one embodiment of the invention, when the producing cell is eukaryotic, the non-conditionally essential genes are selected from FOL1, MED7, RRP40, NOP8, PGI1, NEP1 and homologs thereof of Saccharomyces cerevisiae, and the conditionally essential genes are selected from URA3, LEU2, TRP1, HIS3 and homologs thereof of Saccharomyces cerevisiae.

[0049] In one embodiment of the invention, when the production cell is a filamentous fungus such as Aspergillus, the essential genes are selected from ARG3, adeA, ERG10, PFS2 and TUB1 and homologs thereof.

[0050] In one embodiment of the invention, the at least one, and preferably two, essential genes are conditionally essential genes causing the synthesis of a product required for auxotrophic growth or the synthesis of a protein product required for resistance to growth inhibitors, such as antibiotics, specific toxins or protoxins.

[0051] When the at least one, and preferably two, essential genes are conditionally essential, it is necessary to adjust the composition (configuration) of the growth medium / growth conditions of the producing cells, for example, by using a growth medium that does not contain a specific nutrient or that contains a growth inhibitor.

[0052] In a further embodiment, the production cell may contain a combination of non-conditionally essential and conditionally essential genes.

[0053] [Table 1] TIFF2024531241000003.tif242150TIFF2024531241000004.tif118159

[0054] Gene essentiality can be determined by generating cells in which the respective gene is knocked out, where a non-conditional gene knockout results in zero cell viability or inability to grow regardless of growth conditions; whereas a conditional gene knockout results in inability to grow or zero cell viability depending on the media composition / culture conditions.

[0055] A suitable essential gene is one that is capable of expressing the native promoter of the essential gene to be assayed using an inducible promoter (L-arabinose inducible promoter). BAD (In the case of E. coli), xylose-induced p XYL(in the case of Bacillus subtilis or Bacillus licheniformis) or galactose-inducible p GAL (for Saccharomyces cerevisiae or Pichia pastoris), or plac (for Escherichia coli or Lactobacillus) or p thiA In such an essential gene assay, an integrating DNA construct containing the inducible promoter is integrated into the chromosome of a host microbial cell using standard methods. In the case of prokaryotes, the integrating DNA preferably contains an RBS catalog (e.g., Table 3) that directs the translation of the essential gene at different rates, which constitute the different baseline expression levels of the different promoters as well as the different baseline expression levels required for the essential gene to support growth at the corresponding wild-type specific growth rate. After targeted integration of the integrating DNA, the integrating 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 in a standard growth curve assay for the dependence of the cells' synthesis on the inducer conditions, and suitable essential genes can be identified as genes with identifiable inducer-dependent growth rates. Inducer-dependent growth can be observed as a greater than 5% decrease in growth rate in the absence of inducing conditions. Suitable essential genes are characterized by a decrease in log-phase specific growth rate under optimal inducer conditions in production-relevant media of less than 5%.

[0056] Methods for identifying essential genes are described in detail in "Gene Essentiality: Methods and Protocols", January 2015 [DOI 10.1007 / 978-1-4939-2398-4]; these methods allow a person skilled in the art to determine whether a gene is essential for microbial cell growth under specific growth conditions; and 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, mainly by screening transposon-tagged libraries. Furthermore, the various "computational tools" described in the above documents make the widespread availability of microbial whole genome sequences and structural features of many known essential genes readily available to a person skilled in the art to directly predict and identify genes encoding essential proteins in microbial genomes. Thus, the skilled artisan is provided with both databases of essential genes and various freely available online tools and algorithms that allow good and reproducible identification of a large number of essential genes present in the genomes of microbial cells without undue burden. It should be noted that 82% of essential genes in the budding yeast Saccharomyces cerevisiae code for essential proteins that are similar to proteins in other organisms [Giaever G et al., 2002], suggesting that most essential genes known or identified in one microorganism (e.g. yeast) have homologs in other microorganisms and can therefore be identified in the microorganism of interest by a simple BLAST search.

[0057] (II.iii) Load-sensitive promoter As described above, each of the producing microbial cells of the invention comprises at least one, preferably two load-sensing promoters operably linked to at least one, preferably two essential genes encoding at least one, preferably two proteins required for cell growth and / or survival. The load-sensing promoters are induced in producing microbial cells that are subject to a load and / or fitness cost due to the production or overproduction of a product. Typically, the induction of the load-sensing promoter occurs when the production of a product by the microbial cell results in a decrease in the cell growth rate due to a fitness cost of production. Importantly, the induction of the at least one, preferably two load-sensing promoters of the invention occurs due to the production of a product that puts the producing cell under "load" and not due to the product itself. The load dependency imposed by the invention encourages a wide range of producing microbial cell applications, since the use of the at least one, preferably two load-sensing promoters and essential genes to increase the productivity of the producing microbial cells is largely independent of the product being engineered.

[0058] Since the essential gene is operably linked to a load-sensing promoter, its transcription and expression level increases above basal level only when the load-sensing promoter is induced. A suitable load-sensing promoter is a promoter that, in the non-induced state, causes the expression of the operably linked essential gene to a basal level that significantly limits or suppresses cell growth and / or survival. The basal level expression of an essential gene is a level sufficient to support cell growth under production conditions (or logarithmic growth phase) and is equal to or less than 10%, 20%, 50%, 90%, or 95% of the growth rate of the corresponding cell in which the essential gene is operably linked to its native promoter. In load-dependent cells, the basal level of essential gene expression is such that the lack of activation of the load-sensing promoter by the load of efficient product synthesis and the resulting reduced growth rate constitute a selective disadvantage in the absence of product synthesis.

[0059] Cells containing a load-sensing promoter in an uninduced state will grow much slower than cells in which the essential gene is operably linked to its native promoter (Example 1, Figure 2). In contrast, productive microbial cells containing a load-sensing promoter linked to an essential gene show a significant increase in 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 a product, the productive microbial cells of the present invention can surprisingly achieve an initial growth rate that is the same as the growth rate of the parental microbial cells from which the productive microbial cells are derived, which lack the genes encoding the product or the metabolic pathway for the biosynthesis of the product (Example 1, Figure 4). Thus, under production conditions, the productive microbial cells of the present invention will have a selective advantage over escape variants of the productive cells that have ceased to synthesize the product and therefore have a reduced escape rate.

[0060] In order to increase the production of the desired product and further extend the period during which the producing cells are productive, two or more load-sensing promoters may be advantageously utilized to promote the growth of beneficial cell variants. By using two or more different load-sensing promoters to make the producing cells dependent, the highly productive cell variants can be more uniquely differentiated from less productive (genetic / non-genetic) cell variants that may arise over time from the same ancestral starting cell. For example, environmental factors (e.g., those resulting from external stress in the production culture) may activate the less productive cells to some extent, allowing them to grow.

[0061] By using two loading sensors controlling essential genes, the transcriptional space that dictates the selection regime based on the imposed dependency can be further tuned, which may be important to limit potential cellular escape modes where the loading-dependent response of a single loading biosensor may allow sufficient proliferation to cause productivity loss.

[0062] Table 2 provides a non-exhaustive list of potential load-sensing promoters.

[0063] In one embodiment, the load-sensitive promoter is heterologous with respect to the essential gene, but may be a native promoter with respect to the producing microbial cell, or may be a modified native promoter.

[0064] In one embodiment, the promoter comprises a natural TF / sigma factor binding site. A suitable load-sensing promoter is the promoter of the σ32 regulon of E. coli, since it is 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, while further suitable promoters are described in Nonaka et al., 2006.

[0065] In one embodiment, the load-sensitive promoter is a non-nutrient growth sigma factor regulated promoter. In a preferred embodiment, the load-sensitive promoter is a sigma factor regulated promoter. 32 The promoters are activated via a regulon and can be selected from the promoters of the E. coli genes htpG, ibpA, clpB, yccV, grpE, ycjX, ldhA, mutM, ybbN, prlC, groES, fxsA, and htpX.

[0066] In one embodiment, the load-sensitive promoter is the promoter of the mutM gene, which encodes a functionally conserved DNA glycosylase that is responsible for initiating the repair of one of the most common oxidative stress-induced DNA lesions, namely the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG) (Jain et al., 2007).

[0067] Other suitable load-sensitive promoters may be selected from the promoters of the E. coli genes rrnB, rrnE, cspD, katE, xthA, uspE, gadB, ahpC, katG, grxA, oxyS, poxB, trxC.

[0068] Load-sensing promoters also include promoters related to the carbon nutrient status and growth phase of cells.Thus, in one embodiment, the load-sensing promoter is the promoter of the toxin gene cspD, which is activated by growth rate slowdown and carbon starvation (Uppal et al., 2014; Yamanaka and Inouye, 1997);thus, it is expected to be differentially upregulated in cells that are burdened or subject to fitness costs by the production of metabolites.

[0069] In one embodiment, the load-sensitive promoter is a ribosomal RNA promoter, in particular the promoter of the rrnB and rrnE genes, which respond to nutrient supply, are induced by the Fis protein, and are inhibited by the alarm substance ppGpp. Rrn promoter activity is induced in cells with high protein production, observed in early logarithmic growth phase (Nonaka et al., 2006).

[0070] In one embodiment, the load-sensing promoter is a promoter responsive to oxidative stress associated with metabolic stress and heterologous production of microorganisms (Dragosits and Mattanovich, 2013). Examples of oxidative stress-responsive promoters include promoters controlled by E. coli OxyR or its homologues (such as PerR in Bacillus subtilis), which activate several genes related to cellular conditioning and protection in response to oxidative damage, and rpoS (σ S ) promoters.

[0071] For B. subtilis, suitable load-sensing promoters may be selected from HrcA-regulated promoters, including, for example, the promoter of groES. Other suitable load-sensing promoters may be selected from the promoters of the B. subtilis genes ctsR, dnaK, perR, hrcA, spx, sigB, yflT, mutM.

[0072] For Corynebacterium, such suitable load-sensing promoters may be selected from katA, cplX, mutM and groES.

[0073] For Saccharomyces, suitable load-sensitive promoters may be selected from the promoters of the genes KAR2, PDI1, SAA1, FPR2, RPL3, RPL6A, RPL28, OGG1, RAD51, RAD54.

[0074] For Aspergillus, such suitable load-sensing promoters may be selected from bipA, PDI, clxA, and homologs of OGG1, RAD51 and RAD54 of Saccharomyces cerevisiae.

[0075] [Table 2] TIFF2024531241000006.tif242148TIFF2024531241000007.tif242147TIFF2024531241000008.tif200159

[0076] The different load-sensing promoters (e.g., Table 2) have different basal expression levels and specific response curves upon induction / activation, which can be used to select the load-sensing promoter that is most compatible (in terms of strength) with a specific producing microbial cell and its product.

[0077] The gene of interest may be part of an operon, in which case preferably the first gene of the operon is used to define the relevant promoter sequence.

[0078] Suitable load-sensing promoters, induced by load and / or fitness costs in the producing microbial cell during production, can be selected from public databases known to the skilled artisan, e.g., for E. coli promoters, https: / / ecocyc.org; for Bacillus subtilis promoters (which also function as Corynebacterium promoters) and their respective cognate prokaryotes, https: / / bsubcyc.org; for Saccharomyces cerevisiae promoters and their cognate fungi, https: / / yeastgenome.org; and https: / / aspergillusgenome.org, which is a genome database (AspGD) of related Aspergillus species.

[0079] Suitable load-sensing promoters can also be verified 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 incorporated into product-synthesizing engineered producing microbial cells and corresponding non-producing cells (controls) isolated after serial dilution culture experiments in production medium for 50-150 cell generations or isolated after completion of a large-scale fermentation culture. The load-sensing promoter induces transcription of the candidate load-sensing promoter operably linked to fluorescent protein expression in the producing cells compared to the non-producing cells (controls), where the production cells are at least 5%, 7.5%, 10%, 15%, 25%, 60%, 150%, 300% induced transcription compared to the activity in the isolated corresponding non-producing cells.

[0080] (II.iv) Matching load-sensing promoters to producing microbial cells A producing microbial cell exhibits a transcriptional state characterized by the expression of a specific gene in response to the load or fitness cost of producing a product, but the expression of that specific gene is not induced in the corresponding non-producing parent cell or in the non-producing escape cells that arise from the producing microbial cell during productive fermentation. The physiological nature of the load or fitness cost will depend on the product synthesized by a given producing microbial cell; it will also be reflected in the type of gene whose expression is induced. Gene promoters that are induced in cells producing a given product can be identified by techniques well known in the art (e.g., transcriptomics; see Example 7). Many of the induced gene promoters are related or correspond to those listed in Table 2, so they provide a starting point for finding a load-sensing promoter that is compatible with a given producing microbial cell. Identifying 5-15 promoters of genes that are specifically upregulated in a producing strain of interest can broaden the promoter selection. As illustrated in the Examples herein, the method of using a load-sensing promoter operably linked to an essential gene to tailor a product-specific microbial cell is experimentally rapid and can be performed with simple laboratory equipment.

[0081] (II.v) Translational control elements To control the growth of the producer microbial cells of the invention by expression of an essential gene, the response threshold and response curve of the load-sensing promoter and the expression level of the essential gene must be balanced such that a basal level of essential gene expression supports limited or no growth, while in highly productive cells, the induced load-sensing promoter expresses the essential gene sufficiently to support a significant increase in growth rate, preferably a growth rate comparable to or no more than 5% lower than that of a producer cell lacking the load-sensing promoter of the invention, when measured during logarithmic growth phase.

[0082] One suitable approach to balance the load response of a load-sensing promoter to the expression level of an essential gene is to modify the translation strength of the essential gene. In bacteria, the translation strength is defined by the Shine-Dalgarno sequence / ribosome binding site (RBS) sequence directly upstream of the start codon, whereas in eukaryotic translation initiation regions, the translation strength can be modified using a translation initiation site (TIS) or Kozak element. RBSs of E. coli that provide a wide range of translation strengths are shown in Table 3.1, while further examples can be found in the literature (e.g., Bonde et al., 2016). A person skilled in the art can balance the translation strength of the essential gene to be controlled by constructing four variants of the ribosome binding site of each load-sensing promoter and testing which variant effectively controls the growth rate of the selected essential gene (Rugbjerg et al., 2018, PNAS). Exemplary RBSs for Bacillus licheniformis or Bacillus subtilis are shown in Table 3.2. An exemplary TIS for use with Pichia is shown in Table 3.3.

[0083] [Table 3]

[0084] [Table 4]

[0085] [Table 5]

[0086] (II.vi) Engineering of Load-Sensitive Promoters and Translational Control Elements The selected natural load-sensitive promoter is generally contained in the region -1 to -300 bp (upstream) of the natural regulatory ORF in prokaryotes and in the region -1 to -500 bp in eukaryotes. The sequence boundaries of the core promoters and their regulatory sites (e.g., transcription factor binding sites) that must be contained are common and well known (e.g., for σ32) (Nonaka et al., 2006). To avoid altering the regulatory properties of the selected promoter sequence, translational control elements / RBS can also be added downstream of the promoter.

[0087] (II.vii) Improved production levels In addition to the increased initial growth rate compared to non-producing cells, an unexpected advantage of the producing microbial cells of the present invention is that the producing cells have significantly improved productivity during large-scale fermentation over multiple cell divisions (simulated in Example 2; FIG. 6) when compared to producing microbial cells lacking engineered load dependency. As can be seen from the diversity of product types synthesized in the producing microbial cells shown in Examples 2-5, the unexpected advantage conferred by the load dependency in the producing microbial cells of the present invention is obtained regardless of the type of product synthesized.

[0088] In one embodiment, the producer microbial 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 generations of cell division from a single cell, as compared to a non-load-dependent producer cell with the same number of generations of cell division, where product yield is measured as moles or grams of product produced per unit substrate.

[0089] In one embodiment, after at least 50 generations of cell division from a single cell, the production level is increased by at least 10%, 25%, 50%, or 80% compared to a non-load-dependent producing cell with the same number of generations of cell division.

[0090] A common objective for which microbial cell factories are engineered is to minimize the known burdens caused by the maintenance and expression of heterologous pathways. The present invention provides a counter-empirical solution for improving cell factory yields, since it makes the growth and survival of microbial cells dependent on the cells producing a product, while imposing a certain burden or fitness cost. Without being limited by theory, it is speculated that the selective pressure on the productive microbial cells of the present invention and their progeny, where only the productive cells in the loaded state are able to grow / survive, serves to gradually select for high-load cell subpopulations during the long-term culture course of a starting population of originally genetically homogeneous cells.

[0091] [III] Methods for preparing and identifying cells of load-dependent producing microbial strains In a preferred method of carrying out the present invention, the load-dependent production microbial strain is prepared and identified by the following steps: To obtain the best-functioning relationship between the load-sensing promoter and the essential gene, it may be reasonable to prepare and test different candidate load-sensing promoters with different RBSs for controlling the selected essential gene.

[0092] (III.i) Identification of load-sensitive promoter candidates in specific production strains Candidate load-sensing promoters listed in Table 2 may be tested. Alternatively, unique positively differentiating gene transcripts detected in a particular production strain of interest may be identified, each of which promoters being a source of candidate load-sensing promoters. Preferably, such differentiating gene transcripts are identified by comparing the transcript profile of the production microbial strain (in production) with the isolation of genetic or non-genetic escape mutant variants from the corresponding production strain, such escape mutant variants being characterized by up to 50% slower production rates. Positively differentiating gene transcripts can be identified by utilizing transcriptomics; for example, by performing RNA sequencing of the transcribed RNA extracted from the producing microbial cells and comparing it with non-producing / low-producing escape mutant variants. The identified promoters can be tested in the producing microbial cells in combination with a given essential gene.

[0093] A method for identifying at least one, and preferably two, load-sensing promoters is exemplified in Example 7, where the promoters are operably linked to essential genes in the producing microbial cells selected for testing.

[0094] (III.ii) Introduction of load-sensitive promoters into the growth control process At least one, preferably two promoters selected from the list of general candidate load-sensing promoters (Table 2) or from the identified specific load-sensing promoters (see IIIi) are operably linked to essential genes and tested in the producing microbial cells. Different translation strengths of the essential genes can be tested simultaneously by using different RBS sequences for the homologous essential genes and testing different essential genes (as described in section IIIi). The essential genes can be native or heterologous with respect to the producing microbial cells.

[0095] If the essential gene is a native gene, its native homologous promoter may be destroyed (i.e., rendered non-functional), for example by mutation or deletion, and then a load-sensitive promoter (heterologous to the essential gene) is operably linked to the native essential gene by targeted introduction. In another embodiment, the native essential gene promoter is replaced by the load-sensitive promoter by targeted introduction.

[0096] Suitable methods for targeted introduction of genetic sequences in microorganisms include recombination in bacteria, such as lambda red recombination in E. coli; on the other hand, homologous recombination can be used in yeast and filamentous fungi. Both concepts can be optionally combined with CRISPR to increase gene replacement efficiency.

[0097] (III.iii) Screening for balanced load sensing and growth control The producer microbial cell clones containing an inserted load-sensing promoter operably linked to an essential gene are then screened to identify clones in which the load-sensing promoter controls the essential gene in a growth-regulated manner. For example, a number of such clones (e.g., 8-96 clones) are compared to cells of the corresponding non-load-dependent producer microbial strain for growth under conditions that control the production of the product (e.g., using a producer microbial strain in which the production gene is inducible).

[0098] When cell growth is measured under conditions of low / no product synthesis by both strains, a suitable load-dependent production clone is one that exhibits a growth rate that is significantly lower than the non-load-dependent production strain, and preferably at least 5% slower (Example 1, Figure 2). In contrast, when production is high / occurring, upregulation of essential gene expression in a suitable load-dependent production clone means that the difference in growth rate is significantly reduced or even completely eliminated, compared to the corresponding non-load-dependent production strain.

[0099] (III.iv) Verification that central and product metabolism are not perturbed Changes in transcriptional regulation or expression of essential genes may result in unnecessary indirect perturbation of production genes and central carbon or nitrogen metabolism, resulting in reduced product production, thereby reducing the load of the load-dependent producing microbial cells. To exclude such clones, cell growth is measured under conditions in which both strains synthesize the product (i.e., under conditions in which the suitable load-dependent producing clone is a clone that shows a growth rate equal to or slower than the non-load-dependent producing strain) (as shown in Example 2, Figure 7).

[0100] (III.v) Growth rate stability screening Clones that meet the above criteria are tested for growth rate stability under production-mimicking conditions, for example by serial passage and measuring growth rate, or by sampling at each stage of the large-scale production process, through at least 20 generations of cell division. Suitable load-dependent producer microbial cells maintain a slower growth rate over time than non-load-dependent producers (e.g., Example 2, Figure 7).

[0101] [IV] Methods for Producing Desired Products Using Cells of Load-Dependent Producing Microbial Strains A second aspect of the present invention relates to a method for producing a desired product, the method comprising the steps of: (a) providing a producer microbial cell genetically engineered to synthesize at least one product; wherein said at least one cell further comprises at least one, and preferably two, essential genes operably linked to at least one, and preferably two, load-sensitive promoters; and wherein said promoter is heterologous with respect to said essential gene; (b) introducing at least one genetically modified microbial cell into a medium containing a substrate for producing the product; (c) recovering the product synthesized by the culturing; wherein synthesis of said product imposes a burden and / or fitness cost on said at least one cell; and wherein when said at least one, preferably two load-sensitive promoters are induced by said load and / or fitness cost, expression of said at least one, preferably two essential genes is upregulated compared to basal level expression of said at least one, preferably two essential genes; and wherein lack of synthesis of said product in said at least one producing cell or a progeny thereof reduces the proliferation rate of said at least one cell.

[0102] The method for producing the desired product may include providing a cell culture of a producing microbial cell genetically engineered to synthesize at least one product, and introducing the cell culture of the at least one genetically engineered microbial cell into a culture medium containing a substrate for the production of the product. The cells are cultured in a medium that allows the cells to produce the desired product and supports growth. The culture time may be optimized according to the desired product.

[0103] The method of producing the desired product may further comprise the step of isolating the product and / or formulating the product into a composition, such as a nutritional composition, a pharmaceutical composition, a cosmetic composition, a surfactant composition, a lubricant composition, or a fuel composition.

[0104] [V] Use of Load-Dependent Producer Microbial Strain Cells to Produce Desired Products A third aspect of the present invention relates to the use of a load-dependent producing microbial cell of the present invention to produce a desired product, where lack of synthesis of the product in said load-dependent producing strain or its progeny reduces the growth rate of said strain, even if the product is present in the intracellular or extracellular environment.

[0105] Working Example Example 1: Engineering a load-dependent E. coli strain producing recombinant human growth hormone (1.1) The maintenance and expression of heterologous genes by cell factories constitutes a non-native burden on the cells The growth rate of parental E. coli BL21(DE3) cells was compared with that of engineered derivatives transformed with plasmid pEG34, which contains the gene encoding recombinant human growth hormone (hGH) fused to green fluorescent protein (GFP).

[0106] 1.1.1 Materials and Methods Cells of the host E. coli strain BL21(DE3) (Yale University E. coli Stock Center) were treated to make them electrocompetent and transformed with plasmid pEG34 by standard electroporation methods (1800 V, 25 μF, 200 ohms, 1 mm cuvette width) and plated on LB agar plates containing chloramphenicol.

[0107] [Table 6]

[0108] Single colony transformant precultures were grown in 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 in 96-well microtiter plates with fast horizontal shaking in an Elx808 plate reader (Biotek) at 37° C., reading OD630 every 10 min. The initial OD630 reading was used to subtract background from the OD630 values.

[0109] (1.1.2) Results E. coli cells containing the hGH-GFP expression plasmid pEG34 grew more slowly than the parental E. coli host cells from which they were derived (Fig. 1 ), indicating a burden or strain imposed on the cells as a result of allocating cellular resources to the maintenance and expression of the synthetic construct.

[0110] (1.2) Engineering load-dependent cell factories A load-dependent strain of the parent E. coli host was genetically engineered to incorporate a genetic circuit designed to confer a selective fitness advantage to the cell factory's productive cells. Specifically, the native promoter of the essential gene operon folP-glmM in the E. coli BL21(DE3) chromosome was replaced by a load-sensitive promoter (p mutM , p yccV , or p ycjX ) was substituted for the gene circuit, which further contained an RBS between the promoter and the essential gene; here, four different RBSs (Table 3) were tested with the aim of modulating the expression level of the essential gene. The growth rate of the strain containing the gene circuit was compared to the parent host E. coli strain BL21(DE3).

[0111] 1.2.1 Materials and Methods Load-sensitive promoters: Promoter p was synthesized by amplifying a 0.3 kb region immediately upstream of each gene (see Table 3) by PCR using the specific primers in Table 5 and genomic DNA from lysed E. coli BL21(DE3) cells as template. mutM , p yccV , or p ycjX 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 (1x); 95°C for 20 s, 68–58°C (touchdown) for 30 s, 72°C for 60 s (35x); 72°C for 300 s (1x); stand at 15°C.

[0112] [Table 7]

[0113] USER cloning was used to create an integration sequence that contains an amplified promoter region fused to a linear 1.5 kb DNA fragment containing a 221 bp targeting sequence identical to the 221 bp immediately upstream of the folP gene and the kanR gene for selection of the correct recombinant product. The amplified promoter region and the kanR-folP fragment were mixed in equimolar amounts and fused together by adding 1 μl of 10xT4 ligation buffer (Thermo Scientific) and 0.75 μl of USER enzyme (New England Biolabs) to adjust the total reaction volume to 10 μl. The USER reaction was then placed at 37°C for 30 minutes. The reaction was then placed at room temperature for 15 minutes, after which 0.75 μl of T4 DNA ligase (Thermo Scientific) was added and incubated at room temperature for 30 minutes. An example of such an integration sequence containing a promoter, a KanR resistance gene, and a folP targeting sequence can be given in SEQ ID NO: 140 (s9_pmutM_folP) in the Sequence Listing. The ligated products were then amplified at approximately 250 ng / μl with primer "rev" (according to the specific promoters in Table 5) and P493 (Table 5) using the following PCR reaction protocol: 98°C for 180 sec (1x); 85°C for 20 sec, 72-68°C (touchdown) for 30 sec, 72°C for 60 sec (35x); 72°C for 5 min (1x); stand at 15°C. The primer overhangs provided a 50 bp targeting sequence identical to folP that targets direct recombination into the folP locus.

[0114] Chromosomal integration of a load-dependent promoter: A promoter was integrated upstream of the folP gene in the genome of E. coli BL21(DE3) cells as follows. 600 μl of a culture of BL21(DE3) previously transformed with pSIM5-tet (Koskiniemi et al., 2011) and grown overnight was added to 100 ml of 2xYT medium containing tetracycline. The cells were grown at 30°C and when they reached OD600=0.20, the culture was transferred to a shaking bath at 42°C for 15 min to allow expression of the recombinant enzyme on pSIM5-tet. The culture was then transferred to 2× cold 50 ml centrifuge tubes and centrifuged at 4000g for 10 min, 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 4000g for 6 min, 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 4000g for 6 min and each cell pellet was carefully resuspended and pooled in 495 μl of ice-cold 10% glycerol. 90 μl of the resuspended cells were added to electroporation cuvettes, each containing 1 μl (>250 ng) of one of the load-sensitive promoter integration sequences. The cells were electroporated with the following settings: 1800V, 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 left to recover at 37°C for 1.5 hours in a 1.5 ml Eppendorf tube. An overnight incubation at room temperature was allowed for recombination of the electroporated cells, which were then plated on LB agar plates containing 50 mg / L kanamycin and grown overnight at 37°C to ensure recovery of pSIM5-tet. The precise targeting of the essential gene locus was verified using primers P525 and P526. R Colonies were selected that had a size at or below the average size of the colony population.

[0115] The integration of the loading-dependent promoter in the genome of kanR-selected colonies was verified by colony PCR using Taq DNA polymerase and primers targeting the folP promoter region, and the identification of the RBS in the selected colonies was performed by Sanger sequencing.

[0116] The growth rate of strains containing the gene circuits was then compared to the parent host E. coli strain BL21(DE3) by culturing in 200 μl of 2xYT containing 500 mM IPTG at 37°C with horizontal shaking.

[0117] (1.2.2) Results The growth rates of the selected load-dependent E. coli strains were slower to various degrees compared to the parental E. coli host from which they were derived (Figure 2; shows the growth of load-dependent strains s5.0#3, s7.0#8, and s9.0#8, in which the essential genes folP-glmM are, respectively, driven by the load-sensing promoter p yccV , p ycjX , and p mutM Since cell growth depends on the expression levels of the essential folP-glmM genes, we conclude that the expression levels of the essential genes driven by their respective load-sensing promoters in the non-productive, load-dependent strains are insufficient to support growth at the level of the wild-type parent strain.

[0118] The reduced growth rate observed in these load-dependent strains is a measure of the penalty imposed on non-producing cells that naturally evolve during the culture of a cell factory population containing a load-dependent gene circuit. The magnitude of this penalty was determined by selecting for a load-sensitive promoter in combination with the strength of the selected RBS. The greater the penalty, the wider the "negative selection" window for low- or non-producing variants that naturally arise during culture (e.g., due to mutations in a productive gene).

[0119] (1.3) Production increases the growth rate of load-dependent cell factories A load-dependent E. coli strain with a non-productive growth penalty (FIG. 2) was used as a host strain to demonstrate the selective fitness advantage of productive load-dependent cells.

[0120] 1.3.1 Materials and Methods The slowest growing clones of the generated load-dependent E. coli strains (s9.0#8; s5.0#3; and s7.0#8) were made electrocompetent and transformed with pEG34 or pEG0 (Table 4) by standard electroporation (1800V, 25μF, 200 ohms, 1mm cuvette width), respectively, and plated on 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 200μL of 2xYT medium (500μM IPTG, chloramphenicol); grown at 37°C with fast horizontal shaking and OD630 readings every 10 minutes in an Elx808 plate reader (Biotek). The initial OD630 reading was used to subtract background from the OD630 values.

[0121] The E. coli strains were transformed with plasmids pEG34 or pEG0 (Table 4), respectively, to induce the cells to synthesize recombinant hGH protein fused to GFP, and the cell growth rate characteristics upon induction were measured.

[0122] (1.3.2) Results The load-sensing promoter p controls the expression of the folP-glmM essential genes mutM E. coli strain s9.0#8, a load-dependent strain containing the β-glucosidase inhibitor ... mutMdemonstrate that the synthesis of hGH-GFP recombinant protein is induced by the stress or burden placed on the cells, which in turn leads to upregulated expression of the folP-glmM genes and enhanced logarithmic growth.

[0123] hGH-GFP synthesis in each of the load-dependent E. coli strains s5.0#3, s7.0#8, and s9.0#8 containing the pEG34 plasmid not only abolished the decline in logarithmic growth rate, but furthermore, the growth rates of these strains were comparable to that of the parental E. coli BL21(DE3) (Figure 4).

[0124] In summary, this example demonstrates that a load-dependent genetic circuit can be used to confer a selective fitness advantage to cells in a cell factory whose synthesis of a protein or metabolite is high enough to constitute a load or burden, and that this load or burden can be detected by a load-sensitive promoter operably linked to an essential gene in the cell. In contrast, non-productive variant cells (e.g., resulting from mutations in productive genes) that arise naturally during the culture of a load-dependent cell factory are often subjected to negative selection pressure because their growth slows down to a rate dictated by the basal expression of essential genes. The reduced growth rate itself is slow enough to slow the increase in frequency of such variant cells in the cell factory, thereby slowing the decline in productivity of the cell factory over time.

[0125] Example 2: Load-dependent E. coli strains producing human growth hormone show enhanced long-term production stability The load-sensing promoter is shown to be a promoter that senses and is activated by the cellular load-induced state resulting from cellular synthesis of hGH-GFP recombinant protein. Once activated, the load-sensing promoter is shown to elevate expression of the essential genes folP-glmM to a level sufficient to confer a selective growth advantage to the cells when compared to non-producing cells. To maximize the dynamic range of essential gene expression in response to its cognate load-sensing promoter, the load-sensing promoter is randomly combined with variant RBS coding sequences (Table 3) that confer different translational strengths.

[0126] Promoters with suitable load-sensing properties for use in load-dependent gene circuits are shown to include promoters responsive to heat shock, DNA damage, oxidative stress, as well as rRNA promoters, as exemplified by the following engineered production strains grown under simulated large-scale production conditions:

[0127] First, for each of the load-sensitive promoters, a number of clones containing random variant RBS coding sequences were selected and their hGH-GFP synthesis was followed over multiple cell divisions in order to demonstrate their relative abilities to increase / sustain recombinant hGH-GFP synthesis over time.

[0128] (2.1) Materials and Methods Load-sensitive promoters: PCR and USER cloning using the specific promoters in Table 6 was performed by the method described in Example 1.2.1 to obtain promoter p yccV , p ycjX , p ibpA , p grpE , p ldhA and p ybbN , and their respective integration sequences were generated; whereas the products joined by ligation were amplified with primers "rev" (for the specific promoters in Table 6) and P493 (Table 5).

[0129] [Table 8]

[0130] Chromosomal integration of load-dependent promoters: Each of the promoters was integrated upstream of the folP gene in the genome of E. coli strain BEG34 (corresponding to E. coli BL21(DE3) containing plasmid pEG34) in the following manner: Cells of E. coli strain BEG34 previously transformed with recombinant plasmid pSIM5-tet were prepared and transformed with each of the promoter integration sequences by electroporation as described in Example 1.2.1. After the described recombination and pSIM5-tet recovery steps, 8 colonies were selected from each plate, corresponding to 8 clones with the same promoter integration but with random variant RBS sequences (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 the maintenance of plasmid pEG34. Before freezing the 96-well plates, 2 μl of each culture clone was used to verify the promoter integration by colony PCR as described in Example 1.2.1.

[0131] Short-term hGH-GFP production screening assay: Frozen 96-well plates were thawed and cells were transferred to new 96-well plates containing 200 μL of 2xYT supplemented with chloramphenicol using a pin replicator. The plates were sealed with a BreathEasy sealing membrane (Sigma-Aldrich) and placed overnight (20 h) at 37°C with reciprocal shaking at 754 rpm on a SynergyH1 plate reader (Biotek); OD (600 nm) and GFP fluorescence (ex / em 485 nm / 528 nm) of each well were measured every 10 min during the 20 h period. The plates were then shaken at room temperature for 4 h on a standard benchtop plate shaker, after which 2 μL of culture was removed from each well and transferred to new 96-well plates containing 200 μL of 2xYT supplemented with chloramphenicol and 0.5 mM IPTG. The new 96-well plate was similarly sealed with a BreathEasy sealing membrane (Sigma-Aldrich) and placed overnight (20 hours) at 37°C with reciprocal shaking at 754 rpm on a SynergyH1 plate reader (Biotek); during the 20 hours, the OD and GFP fluorescence of each well was measured every 10 minutes at 600 nm and ex / em 485 nm / 528 nm. The next day, 2 μL of 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.

[0132] Long-term hGH-GFP production assay (Figure 6): A single selected colony of each load-dependent hGH-GFP producer was used to inoculate a 15 mL Greiner culture tube containing 4 mL of 2xYT supplemented with chloramphenicol and 0.5 mM IPTG. The culture was grown for 23 hours at 37°C on a shaking table at 250 rpm. 2 μL of each culture was then diluted 2000-fold (corresponding approximately to 11 cell division generations) and inoculated into a new Greiner culture tube under identical conditions; in this case, the steps of the method were repeated for a total of 12 seeds. After each passage, a 200 μL sample was taken from the culture and hGH-GFP synthesis was determined by measuring OD600 and GFP fluorescence (ex / em 485 nm / 528 nm) in a SynergyH1 plate reader (Biotek).

[0133] Long-term hGH-GFP production assay (Figure 8): A single selected colony from each load-dependent hGH-GFP producer was used to inoculate 1.8 mL of 2xYT with chloramphenicol and 0.5 mM IPTG in a 24-well deep-bottom plate. The cultures were grown for 23 hours at 30°C on a shaking table at 200 rpm. 2 μL of each culture was then diluted 1000-fold (corresponding to approximately 10 generations of cell division) and inoculated into a new deep-well plate under the same conditions. In this case, the steps of the method were repeated for a total of 9 seeds. After each passage, a 200 μL sample was taken from the culture to determine hGH-GFP synthesis by measuring OD600 and GFP fluorescence (ex / em 485 nm / 528 nm) in a SynergyH1 plate reader (Biotek).

[0134] Measurement of growth rate of selected strains: The selected h-hGH high-producing strains s3.6#2, s6.6#6, s7.6#8 and s10.6#7 were plated on LB agar plates containing chloramphenicol and kanamycin. BEG34 and BEG0 strains were plated on LB agar plates containing chloramphenicol. Seven colonies from each plate were used to inoculate a 96-well plate containing 200 μl of 2xYT containing chloramphenicol. The plate was sealed with a BreathEasy sealing membrane (Sigma-Aldrich). Their growth was measured overnight at 37°C with reciprocal shaking at 754 rpm on a SynergyH1 plate reader (Biotek). A 2 μl sample was taken from each well and transferred to a well of a new 96-well plate containing 200 μl of 2xYT containing chloramphenicol and 0.5 mM IPTG. The 96-well plates were sealed with Breatheasy membrane and incubated at 37° C. with reciprocal shaking at 754 rpm for 20 hours, with growth (OD600nm) measured every 10 minutes using a Synergy H1 plate reader.

[0135] Strain catalogue: BEG34 = E. coli BL21(DE3) harboring pEG34; BEG0 = E. coli BL21(DE3) carrying the empty plasmid pEG0 sX.Z#Y = E. coli BL21(DE3) carrying pEG34 with the wild-type folP promoter substituted for the load-dependent promoter X; where X represents the promoter ID in Table 2. The number Y after the # represents a clone selected from a pool of four RBS variants introduced by degenerate primers (Table 7). Z represents the production genes for hGH-GFP (0 ​​or 6), mevalonate (1), and lysostaphin (4), respectively.

[0136] [Table 9]

[0137] (2.2) Results (2.2.1) Short-term hGH-GFP productivity screening Heat shock promoter (p ibpA , p grpE , p ycjX , and p ybbN The productivity of four hGH-GFP producing strains containing a load-sensing promoter selected from the group of s3.6#2 (pG34) and one of four RBS coding sequence variants was tested under simulated large-scale production conditions by the following method. The strains were serially passaged daily for six consecutive days by 100-fold back dilution, corresponding to approximately six generations per seed. By day 6 (seed 6), multiple strains had elevated hGH-GFP synthesis compared to the non-load-dependent producer BEG34 in both seed 1 and seed 6. Compared to the BEG34 strain, the strain with short-term high hGH-GFP productivity was s3.6#2 (pG34). ibpA ), s6.6#6(p grpE ), s7.6#8(p ycjX ), and s10.6#7(p ybbN ) (see Figure 5).

[0138] (2.2.2) Improving long-term hGH-GFP productivity The productivity of hGH-GFP in the load-dependent strains s3.6#2, s6.6#6, s7.6#8, s10.6#7, as well as the non-load-dependent strain BEG34 and the non-producing strain BEG0, was monitored by sequential passaging under simulated large-scale fermentation. After seed 2, the load-dependent strains s3.6#2, s6.6#6, s7.6#8, s10.6#7 and the non-load-dependent control strain BEG34 were all equally good at synthesizing hGH-GFP (Figure 6). However, after about 20 more generations (seed 4), the productivity of hGH-GFP in the control strain BEG34 dropped significantly, and by seed 6 this strain had essentially stopped producing. Unexpectedly, the productivity of the load-dependent strain s7.6#8 (p controlling folP-glmM) was significantly higher than that of the control strain BEG34 (Figure 6). ycjX The load-dependent strain s3.6#2 (containing the folP-glmM controlling p ibpA s10.6#7 (having folP-glmM controlling p ybbNs6.6#6 (having folP-glmM controlling p grpE (having the same structure as the load-independent strain BEG34) also showed significantly better long-term productivity compared to the non-load-dependent strain BEG34.

[0139] (2.2.3) Growth rate 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 faster than the non-load-dependent hGH-GFP producing strain BEG34, and therefore their improvement in hGH-GFP production levels over time was not simply due to lower initial production levels and therefore essentially lower loading (Figure 7).

[0140] (2.2.4) Dependence of load-dependent gene circuits on selected essential genes The heat shock promoter p controls the essential genes folP-glmM fxsA We compared the hGH-GFP-producing load-dependent strain (s13.6#2(fxsA)) containing folP with a mutant derivative strain s13.6#2evo(fxsA) containing a frameshifted folP that abolishes folP expression, whose growth is dependent only on glmM expression. Although the loss of folP expression resulted in a reduced growth rate in 2xYT complex medium (data not shown), the essential gene glmM alone is sufficient to enhance the long-term stability of hGH-GFP production in the load-dependent strain s13.6#2evo(fxsA) to a similar level as that of strain s7.6#8(pycjX) when compared with the non-load-dependent hGH-GFP-producing strain BEG34 (Figure 8).

[0141] (2.2.5) Ribosomal RNA promoter utilization in load-dependent gene circuits In addition to the heat shock promoter, p rrnB and p rrnE These results indicate that ribosomal RNA promoters such as ribosomal RNA promoters can sense and respond to the load-dependent state of cells to control the expression of essential genes, such as enhancing the long-term productivity of hGH-GFP producing strains. As can be seen from Figure 9, the s15.6.7 strain (p rrnB) and strain s16.6.6 (p rrnE ) was significantly improved in comparison with the non-load-dependent hGH-GFP producing E. coli strain BEG34 under simulated large-scale production conditions corresponding to approximately 110 generations of cell division.

[0142] (2.2.6) Use of oxidative stress-sensing promoters in load-dependent gene circuits p poxB These results indicate that oxidative stress-sensing promoters such as s29.6#3 (p poxB Both the production level and long-term stability of hGH-GFP production by pEG34 were significantly increased compared to the non-load-dependent hGH-GFP producing E. coli strain BEG34 under simulated large-scale production with 7 successive passages corresponding to approximately 90 generations and long-term cultivation of the final seed. As can be seen in Figure 10, p poxB Production using a load-dependent genetic circuit based on surprisingly resulted in increased production over time suggesting enrichment of non-genetic high-performing variants in the population.

[0143] In summary, this example shows that coupling the transcription of the essential genes folP-glmM to one of the E. coli heat shock-, oxidative stress- and DNA damage-responsive promoters, and rRNA promoters improves the long-term stability and productivity of the load-dependent engineered E. coli strain synthesizing human growth hormone fused to GFP.

[0144] Example 3: Load-dependent E. coli strains producing lysostaphin exhibit enhanced long-term production stability Lysostaphin is a 27 kDa endopeptidase that cleaves the pentaglycine-linked bridges of the cell wall peptidoglycan of Staphylococcus aureus obtained by cell lysis. This antibacterial substance can be synthesized in E. coli by recombinant techniques. As shown by the load-dependent E. coli strains below that produce lysostaphin under simulated large-scale production conditions, it is shown that a load-dependent gene circuit containing a promoter with load-sensing properties enhances the production stability of lysostaphin synthesis in engineered E. coli strains and others. It is further shown that the productivity of load-dependent production strains is optimized by strain selection that combines a load-sensing promoter with an RBS sequence (Table 6) that confers optimized translation strength of the cognate essential gene. These advantages are further shown by the load-dependent strain s6.4#5 (p grpE ), which shows that this strain is more resilient to loss of productivity due to its dependency on the burden or fitness costs due to lysostaphin expression.

[0145] (3.1) Materials and Methods Load-sensitive promoter: p shown in Table 6 grpE PCR was performed using specific primers according to the method described in Example 1.2.1. grpE The promoter was amplified to obtain promoter p grpE A promoter integration sequence containing the following was created.

[0146] Chromosomal integration of a load-dependent promoter: In the genome of the E. coli strain BENDU5cam (corresponding to E. coli BL21(DE3) containing the lysostaphin-producing plasmid pENDU5cam), the grpE promoter was integrated upstream of the folP-glmM gene in the following manner. Cells of the E. coli strain BENDU5cam that had previously been transformed with the recombinant plasmid pSIM5-tet were prepared, and p grpEThe promoter integration sequence was used to transform by electroporation as described in Example 1.2.1. After the steps of recombination and recovery of pSIM5-tet described, 5 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 the maintenance of the plasmid pENDU5cam. Before freezing, 2 μl of each cultured clone was used to verify the integration of the promoter as described in Example 1.2.1.

[0147] Lysostaphin production screening: Using a screening assay, the E. coli strain s6.4#5 (p grpE ) was identified, but the assay showed that the RBS and p grpE The other three potential combinations of were cultured in 2xYT containing chloramphenicol using a 96-well format for six successive passages of 1000-fold dilutions (corresponding to 60 generations). The strains were grown in 3 mL of 2xYT medium containing 30 mg / L chloramphenicol in 15 mL culture tubes at 37° C. for 21 hours. 150 μL of culture was mixed with 50 μL of 50% glycerol and stored at −80° C. in a 96-well plate. Additionally, 30 μL of culture was transferred to 3 mL of fresh 2xYT medium supplemented with chloramphenicol and grown under the same conditions for another 21 hours. The above passages were repeated a total of five times, with frozen stocks of overnight cultures being made for each passage.

[0148] Lysostaphin Expression and Detection Assay: A 96-well plate containing all frozen stocks from passage 5 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 grown on a plate reader at 37° C. until most wells reached OD630=0.35; then 10 μl of 2xYT supplemented with 30 mg / L chloramphenicol and 20 mM IPTG was added to each well to bring the final concentration of IPTG to 1 mM, sufficient for induction of lysostaphin gene expression. The induced culture was grown at 37° C. for 4 hours, and then the plate was centrifuged at 4000 RPM for 10 minutes. 100 μl of the supernatant was added to a new 96-well plate containing 100 μl of S. aureus from an overnight culture. The lysis of S. aureus in each well was monitored by OD630 every 10 minutes for 2 hours at 37° C. using a plate reader. The rate of lysis of S. aureus was quantified by dividing the change in OD of S. aureus by the 60 minute time frame in which the change occurred, using the following equation:

number

[0149] (3.2) Results p grpE Load-dependent lysostaphin-producing strains harboring one of four RBS coding sequence variants controlling the expression of the promoter and essential genes folP-glmM were cultivated under simulated large-scale production conditions; the simulated large-scale production conditions were achieved by sequential passaging. As can be seen in FIG. 11, there was a sharp decline in production in strain s6.4#5 (p grpE ) maintained a fairly high lysostaphin production level. grpE ) and BENDU5cam strains had the same initial lysostaphin production rates. grpE) is not due to the inherently lower initial load from lysostaphin production.

[0150] In summary, the transcription of the essential genes folP-glmM was regulated by the heat shock promoter p grpE By coupling to , it was demonstrated that load-dependence improves long-term stability and production in E. coli engineered to synthesize secreted lysostaphin.

[0151] Example 4: Load-dependent E. coli strains producing mevalonate show 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 an acetyl-CoA pool. A load-dependent gene circuit containing a promoter with load-sensing properties and controlling the transcription of essential genes is shown to enhance the production stability of such mevalonate-producing E. coli strains under simulated large-scale production conditions. Productivity of the load-dependent production strains is shown to be further optimized by strain selection of a load-sensing promoter combined with an RBS coding sequence (Table 5) that confers optimized translation strength of the cognate essential genes. Specifically, the load-dependent promoter pMevT controlling folP-glmM transcription is shown to enhance the production stability of such mevalonate-producing E. coli strains under simulated large-scale production conditions. cspD (oxidative stress and glucose starvation-sensing promoter), or p mutM Mevalonate-producing strains containing (heat shock / DNA damage-sensing promoters) are shown to be more resilient to loss of productivity than non-load-dependent strains, both due to their dependence on transcriptional signals generated by mevalonate production.

[0152] (4.1) Materials and Methods Load-sensitive promoter: The promoter shown in Table 8 was prepared by the method described in Example 1.2.1. mutM Specific and p cspD PCR using specific primers mutM Promoter and p cspDThe promoter was amplified to obtain promoter p mutM and p cspD A promoter integration sequence containing the following was created.

[0153] [Table 10]

[0154] Chromosomal integration of a load-dependent promoter: In cells of the E. coli strain BL21(DE3)pMevT containing the lysostaphin-producing plasmid pMevT, the promoter was integrated upstream of the folP-glmM gene in the genome using the following method. mutM and p cspD were individually integrated. Cells of E. coli strain BL21(DE3)pMevT, 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 recombination and pSIM5-tet recovery steps, 5 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 and cultured in a well of a 96-well plate containing 200 μl of 2xYT supplemented with chloramphenicol for the maintenance of plasmid pMevT. Before freezing the 96-well plate, 2 μl of each cultured clone was used to verify the integration of the promoter as described in Example 1.2.1.

[0155] Mevalonate production screening: Using a screening assay, the best mevalonate-producing E. coli strain s19.1.1(p cspD ) and s9.1.4(p mutM) was identified. In parallel with the non-load-dependent producing E. coli strain BL21(DE3)pMevT, strains containing the other four potential combinations of RBS and promoters were grown in 96-well plates over six successive passages of 1000-fold dilutions (corresponding to 60 generations) in the following manner. The above strains were grown in 200 μL of 2xYT medium containing 30 mg / L chloramphenicol and 0.5 mM IPTG in microtiter plates sealed with a BreathEasy sealing membrane for 21 h at 37°C with horizontal shaking. 150 μL of culture in the 96-well plate was mixed with 50 μL of 50% glycerol and stored 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 another 21 h under the same conditions. The above passages were repeated a total of five times, and frozen stocks were prepared from overnight cultures for each passage.

[0156] Mevalonate synthesis and detection assay: 96-well plates containing frozen stocks of passages 2, 5, and 6 were thawed on ice and used to inoculate 10 mL of 2xYT containing 0.5 mM IPTG and 30 mg / L chloramphenicol and grown at 37°C for 54 h with horizontal shaking (250 rpm). 300 μL of each culture was treated with 23 μL of 20% sulfuric acid and vigorously shaken. Then, the mixture was centrifuged at 13,000×g for 2 min to collect at the bottom. Supernatant (medium) samples were injected onto an Aminex HPX-87H ion-exclusion column (300 mm×7.8 mm, Bio-Rad Laboratories) in a high-performance liquid chromatography Ultimate3000 with a 5 mM sulfuric acid mobile phase (0.6 mL / min) at 50°C. A refractive index detector was used for detection. A standard curve for mevalonic acid was generated using mevalonolactone (Sigma-Aldrich) dissolved in 2xYT medium supernatant of a non-producing E. coli strain incubated under identical conditions.

[0157] (4.2) Results Promoter p cspD or p mutMand one of four RBS coding sequence variants controlling the expression of the essential genes folP-glmM were cultivated under simulated large-scale production conditions; the simulated large-scale production conditions were achieved by sequential 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 a significantly higher mevalonate production level compared to the rapid decline in production observed in the non-load-dependent mevalonate-producing strain BL21(DE3)pMevT. The initial mevalonate production rate of each load-dependent strain was identical to that of the control BL21(DE3)pMevT strain at seed 0, indicating that strain s19.1.1 (p cspD ) and strain s9.1.4 (p mutM ) is not due to the inherently lower initial load from mevalonate production.

[0158] In summary, transcription of the essential genes folP-glmM was regulated by the E. coli oxidative stress- and glucose starvation-sensing promoter p cspD , as well as the heat shock / DNA damage sensitive promoter p mutM By coupling to , it was demonstrated that load-dependence improves long-term stability and production in E. coli engineered to synthesize mevalonate.

[0159] Example 5: Evaluation of promoters as load sensors to render yeast cells dependent on the load of engineered recombinant protein production

[0160] Promoters capable of inducing expression of essential genes after sensing cellular load or strain caused by recombinant expression of a protein or biosynthetic pathway can be used to create load-dependent genetic circuits for use in yeast. Methods for evaluating candidate promoters are exemplified with yeast cells genetically engineered to synthesize recombinant human serum albumin (hSA) or insulin precursor (IP), optionally translationally fused to green fluorescent protein (GFP).

[0161] To render growth responsive to the activity of a candidate load-sensing promoter, homologous recombination of a linear DNA construct transduced into yeast cells with a standard selectable marker is used to genetically replace the native promoter of an essential gene in the yeast Saccharomyces cerevisiae with a candidate promoter. As an example, the candidate promoter may 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 response (UPR) promoters that are upregulated by the HAC1 transcription factor (Kimata et al., 2006). The expression level of the essential gene may need to be fine-tuned to allow the activated load-sensing promoter to confer a selective growth advantage on the cell compared to a non-sensing promoter. A range of translational strengths can be engineered by varying the translation initiation region introduced together with the load-sensing promoter.

[0162] Clones harboring potentially different combinations of candidate load-sensitive promoters were selected and evaluated for sustained protein production over 30-100 generations of cell division.

[0163] (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 yeast nitrogen base without amino acids and ammonium sulfate, but lacks uracil.

[0164] Chromosomal integration and verification of promoter constructs: Chromosomal integration constructs will contain 300-600 bp upstream of the naturally regulated gene. Chromosomal integration of the promoter constructs will be achieved by transformation of Saccharomyces cerevisiae and homologous recombination using standard electroporation procedures. Correct integration of the promoter into the chromosome was verified using colony PCR.

[0165] Long-term cultivation and production: A single colony of each strain is transferred to a 24-well deep-well plate and cultivated in 1.8 mL of YPD medium under recombinant protein production-inducing conditions at 250 rpm and 30°C. After 48 hours of cultivation, the cells are passaged to a new deep-well plate by 1000-fold back dilution under the same conditions. Samples are analyzed for production using a recombinant protein-specific assay (e.g., GFP detection) and cell density is monitored by OD600.

[0166] Growth rate measurements of selected strains: The growth rate of the individual strains is compared to a non-load-dependent producer. The 96-well plates are sealed with Breatheasy membranes and growth is measured for 20 hours on a Synergy H1 plate reader with reciprocal shaking at 754 rpm at 37° C. The OD600 is measured every 10 minutes.

[0167] (5.2) Results (5.2.1) HAC1 upregulated promoter In recombinant protein-producing Saccharomyces strains producing human insulin precursor or human serum albumin linked to optional GFP, the HAC1 upregulated promoter containing an unfolded protein response (UPR) element, e.g., KAR2 (SEQ ID NO: 91), PDI1 (SEQ ID NO: 92), SSA1 (SEQ ID NO: 93) or FPR2 (SEQ ID NO: 94), is shown to be useful for growth control when inserted before a native growth control gene (e.g., URA3, a conditionally essential gene encoding orotidine 5'-phosphate decarboxylase essential for pyrimidine biosynthesis). Production stability is followed under simulated long-term production by sequential passaging corresponding to 60-80 generations of cell division. In Saccharomyces strains containing the HAC1 upregulated promoter controlling transcription of the growth control gene (URA3), production is expected to be more stable than the corresponding recombinant protein-producing Saccharomyces parent strain.

[0168] (5.3.2) RNA polymerase I upregulated promoters In yeast, RNA polymerase I transcribes ribosomal RNA genes. RNA polymerase I upregulated promoters, such as the promoters of genes RPL3 (SEQ ID NO: 95), RPL6A (SEQ ID NO: 96) and RPL28 (SEQ ID NO: 97), are useful for growth control of yeast essential genes. In recombinant protein overproducing strains producing human insulin precursor or human serum albumin potentially linked to GFP, such upregulated promoters are inserted in front of the native growth controlled gene (e.g., the conditionally essential gene URA3). Production stability is experimentally tracked in simulated long-term production by sequential passaging corresponding to 60-80 generations of cell division. In Saccharomyces strains containing an RNA polymerase I upregulated promoter controlling transcription of the conditionally essential gene URA3, production will be more stable than in the corresponding recombinant protein producing Saccharomyces parent strain.

[0169] (5.3.3) DNA damage responsive promoters The heterologously expressed yeast DNA damage response broadly induces the transcription of DNA repair systems including OGG1 (SEQ ID NO:98), RAD51 (SEQ ID NO:99) and RAD54 (SEQ ID NO:100). The promoters of the genes encoding OGG1, RAD51 or RAD54 are useful for growth control of the yeast production cells of the invention when operably linked to essential genes. Such promoters are inserted in front of a native essential gene (e.g. URA3, which encodes a growth control gene) in cells of a yeast protein production strain producing human insulin precursor or human serum albumin, optionally fused to GFP.

[0170] The production stability is tracked under simulated long-term production by sequential passaging corresponding to 60-80 generations of cell division. RAD51 and / or p RAD54 In strains with upregulated essential genes, production will be more stable.

[0171] In summary, by coupling the transcription of essential genes to load-sensitive promoters selected from promoters activated during recombinant protein production load or promoters associated with ribosomal RNA promoters (Table 2), load-dependence enhances long-term stability and production in Saccharomyces cerevisiae cells engineered to synthesize human serum albumin or insulin precursor production.

[0172] Example 6: Examples of load-dependent yeast strains producing human serum albumin that exhibit enhanced long-term production stability The load-dependent system used in the yeast strain comprises promoters derived from genes encoding (1) to (3) the following: (1) Protein isomerase PDI1, which encodes a chaperonin belonging to the unfolded protein response in yeasts such as Saccharomyces cerevisiae and Pichia pastoris, and whose abundance is frequently upregulated in response to overexpression of recombinant proteins; (2) ribosomal subunits encoded by the RPL6A and RPL3 genes; and (3) FPR2-encoded peptidyl-prolyl cis-trans isomerase, known to be activated upon DNA replication stress. We introduced these promoter-based load-dependent systems into Pichia pastoris strains engineered to express and secrete human serum albumin; the aim was to determine their effect on long-term human serum albumin (hSA) production stability.

[0173] (6.1) Materials and Methods Pichia pastoris (Komagataella phaffii) strain EGS31 is a derivative of strain CBS7435 (NRRL-Y11430 or ATCC76273) engineered to secrete hSA by genomically integrated cDNA version of the ALB1 gene encoding hSA under the control of the AOX1 promoter.

[0174] Strain Construction: Load-dependent versions of EGS31 strains were created by genetically integrating constructs containing the kanMX conditionally selectable G418 resistance gene operably linked to either the load-responsive promoters pPDI1, pFPR2, pRPL3, or pRPL6A. These load-dependent constructs were integrated into the KU70 genomic locus of Pichia pastoris strains by transforming EGS31 cells with linear integrated DNA flanked by >750 bp homology arms (sequences N1-N3, respectively). Transformation of logarithmically growing cells pretreated with lithium acetate and dithiothreitol was performed using a standard electroporation procedure (Wu and Letchworth, 2004).

[0175] [Table 11]

[0176] Growth media: BMGY and BMMY liquid media (1 L) were prepared as follows: 10 g yeast extract and 20 g peptone were added to 700 mL HO, stirred with a magnetic stir bar, and then autoclaved. After cooling to room temperature, the following was added to the solution: 100 ml 1 M potassium phosphate buffer (pH 6.0); 100 ml 13.4% (w / v) yeast nitrogen base (no amino acids, with ammonium sulfate); 2 ml 0.02% (w / v) biotin, and in the case of BMGY, 100 ml 10% (v / v) glycerol; and in the case of BMMY, 100 mL 5% (v / v) methanol was added.

[0177] Culture: EGS31 and load-dependent EGS31 strains were plated on YPD (1% yeast extract, 2% peptone, 2% D-glucose) agar plates and incubated overnight at 30°C. Single colonies were selected and precultured in 2mL of BMGY; 50μg / mL of G418 was added to the culture of the load-dependent strain and incubated overnight at 30°C with horizontal shaking at 300 rpm. Expression cultures were inoculated with 1μL of preculture into 500mL of BMMY medium containing different concentrations of G418 (0mg / mL, 750mg / mL) in 96-well deep-bottom plates with vented lids to create "Seed 1".

[0178] Cultures were incubated at 30°C for 72 hours (with horizontal shaking at 300 rpm). To generate the next seed, the growing cultures were serially passaged (500-fold dilution) four more times into fresh 500 mL BMMY medium containing different concentrations of G418 (0 mg / mL, 750 mg / mL) in 96-deep well plates with vented lids. At each serial passage, glycerol stocks (20% glycerol) of the growing cultures were stored at -80°C.

[0179] To quantitate the production of secreted hSA, quantitative cultures from glycerol stocks were regrown in 500 mL of BMMY medium containing different concentrations of G418 (0 mg / mL and 750 mg / mL) in 96-deep-bottom well plates with vented lids for 72 h.

[0180] The cultures were centrifuged at 3000 g for 15 min and the concentration in 50 mL of supernatant was quantified using a hSA-specific ELISA kit (Abcam catalogue number: ab179887: Human Albumin SimpleStep ELISA® Kit) according to the manufacturer's instructions.

[0181] (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 G418 was added to activate the strain's load-dependent system (Figure 13). Furthermore, improvements in hSA production were observed for each of the other load-dependent promoters tested after approximately 30 cell divisions at higher selection (750 mg / mL G418) (Figure 14A; seed 1). When cultured for approximately 10 additional cell divisions (Figure 14B; seed 2), the above strains with activated load-dependence (150-750 mg / mL G418) show increased levels of hAS production compared to the non-load-dependent control and strains without activated load-dependence (0 mg / mL G418).

[0182] In conclusion, yeast cell cultures containing exemplary load-dependent systems of the invention, under conditions that activate their respective load-dependent systems, are expected to result in enrichment of highly productive yeast variants within the culture population, and while an increase in hSA production may be detectable after a relatively short period of culture (30 cell divisions), the enrichment of highly productive yeast variants is maintained or even enhanced over longer culture periods than traditional cultures usually exhibit a significant loss of productivity.

[0183] Example 7: Identification of suitable load-sensitive promoter candidates Different engineered production genes and pathways induce different transcriptional responses that are indicative of the production process. To identify suitable promoter candidates for use as loading sensors, the following experiments were performed.

[0184] Methods: Exemplary genetic escape cells were isolated from long-term cultures of engineered producing microbial cells of interest in the intended fermentation medium. Suitable genetic escape cells are characterized by having at least a 5% higher log phase growth rate and at least a 30% lower production rate or product yield than the original engineered producing cells.

[0185] Producer cells and corresponding escape cells were cultured under intended fermentation conditions, under scaled-down conditions, or under shake flask conditions mimicking intended fermentation conditions. Samples for RNA sequencing were taken at the time point corresponding to the maximum production rate of the producer cells. Total RNA was purified using the Purelink RNA Mini kit (Thermo Fischer) and prepared using the TruSeq Stranded mRNA kit (Illumina) according to the kit manufacturer's instructions. Reads were mapped and analyzed against the reference genomes of the above strains, and then differential expression between the producer cells and the corresponding escape cells was analyzed.

[0186] Results: Suitable candidate promoters were identified that drive gene expression that exhibited differential expression of more than three-fold higher in producer organisms compared to at least one isolated genetic escape strain.

[0187] Example 8: Load-dependent Bacillus subtilis strains producing IgA fragments show enhanced long-term production stability The load-sensing promoter is shown to be a promoter that can sense and be activated by an intracellular load-induced state triggered by cellular synthesis of an IgA fragment fused to GFP at its C-terminus (IgA-GFP) recombinant protein. Upon activation, the load-sensing promoter is shown to elevate expression of the essential genes (operons) iscU or accC to a level sufficient to confer a selective growth advantage to the cells compared to non-producing cells. To maximize the dynamic range of essential gene expression in response to the essential gene's cognate load-sensing promoter, variant RBS coding sequences (Table 3.2) that confer different translational strengths were randomly combined with that load-sensing promoter.

[0188] Suitable promoters with load-sensing properties for use in load-dependent gene circuits are shown to include heat shock promoters, DNA damage responsive promoters, and oxidative stress responsive promoters, as demonstrated in engineered production strains cultured under simulated large-scale production conditions below.

[0189] (8.1) Materials and Methods Load-sensitive promoter: Promoter P hrcA and P perR , and sequences for integrating them into the target essential genes, were generated by PCR and USER cloning to obtain integration vectors (Table 10).

[0190] [Table 12]

[0191] [Table 13]

[0192] Chromosomal integration of loading-dependent promoters: To generate candidate loading-dependent strains (Table 11), integration vectors (Table 10) carrying each of the promoters fused to RBS variants (Table 3.2) were integrated by homologous recombination upstream of an operon containing either iscU or accC in the genome of the IgA-GFP-producing EGS084 strain (corresponding to B. subtilis KO7 containing the expression cassette of pEG062 inserted at the amyE locus) as follows: Competent cells were prepared and transformed with pEG151 to pEG166 by standard B. subtilis transformation methods according to previously reported protocols (dx.doi.org / 10.17504 / protocols.io.bdmti46n). Transformants were selected on LB agar plates supplemented with spectinomycin (200 μg / mL) and confirmed by colony PCR using primers E257 / E258 (iscU) or E261 / E262 (accC).

[0193] [Table 14]

[0194] Long-term IgA-GFP production assay: A single selected colony of each load-dependent IgA-GFP producing strain was inoculated into a 96-well deep-bottom plate containing 500 μL / well of Cal18-2 medium (dx.doi.org / 10.17504 / protocols.io.bdmui46w) supplemented with kanamycin (10 μg / mL). The cultures were grown for 24 hours on a horizontal shaking incubator (Innova, 2-inch strength) at 30°C / 250 RPM. Then, 1 μL of each culture (as a successive passage) was transferred to fresh 499 μL of Cal18-2 medium (corresponding to approximately 10 generations per passage) in a new deep-well plate under the same culture conditions. The steps of the above method were repeated a total of 13 times. At each successive passage, 100 μL of each strain was mixed with an equal volume of 50% glycerol and stocked for storage and follow-up testing. For quantification of IgA-GFP expression after each passage, each culture was grown for an additional 24 hours under the above growth conditions. The cultures were harvested by centrifugation at 2000g for 5 minutes and then washed twice with 1 volume of PBS. 20 μL of culture sample was added to 180 μL of PBS in a 96-well plate (10-fold dilution) and OD600 and GFP fluorescence (λ) were measured using a Synergy H4 plate reader (Biotek). ex / λ ex Cell density and IgA-GFP production were quantified by measuring the OD600 (OD600 = 485 nm / 528 nm). The specific production level of each culture was quantified as the GFP signal normalized to the OD600 value after subtraction of a similar value measured in the non-GFP producing B. subtilis KO7 strain.

[0195] (8.1) Results After prolonged culture over five serial passages of 500-fold dilutions (approximately 65 cell generations), IgA-GFP production was quantified and showed improved IgA-GFP production in the load-dependent lines (FIGS. 15 and 16).

[0196] Based on the above methodology, similar load-dependent strains can be generated using other candidate load sensors that may be compatible with the transcriptional loading response of other production strains, and their suitability can be easily evaluated using the long-term production assays devised above or, for example, continuous small-scale (e.g., 400 mL) production cultures.

[0197] Example 9: Load-dependent regulation of multiple essential genes improves production In this example, we demonstrate how to introduce two (and by analogy even more) load-sensing promoters (operons) controlling different essential genes into a single producer cell, with the goal of increasing production of the desired product and further extending the period during which the producer cell is productive. Furthermore, by using two or more load sensors controlling essential genes, the transcriptional space for the imposed dependency-based selection regime can be further controlled.

[0198] The native promoter of the accC essential gene operon was compared with the candidate loading sensor P ctsR , P dnaK and P hrcA The loading-dependent IgA-GFP producing strain EGS340 (P controlling the iscU essential gene operon) was hrcA ) was genetically transformed.

[0199] (9.1) Materials and Methods Chromosomal integration of load-dependent promoters: The spectinomycin resistance marker in pEG159-pEG166 was replaced with a chloramphenicol resistance marker using primers E372 / E373 for the vector backbone and E374 / E375 for the chloramphenicol resistance marker of pDG1662 (Guerout-Fleury et al., 1996) to generate pEG204-pEG211 (Table 10). Each of the above promoters was integrated upstream of the essential gene operon containing accC in the genome of strain EGS340 (corresponding to EGS084, which contains PperR controlling the iscU essential gene operon) as shown below. Competent cells were prepared according to a previously published method (dx.doi.org / 10.17504 / protocols.io.bdmti46n) and transformed with pEG204-pEG211. Transformants were selected on LB agar plates supplemented with chloramphenicol (5 μg / mL) and confirmed by colony PCR using primers E261 / E262.

[0200] [Table 15]

[0201] Long-term IgA-GFP production assay: A single selected colony of each load-dependent IgA-GFP producing strain was inoculated into Cal18-2 medium (dx.doi.org / 10.17504 / protocols.io.bdmui46w) supplemented with kanamycin (10 μg / mL) at 500 μL / well in a 96-well deep-bottom plate. The cultures were grown for 24 hours on a 30°C / 250 RPM horizontal shaking incubator (Innova, 2-inch strength). 1 μL of each culture was then transferred (as a successive passage) to 499 μL of fresh Cal18-2 medium (corresponding to approximately 10 generations per passage) in a new deep-bottom well plate under the same culture conditions. The steps of the above method were repeated a total of 13 times. At each successive passage, 100 μL of each strain was mixed with an equal volume of 50% glycerol and stocked for storage and follow-up testing. For quantification of IgA-GFP expression after each passage, each culture was grown for an additional 24 hours under the above growth conditions. The cultures were harvested by centrifugation at 2000g for 5 minutes and then washed twice with 1 volume of PBS. 20 μL of culture sample was added to 180 μL of PBS in a 96-well plate (10-fold dilution) and OD600 and GFP fluorescence (λ) were measured using a Synergy H4 plate reader (Biotek). ex / λ ex Cell density and IgA-GFP production were quantified by measuring the OD600 (OD600 = 485 nm / 528 nm). The specific production level of each culture was quantified as the GFP signal normalized to the OD600 value after subtraction of a similar value measured in the non-GFP producing B. subtilis KO7 strain.

[0202] Cell disruption and ELISA: After washing with PBS, the cultures were harvested by centrifugation at 2000g for 5 min. The pellet was resuspended in 1 volume of lysis buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 500 μM EDTA); harvested again by centrifugation at 6000g for 5 min and resuspended in 1 volume of lysis buffer containing lysozyme (10 mg / mL). The reaction was incubated at 37°C for 30 min to disrupt the cells. The reaction was then briefly vortexed and harvested by centrifugation at 12000g for 30 min at 4°C. The supernatants were measured by ELISA using Abcam's Pig IgA ELISA Kit (ab190536) according to the manufacturer's protocol (except that the absorbance of each well was read at 600 nm every 40 s for 10 min after addition of chromogenic substrate). Relative IgA concentrations were calculated as the slope of the values ​​relative to the OD600 of the original culture.

[0203] (9.2) Results The single load sensor strain EGS340 controls the transcription of the iscU essential gene operon (EGS340), p perR EGS340 and its derivatives (the transcription of the accC essential gene operon is also restricted to p ctsR Load sensor based on EGS460, p dnaK Based on the load sensor (EGS462), and hrcA After prolonged culture for approximately 75 cell generations, IgA-GFP production was quantified and improved IgA-GFP production was observed in the load-dependent lines (Figures 17 and 18).

[0204] Based on the above methodology, similar load-dependent strains can be easily generated using other candidate load sensors that may be compatible with the transcriptional loading response of other production strains, and their suitability can be easily evaluated using the above-devised long-term production assays or continuous small-scale (e.g., 400 mL) production cultures.

[0205] Example 10: Alternative methods for identifying suitable load-sensitive promoters In this example, suitable candidate load-sensing promoters are identified for use in load-dependent sustained long-term heterologous productivity in a particular production strain.

[0206] The above method is divided into optional Track 1, which involves denovo discovery (RNA sequencing), and Track 2, which involves validation of putative load-sensitive promoters (q-PCR).

[0207] In track 1, RNA sequencing is used to compare the transcriptional activity of coding genes in a producing organism under typical production conditions (e.g., with respect to temperature, agitation, product / by-product / substrate concentration / growth phase) with that of an isolated non-producing / low producing organism under the same typical production conditions. Cultures of the isolated non-producing / low producing organisms may optionally be spiked with product to a concentration approximately found at a given time in the corresponding culture of the producing organism. Non-producing / low producing isolates can typically be isolated after (preferred) more than 50-100 divisions, although suitable isolates can also be engineered by genetically inactivating one or more key heterologous production genes.

[0208] RNA sequencing is preferably performed using Illumina short read sequencing according to standard methods known in the art, preferably in at least three replicates.

[0209] Using standard bioinformatics analysis for differential gene expression (e.g., include the edgeR workflow (DOI: 10.18129 / B9.bioc.edgeR)), identify suitable candidate load-sensing promoters from genes that are upregulated at least 5-10 fold in high-producing cultures compared to non-producing / low-producing cultures under identical typical production conditions. By identifying candidate load-sensing promoters using this methodology, it is possible to detect promoters that are selectively activated by the load of product production (compared to simply production stress).

[0210] Next, in Track 2, q-PCR of samples is used to confirm putative load-sensing promoters (from Track 1 or other lists), also comparing one non-producing / low-producing organism with a high-producing isolate under the same typical production conditions (e.g., for temperature, agitation, product / by-product / substrate concentration / growth phase). Suitable candidate load-sensing promoters are identified from genes that are upregulated at least 5-10 fold in the high-producing cultures compared to the non-producing / low-producing cultures under the same typical production conditions.

[0211] Example 11: Load-dependent regulation of multiple essential genes improves production of recombinant human growth hormone in E. coli Integration of an additional, different load-sensing promoter, e.g., selected from the list and screened as described above, controlling the transcription of a second essential gene improved the long-term production stability of the load-dependent E. coli strain s7.6#8.

[0212] (11.1) Materials and Methods Single colonies of s7.6#8 were transformed with recombinant plasmids such as pKD46 using standard electroporation. Using recombination, candidate load-sensing promoters (Table 2) fused to different RBSs (Table 3.1) were integrated into the chromosome directly upstream of the essential gene murI or a similar essential gene, and selection was performed using the spectinomycin resistance gene present in the integration construct.

[0213] After the above steps of recombination and recovery of recombinant plasmid, 5 colonies were selected, which corresponded to clones with the same promoter integration but with random variant RBS sequences. Each clone was then transferred and cultured in a 96-well plate containing 200 μl of 2xYT supplemented with chloramphenicol for the maintenance of plasmid pMevT. Before freezing the 96-well plate, 2 μl of each cultured clone was used to verify the integration of the promoter as described in Example 1.2.1.

[0214] (11.2) Results The different clones obtained were evaluated for long-term production stability by serial passage at 200-fold dilutions. After long-term culture for approximately 75 cell generations, IgA-GFP production was quantified, and the new strains showed improved IgA-GFP production compared to the single-load-dependent strains that only had a single-load-control essential gene.

[0215] Example 12: Modulated expression of essential gene CIA1 with TIS sequence variations shows responsive growth in Pichia pastoris strains This example shows that the variation of the TIS (translation initiation site) sequence of an essential gene controls growth. The translation strength of an essential gene varies with the variation of the TIS sequence, and such variation of the TIS sequence can be used to incrementally examine the load-responsiveness of a load-sensitive promoter to the expression level of the essential gene in eukaryotes; such variation in the TIS sequence is similar to the use of ribosome binding sites in prokaryotes.

[0216] (12.1) Materials and Methods Load-responsive promoters with variations in TIS (translation initiation site) sequence: Promoter P with different TIS sequences increases the strength gsh2Variations of (promoter of Pichia pastoris homolog CDS chr1-4_0496) and cia1 (Pichia pastoris homolog CDS: chr1-3_0207) essential gene integration sequences were created; these were done by PCR and USER cloning to generate integration fragments (Table 14).

[0217] [Table 16]

[0218] Chromosomal integration of a load-responsive promoter with TIS sequence variation: To create a strain that regulates expression of the essential gene cia1 in the IgAL-NanoLuc-producing EGS621 strain, a promoter P with a TIS sequence variation was integrated into the IgAL-NanoLuc-producing EGS621 strain by homologous recombination upstream of the CDS of cia1 as follows: gsh2 Integration of the integration fragments (Table 14) containing the following was performed. Electrocompetent cells were prepared and transformed with the integration fragments INT1, INT3, INT5, and INT9 according to standard Pichia transformation methods (Wu and Letchworth 2018). Transformants were selected on YPD agar plates supplemented with Zeocin® (50 μg / mL) and confirmed by colony PCR using primers E521 (SEQ ID NO: 237) and E627 (SEQ ID NO: 238).

[0219] [Table 17]

[0220] Pichia TIS sequence growth response assay: A single colony of parent strain EGS621 and TIS sequence variants EGS1100, EGS1101, EGS1102, and EGS1104 was each diluted in 30 μL of MilliQ H2O. Precultures were prepared by inoculating 5 μL of diluted colony into 95 μL of YPD medium. G418 (50 μg / mL) was added to EGS621 colonies, and Zeocin® (50 μg / mL) was added to the remaining colonies. Precultures were grown overnight at 30°C with shaking at 300 RPM. The overnight precultures were then diluted 1000-fold into YPD medium supplemented with the appropriate antibiotic. 99 μL of YPD supplemented with the appropriate antibiotic in a 96-well culture plate was inoculated with 1 μL of diluted culture and sealed with a BreathEasy® sealing membrane (Sigma-Aldrich; Darmstadt, Germany). The 96-well plate described above was placed on a Synergy4 plate reader (BioTek, Vermont, USA) and cultures were grown using the following settings: 30°C, high shaking speed, and OD600 measurements every 10 minutes for 48 hours.

[0221] (12.2) Results The growth differentials of all strains are shown (see FIG. 19). cia1 The parent strain (EGS621) carrying the P gene showed the fastest growth. gsh2 Strains with promoter TIS sequence variations (EGS1100, EGS1101, EGS1102, and EGS1104) showed growth consistent with their relative TIS strengths. The strain with the strongest TIS sequence, TIS9 (EGS1104), showed slightly slower growth than the parent strain (EGS621), but was the fastest growing among the TIS strains (EGS1100, EGS1101, EGS1102, and EGS1104). The strain with the weakest TIS sequence, TIS1 (EGS1100), showed the slowest growth of all strains. Strains TIS3 (EGS1101) and TIS5 (EGS1102) showed growth consistent with the relative strengths of the four TIS sequences.

[0222] In summary, the growth of Pichia strains was improved by the P gsh2 It was demonstrated that cia1 is responsive to the strength of the TIS sequence of cia1, supporting the notion that cia1 is an essential gene for the growth of Pichia strains.

[0223] Example 13: Introduction of load-dependency into a production strain of Aspergillus oryzae The load-dependent system of the present invention may also be introduced into other eukaryotic organisms, such as fungi. Herein, an example of load-dependent introduction into an Aspergillus oryzae production strain is described.

[0224] (13.1) Load-Sensitive Promoter Elements Examples of load-sensing promoters include the promoter sequences (750 bp upstream of the start codon) of gene homologs of bipA (e.g., SEQ ID NO: 189), clxA (e.g., SEQ ID NO: 190), and agsA (e.g., SEQ ID NO: 239). To create a load-dependent Aspergillus oryzae production strain, a load promoter is integrated in front of an essential gene such as an ERG10 (e.g., SEQ ID NO: 240), PFS2 (e.g., SEQ ID NO: 242) or TUB1 (e.g., SEQ ID NO: 244) homolog. To modulate the expression of the essential genes, a load-sensing promoter is integrated together with a translation initiation sequence (TIS) library of four different variants as shown in Table 3.3. In these variants, the last six nucleotides of the promoter sequence, i.e., -6 to -1 relative to the start codon, are substituted. Examples of load-dependent integration fragments are shown in Table 16.

[0225] [Table 18]

[0226] (13.2) Creation of Load-Sensitive Integration Constructs To replace the native essential gene promoter with a load-sensitive promoter, three DNA fragments targeting the essential genes are prepared by standard molecular cloning. Approximately 1.5 kbp regions upstream and downstream of each essential gene start codon (ERG10, PFS, and TUB1, respectively) are amplified from Aspergillus oryzae RIB40 genomic DNA. Then, 750 bp promoter regions immediately upstream of the start codons of bipA, clxA, and agsA are similarly amplified from Aspergillus oryzae genomic DNA, respectively. Finally, a synthetic gene fragment containing a selection marker (e.g., amdS or pyrG) is obtained. All fragments are assembled, for example, using Gibson assembly techniques. The full-length knockout construct is amplified from the assembly reaction using PCR.

[0227] According to standard protocols (e.g., Christensen et al., 1988), Aspergillus oryzae protoplasts are prepared, transformed with the load-sensing integrative construct, and selected on an appropriate medium.

[0228] Expression assays are performed using shake flasks containing 10 ml of YPM medium (2 g / l yeast extract, 2 g / l peptone, and 2% maltose) inoculated with spores of the transformed strain and a reference production strain that does not contain a load-sensing promoter element; after 4 days of incubation at 30°C and 200 rpm, the products are sampled.

[0229] Further assays are also carried out in fed-batch fermentation: tank medium (24 g / L sucrose, 10 g / L yeast extract, 5 g / L (NH4)2SO4, 2 g / L MgSO4-7H2O, 2 g / L K2SO4, 1 g / L citric acid, 2 g / L KH2PO4, 0.5 ml / L trace metals solution), temperature 34°C, aeration 1 vvm, and pH adjusted to 6.0 with 10% NH4OH. Seed cultures (spores pregrown for 1 day at 30°C and 250 rpm in shake flasks (20 g / L glycerol, 18 g / L yeast extract)) of transformed and reference strains containing the load-sensing integration constructs are inoculated into the medium. If pH > 6.4, start the feed (400 g / L maltose syrup, 1 g / L citric acid) at a rate of 3.33 g / L / h. Control the agitation speed so that the oxygen pressure does not become too low (< 20%). References Bonde, MT, Pedersen, M., Klausen, MS, Jensen, SI, Wulff, T., Harrison, S., et al. (2016). Predictable tuning of protein expression in bacteria. Nat. Methods 13. doi:10.1038 / nmeth.3727. Christensen, T., Woeldike, H., Boel, E. et al. (1988) High Level Expression of Recombinant Genes in Aspergillus Oryzae. Nat Biotechnol 6, 1419-1422. doi.org / 10.1038 / nbt1288-1419 Falkenberg et al (2022). Protein expression in Bacillus subtilis. Oct 27, 2022. Website: protocols.io. dx.doi.org / 10.17504 / protocols.io.bdmti46n Giaever G et al., Nature. 2002 Jul 25;418(6896):387-91. DOI: 10.1038 / nature00935 Jain, R., Kumar, P. & Varshney, U. A distinct role of formamidopyrimidine DNA glycosylase (MutM) in down-regulation of accumulation of G, C mutations and protection against oxidative stress in mycobacteria. DNA Repair (Amst). 6, 1774-1785 (2007). Guerout-Fleury AM, Frandson N, Stragier P. 1996. Plasmids for ectopic integration in Bacillus subtilis. Gene 180:57-61 Koskiniemi, S., Pranting, M., Gullberg, E., Nasvall, J., & Andersson, D. I. (2011). Activation of cryptic aminoglycoside resistance in Salmonella enterica. Molecular Microbiology, Vol. 80, pp. 1464-1478. https: / / doi.org / 10.1111 / j.1365-2958.2011.07657.x Kimata, Y., Ishiwata-Kimata, Y., Yamada, S., and Kohno, K. (2006). Yeast unfolded protein response pathway regulates expression of genes for anti-oxidative stress and for cell surface proteins. Genes to Cells 11, 59-69. doi:10.1111 / j.1365-2443.2005.00921.x. Laferte, A., Favry, E., Sentenac, A., Riva, M., Carles, C., and Chedin, S. (2006). The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components. Genes Dev. 20, 2030-2040. doi:10.1101 / gad.386106. Maeda, M., Shimada, T., and Ishihama, A. (2015). Strength and Regulation of Seven rRNA Promoters in Escherichia coli. PLoS One 10, 1-19. doi:10.1371 / journal.pone.0144697. Nonaka, G., Blankschien, M., Herman, C., Gross, C. a, and Rhodius, V. a (2006). Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress. Genes Dev. 20, 1776-89. doi:10.1101 / gad.1428206. Pitera, D. J., Paddon, C. J., Newman, J. D., and Keasling, J. D. (2007). Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab. Eng. 9, 193-207. doi:10.1016 / j.ymben.2006.11.002. Rugbjerg, P., Sarup-Lytzen, K., Nagy, M., and Sommer, M. O. A. (2018). Synthetic addiction extends the productive life time of engineered Escherichia coli populations. Proc. Natl. Acad. Sci. 115, 2347-2352. doi:10.1073 / pnas.1718622115. Wu, S. and Letchworth, G.J. (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36, 152-154 Wu an Letchworth (2018) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. BIOTECHNIQUESVOL. 36, NO. 1DRUG DISCOVERY AND GENOMIC TECHNOLOGIES. doi.org / 10.2144 / 04361DD02. Yoon, S. H., Han, M. J., Lee, S. Y., Jeong, K. J., and Yoo, J. S. (2003). Combined transcriptome and proteome analysis of Escherichia coli during high cell density culture. Biotechnol. Bioeng. 81, 753-767. doi:10.1002 / bit.10626. Uppal, S., Shetty, D. M., & Jawali, N. (2014). Cyclic AMP receptor protein regulates cspd, a bacterial toxin gene, in Escherichia coli. Journal of Bacteriology, 196(8), 1569-1577. https: / / doi.org / 10.1128 / JB.01476-13 Yamanaka, K., & Inouye, M. (1997). Growth-phase-dependent expression of cspD, encoding a member of the CspA family in Escherichia coli. Journal of Bacteriology, 179(16), 5126-5130. https: / / doi.org / 10.1128 / jb.179.16.5126-5130.1997

Claims

1. 1. A producing microbial cell genetically engineered to synthesize a product, the microbial cell comprising: (a) a first essential gene operably linked to a first load-sensitive promoter; and (b) a second essential gene operably linked to a second load-sensitive promoter; Further comprising: wherein said first load-sensitive promoter is heterologous to said first essential gene; and said second load-sensitive promoter is heterologous to said second essential gene; wherein synthesis of said product imposes a burden on said cell; and wherein expression of the first essential gene is upregulated when the first load-sensitive promoter is induced by the load, compared to basal level expression of the first essential gene when the first load-sensitive promoter is not induced; and expression of the second essential gene is upregulated when the second load-sensitive promoter is induced by the load, compared to basal level expression of the second essential gene when the second load-sensitive promoter is not induced. Productive microbial cells.

2. 2. The producing microbial cell of claim 1, wherein when the first and second essential genes in the cell are operably linked to their native promoters, the burden imposed by the synthesis of the product has a fitness cost measured as a percentage reduction in the maximum logarithmic phase growth rate of the producing microbial cell selected from the group consisting of ≧5%, ≧10%, ≧15%, ≧20%, ≧25%, ≧35%, and ≧45%, measured compared to a corresponding non-producing microbial cell.

3. A productive microbial cell as described in claim 1 or 2, wherein the load-sensing promoter is not directly induced by the product itself.

4. 10. The producer microbial cell of claim 1, wherein the cell: (i) a bacterium belonging to a genus selected from the genera Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Bacillus, Acetobacter, Acinetobacter, Pseudomonas; Propionibacterium, Bacteroides, and Bifidobacterium; or (ii) a yeast belonging to a genus selected from the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Komagataella, Cryptococcus, Debaryomyces, Hansenula, Yarrowia, Zygosaccharomyces, and Schizosaccharomyces; or (iii) a filamentous fungus selected from the genera Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma, Thermomyces, Streptomyces, and Aspergillus; , a producing microbial cell.

5. 2. The producer microbial cell of claim 1, wherein the first and second essential genes are identical.

6. 2. The producer microbial cell of claim 1, wherein the first and second load-sensing promoters are different.

7. 2. The producing microbial cell of claim 1, wherein the first and / or second essential genes are either (i) genes whose expression is essential for cell growth independent of the nutrient composition of the production conditions of the producing microbial cell, without depending on the presence or absence of specific inhibitors or nutrients, or (ii) genes whose expression is conditionally essential for cell growth due to the removal of nutrients from the production conditions of the producing microbial cell, and wherein knockout of the essential genes results in loss of cell viability.

8. The productive microbial cell of claim 1, wherein the first and second essential genes are: The E. coli genes 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 and their homologs; the B. subtilis genes iscU operon, accC operon, glmM, ylaN, infA, and dapA, and their homologs; the Corynebacterium genes alr, glmM, and their homologs; Saccharomyces genes FOL1, MED7, RRP40, NOP8, PGI1, NEP1, URA3, LEU2, TRP1, HIS3, and their homologs; Aspergillus genes ARG3, adeA, ERG10, PFS2, and TUB1 and their homologs The producing microbial cell is selected from:

9. 2. The producer microbial cell of claim 1, wherein the first and / or second essential genes are operably linked to a synthetic RBS, the sequence of the synthetic RBS altering the translation strength of the first and / or second essential genes independent of induction of the first and / or second load-sensing promoters, respectively, and the first and / or second essential genes are selected such that they can control the growth rate of the cell.

10. 2. The producer microbial cell of claim 1, wherein the first and second load-sensing promoters are: (i) a ribosomal RNA promoter, (ii) a promoter that is upregulated by oxyR or a homolog thereof; (iii) a promoter containing a UPR element that is upregulated by HAC1 or a homolog thereof; and (iv) a DNA damage-sensitive promoter; The producing microbial cell is selected from:

11. The productive microbial cell of claim 1, wherein the first and second load-sensing promoters are: 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, and trxC; promoters of the B. subtilis genes groES, ctsR, dnaK, perR, hrcA, spx, sigB, yflT, and mutM; promoters of the Corynebacterium genes katA, cplX, mutM, and groES; promoters of Saccharomyces genes KAR2, PDI1, SAA1, FPR2, RPL3, RPL6A, RPL28, OGG1, RAD51, and RAD54; Promoters of Aspergillus genes bipA, PDI, clxA, and agsA The producing microbial cell is selected from:

12. 2. The producer microbial cell of claim 1, wherein the cell has increased product yield after at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 generations of cell division from a single cell compared to a parent producer microbial cell lacking the first and second essential genes operably linked to the first and second load-sensing promoters, respectively.

13. 2. The producer microbial cell of claim 1, wherein the cell has at least a 10, 25, 50, or 80% increase in product yield after at least 50 generations of cell division from a single cell compared to a parent producer microbial cell lacking the first and second essential genes operably linked to the first and second load-sensing promoters, respectively.

14. 1. A method for product biosynthesis, the method comprising: (i) providing at least one producer microbial cell of claim 1; (ii) introducing said at least one cell into a substrate-containing culture medium for the production of said product; (iii) recovering the product; A method comprising:

15. 15. The method of claim 14, further comprising isolating the product and / or formulating the product into a composition, such as a nutritional composition, a pharmaceutical composition, a cosmetic composition, a surfactant composition, a lubricant composition, or a fuel composition.

16. 16. A method for biosynthesis of a product according to claim 14 or 15, wherein the product is: the protein is selected from organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, amino acids, peptides, enzymes (such as amylases, lipases, proteases, barnase, β-galactosidase, crystalline proteins, cutinases, pectases, laccases and enzymes acting on carbohydrates (such as xylanases, licheninase, cellulases, lytic polysaccharide monooxygenases and pectases)), therapeutic proteins and their precursors (such as human growth hormone, insulin, glucagon-like peptide 1, monoclonal and polyclonal antibodies, single fragment antibodies and nanobodies), proteins naturally occurring in eggs (such as ovalbumin), milk proteins (such as casein, lactadherin, lactoferrin), immunoglobulin A and immunoglobulin G secreted as secretory components.

17. 1. Use of first and second essential genes operably linked to first and second load-sensitive promoters, respectively, to improve product yield of a productive microbial cell culture population generated after at least 50 generations from a single cell; wherein said first load-sensitive promoter is heterologous to said first essential gene; and said second load-sensitive promoter is heterologous to said second essential gene; wherein synthesis of the product places a burden on the producing microbial cell; and wherein expression of the first essential gene is upregulated when the first load-sensitive promoter is induced by loading compared to basal level expression of the first essential gene when the first load-sensitive promoter is not induced, and expression of the second essential gene is upregulated when the second load-sensitive promoter is induced by loading compared to basal level expression of the second essential gene when the second load-sensitive promoter is not induced.

18. 18. The use of the first and second essential genes operably linked to the first and second load-sensing promoters, respectively, according to claim 17, wherein the product yield after at least 50 generations of cell division from a single cell is increased by at least 10%, 25%, 50%, or 80% compared to a parent producing microbial cell lacking the first and second essential genes operably linked to the first and second load-sensing promoters, respectively.

19. 10. Use of the producer microbial cells of claim 1, comprising: Use for producing biosynthetic products such as organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, amino acids, peptides, enzymes (such as amylase, lipase, protease, barnase, β-galactosidase, crystalline protein, cutinase, pectase, laccase and enzymes acting on carbohydrates (such as xylanase, licheninase, cellulases, lytic polysaccharide monooxygenase and pectase)), therapeutic proteins and their precursors (such as human growth hormone, insulin, glucagon-like peptide 1, monoclonal and polyclonal antibodies, single fragment antibodies), proteins naturally occurring in eggs (such as ovalbumin), milk proteins (such as casein, lactadherins, lactoferrin), immunoglobulin A and immunoglobulin G secreted as secretory components, nanobodies, etc.

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