Pyruvate gene loop mediated saccharomyces cerevisiae ethanol metabolism redirection method

Abstract

The invention discloses a pyruvic acid gene loop mediated saccharomyces cerevisiae ethanol metabolism redirection method. Comprising the following steps: constructing a pyruvic acid response gene loop in saccharomyces cerevisiae host bacteria to obtain modified saccharomyces cerevisiae; the growth of saccharomyces cerevisiae cells, the synthesis of by-product ethanol and the synthesis of products are dynamically regulated and modified by controlling glucose feeding. A low-concentration glucose environment is maintained in the early stage of growth, so that cell growth and ethanol metabolism related genes are normally expressed, and cell growth is recovered; high-concentration glucose is supplemented in the later stage of fermentation, expression of cell growth related ethanol metabolism genes is inhibited, the ethanol synthesis flux is reduced, sufficient pyruvic acid precursors are provided for product synthesis, and the product synthesis flux is maximized. According to the method, the yield of the byproduct ethanol is reduced, the production efficiency of the product is improved, and meanwhile, the fermentation period is shortened.

Description

Technical Field

[0001] The invention relates to a pyruvate gene circuit-mediated ethanol metabolism redirection method for saccharomyces cerevisiae, and belongs to the technical field of microbial fermentation. Background Art

[0002] The imbalance between cell growth and product synthesis is an important factor in the low efficiency of microbial cell engineering synthesis. Static methods such as gene knockout or overexpression can easily cause cellular metabolic disorders. Mimicking nature, gene circuits can sense metabolites or cell states and autonomously regulate downstream gene expression, thereby achieving a dynamic balance of metabolic flux. In the microbial metabolic network, pyruvate is a key hub substance for carbon source assimilation, cell growth, and the synthesis of multiple products. It is an important node in central carbon metabolism. The pyruvate gene circuit is a powerful carbon flux regulation tool that has been used in the design and construction of cell factories such as glucaric acid in Escherichia coli and Bacillus subtilis. However, there is no dynamic regulation method and application based on Saccharomyces cerevisiae.

[0003] Saccharomyces cerevisiae is an important industrial microorganism. However, under high-sugar conditions, Saccharomyces cerevisiae preferentially ferments ethanol through the glycolytic pathway. Ethanol metabolism is one of the core metabolic pathways of Saccharomyces cerevisiae. When producing non-ethanol bioproducts, this characteristic limits the yield of the target product due to the significant carbon flux directed toward ethanol synthesis, resulting in limited cell growth and low fermentation efficiency. To overcome this problem, Maris et al. knocked out the pyruvate decarboxylase gene pdc, completely blocking the metabolic pathway for pyruvate conversion to ethanol. This allowed for the accumulation of pyruvate, providing a crucial microbial substrate for the synthesis of pyruvate and its derivatives, such as malate, succinate, and 2,3-butanediol. However, this static gene knockout strategy also resulted in an imbalance in cofactor reduction and a loss of cytosolic acetyl-CoA, severely impairing cell growth. Although adaptive evolution to glucose feeding restored growth and glucose utilization, strain TAM still exhibited a low growth rate, limiting its widespread application in biofermentation.

[0004] With the rapid development of synthetic biology and metabolic engineering, dynamic regulation based on gene circuits has provided a new approach to addressing these issues. By adopting a growth-production decoupling strategy, when cells reach a certain density, the yield of the target product can be effectively increased by inducing activation of the product synthesis pathway while simultaneously inhibiting growth-related pathways. To avoid the problem of cell lethality caused by knocking out Pdc1, Zhao et al. used optogenetic switches to control growth and production, using a light-activated switch to control Pdc1 expression while simultaneously using a light-inhibited switch to control the product synthesis pathway. A more dynamic regulatory approach utilizes gene circuits that respond to intracellular metabolites to autonomously regulate gene expression and cellular metabolic status in real time. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for redirecting ethanol metabolism in Saccharomyces cerevisiae mediated by a pyruvate gene circuit. To achieve the above object, the present invention adopts the following technical solutions: A method for redirecting ethanol metabolism in Saccharomyces cerevisiae mediated by a pyruvate gene circuit, including: Constructing a pyruvate-responsive gene circuit in the host strain Saccharomyces cerevisiae to obtain modified Saccharomyces cerevisiae; The host bacteria is Saccharomyces cerevisiae in which cell growth-related ethanol metabolism genes are knocked out; the pyruvate-responsive gene circuit comprises the cell growth-related ethanol metabolism genes, genes related to the product synthesis pathway, the transcription factor PdhR, a pyruvate-activated promoter, the xylose transcription repressor XylR, and a pyruvate-repressible promoter; The pyruvate-activated promoter is a constitutive promoter into which the PdhR binding sequence pdhO is inserted; the ketoacid-repressible promoter is a constitutive promoter into which the XylR binding sequence xylO is inserted; the N-termini of the PdhR and XylR are fused with nuclear localization signal peptides respectively; The PdhR inhibits or restores the expression of genes related to the product synthesis pathway by binding to pdhO or pyruvate, and the XylR restores or inhibits the expression of ethanol metabolism genes related to cell growth by binding to xylO or pyruvate; The modified brewer's yeast is fermented and cultured. During the fermentation culture, the initial glucose concentration of the fermentation medium is set, and the remaining glucose is replenished at one time when the fermentation culture cell density reaches a preset range.

[0006] In some embodiments of the present invention, the host bacteria is pyruvate decarboxylase-deficient Saccharomyces cerevisiae; The cell growth-related ethanol metabolism gene is pyruvate decarboxylase.

[0007] In some embodiments of the present invention, the product is malic acid, and genes related to the product synthesis pathway include the pyruvate carboxylase gene pyc2, the malate dehydrogenase gene mdh3, and the malate transporter gene mae1.

[0008] In some embodiments of the present invention, the malate dehydrogenase gene is a malate dehydrogenase gene without a C-terminal peroxisome targeting tag SKL.

[0009] In some embodiments of the present invention, the malate transporter gene is a F253A mutant gene of a malate transporter derived from fission yeast.

[0010] In some embodiments of the present invention, the host strain is Saccharomyces cerevisiae TAM.

[0011] In some embodiments of the present invention, the nucleotide sequence of the nuclear localization signal peptide is shown as SEQ ID NO.2.

[0012] In some embodiments of the present invention, the C-terminal fusion protein degradation tags of the transcription factor PdhR and the xylose transcription repressor XylR are used.

[0013] In some embodiments of the present invention, the nucleotide sequence of the protein degradation tag fused to the C-terminus of the transcription factor PdhR is shown as SEQ ID NO.5, and the nucleotide sequence of the protein degradation tag fused to the C-terminus of the xylose transcription repressor XylR is shown as SEQ ID NO.8.

[0014] In some embodiments of the present invention, the constitutive promoter inserted into the pyruvate-activated gene circuit is P TDH3 The constitutive promoter inserted into the xylO in the pyruvate repressible gene circuit is P TEF1 .

[0015] In some embodiments of the present invention, the PdhO is inserted into P TDH3 -150 site, -128 site or -5 site of the promoter; The XylO is inserted into the P TEF1 The -105 site of the promoter.

[0016] In some embodiments of the present invention, the PdhR expression promoter is selected from P with weak promoter strength. PRM promoter; The expression promoter of the xylose transcription repressor XylR is a pyruvate-activated promoter.

[0017] In some embodiments of the present invention, the initial glucose concentration is ≤10 g / L. Maintaining the glucose concentration below 10 g / L during the early stages of fermentation growth allows for normal expression of ethanol metabolism genes related to cell growth in the strain. Preferably, the initial glucose concentration is ≤5 g / L, as the switching performance of the pyruvate-responsive gene circuit is better at low concentrations.

[0018] The method of the present invention is based on a Saccharomyces cerevisiae gene circuit containing a pyruvate-responsive gene circuit, combined with a phased glucose supply strategy. This utilizes a bifunctional pyruvate gene circuit for positive feedback on product synthesis, while simultaneously providing negative feedback on cell growth and ethanol synthesis, replacing the static modification strategy of knocking out PDC. This method achieves a dynamic balance among cell growth, ethanol synthesis, and product synthesis. Specifically, during the early stages of fermentation growth, the glucose concentration is maintained below a certain threshold, allowing normal expression of cell growth-related ethanol metabolism genes and growth recovery, while maintaining a low product synthesis flux. Glucose is supplemented in the late stages of fermentation, inhibiting the expression of cell growth-related ethanol metabolism genes and reducing ethanol synthesis flux, thereby providing more abundant pyruvate precursors for product synthesis and maximizing product synthesis flux.

[0019] Compared with the prior art, the present invention has the following advantages: (1) The proposed method for dynamic metabolic regulation mediated by a pyruvate gene circuit achieves dynamic regulation of cell growth, ethanol synthesis, and product synthesis fluxes through a bifunctional pyruvate gene circuit. Compared with traditional static modification methods, this method automatically adjusts metabolic flux based on the real-time metabolic state during fermentation, dynamically restores cell growth, and optimizes carbon flux distribution.

[0020] (2) The application of the metabolic dynamic regulation method proposed in the present invention shortens the product fermentation cycle and increases the product production intensity. It has wide applicability and can be applied to the fermentation production of different metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are fluorescence microscopy images of Saccharomyces cerevisiae expressing GFP, PdhR-GFP, and NLS-PdhR-GFP.

[0022] Figure 2 Figure 2 is a performance test diagram of the pyruvate-activated circuit at different pdhO insertion sites.

[0023] Figure 3 This is a performance test diagram of the pyruvate-activated gene circuit at different PdhR expression levels.

[0024] Figure 4 This is a schematic diagram of the design and construction of a bifunctional pyruvate gene circuit.

[0025] Figure 5 This is a test diagram of the orthogonality of the bifunctional pyruvate gene circuit in yeast cells.

[0026] Figure 6 Schematic diagram of the principle of dynamic regulation of malic acid metabolic pathway.

[0027] Figure 7 This is a schematic diagram of the construction of the dynamic regulation of malic acid metabolic pathway.

[0028] Figure 8 is the shake flask fermentation performance of strain TAM1.

[0029] Figure 9 This is a comparison chart of the shake flask fermentation performance of strains TAM2 and TAM3. The changes in the main parameters during the fermentation process include (A) cell growth, (B) ethanol production, (C) pyruvate production, and (D) malic acid production.

[0030] Figure 10 Figure 2 is a graph showing the results of fermentation parameter optimization, including (A) the effect of sugar supplementation on malic acid production at different OD values; (B) the effect of dissolved oxygen levels on malic acid production: the volume of the shake flask was gradually increased when loaded with 50 mL of culture medium. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, which may help to better understand the present invention. The contents described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0032] The Saccharomyces cerevisiae BY4741 and Saccharomyces cerevisiae TAM used in the examples are commercial strains and can be purchased from commercial channels.

[0033] Unless otherwise specified, the experimental materials, reagents, etc. used in the examples of the present invention can be obtained from commercial channels.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0035] Fluorescence detection methods involved: The strain to be tested was inoculated into a SC-Ura tube containing 1 mM xylose and cultured overnight at 30°C. The bacterial solution was washed twice with sterile water and concentrated to obtain the initial OD value. 600 0.5 was transferred to inorganic salt medium, followed by the addition of 10 g / L glucose. After shaking culture at 30°C for 8 h, the cells were detected on a fluorescence microplate reader. Detection parameters included green fluorescence intensity of GFP (excitation: 488 nm; emission: 528 nm), red fluorescence intensity of mCherry (excitation: 550 nm; emission: 620 nm), and cell density (600 nm).

[0036] Example 1: Functional verification of nuclear localization signal peptide Amplification of the constitutively strong promoter P by existing methods TDH3 , green fluorescent protein gene GFP, terminator T CYC1 and transcription factor PdhR (nucleotide sequence shown in SEQ ID NO.1). The GFP expression cassette P was obtained by assembling multiple fragments through fusion PCR overlap extension.TDH3 -gfp-T CYC1 , the expression cassette P of the transcription factor pdhR connected to the reporter gene GFP TDH3 -pdhR-GFP-T CYC1 and the expression cassette P of the transcription factor pdhR connected to the reporter gene GFP and fused to the nuclear localization signal peptide NLS (nucleotide sequence as shown in SEQ ID NO.2) at the N-terminus. TDH3 -NLS-pdhR-gfp-T CYC1 The three expression cassettes were constructed into the centromere expression vector pRS416 to obtain recombinant plasmids pRS416-GFP, pRS416-PdhR-GFP and pRS416-NPdhR-GFP, which were respectively transformed into Saccharomyces cerevisiae BY4741 for the localization verification of PdhR and nuclear localization signal peptide.

[0037] The results are as follows Figure 1 As shown, the unmodified fluorescent protein GFP is uniformly dispersed in the cytoplasm after expression. The PdhR-GFP from E. coli is partially localized to the nucleus, while a portion remains free in the cytoplasm. After modification with the nuclear localization signal peptide, the fluorescence of the NLS-PdhR-GFP is concentrated and single, indicating that PdhR is completely and strictly transported to the nucleus.

[0038] Example 2: Construction and orthogonality verification of pyruvate-responsive gene circuits (1) Construction of pyruvate-activated gene circuit The PdhR binding sequence (pdhO) was inserted into the constitutive promoter P TDH3 The -150, -128 and -5 sites of the pyruvate-activated promoter P TDH3-1 、P TDH3-2 and P TDH3-3 , pdhO, promoter P TDH3-2 The nucleotide sequences are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0039] The expression cassettes P were constructed by overlapping extension PCR and connecting the reporter gene GFP. TDH3-1 -gfp-T CYC1 、P TDH3-2 -gfp-T CYC1 and P TDH3-3 -gfp-T CYC1 The expression cassettes were constructed into the vector pRS416 to obtain recombinant plasmids pRS416-GFP1, pRS416-GFP2 and pRS416-GFP3, which were respectively transformed into Saccharomyces cerevisiae BY4741. The constitutive promoter P was amplified using the genome of Saccharomyces cerevisiae BY4741 as a template. TEF1The transcription factor pdhR N-terminal was fused with the nuclear localization tag NLS, and the C-terminal was fused with the protein degradation tag PEST to obtain NLS-pdhR-PEST. The nucleotide sequence of PEST is shown in SEQ ID NO.5. The expression cassette P was obtained by overlapping extension PCR. TEF1 -NLS-pdhR-PEST-T ADH1 The above expression cassettes were constructed into recombinant plasmids pRS416-GFP1, pRS416-GFP2 and pRS416-GFP3 with different pdhO insertion sites, respectively, to obtain recombinant plasmids pRS416-TPdhR-GFP1, pRS416-TPdhR-GFP2 and pRS416-TPdhR-GFP3, which were respectively transformed into Saccharomyces cerevisiae BY4741 to construct a pyruvate-activated gene circuit. The transcription factor PdhR can specifically recognize pdhO and bind to the modified pyruvate-activated promoter, inhibiting the transcription of the downstream reporter gene gfp; when pyruvate is present, the transcription factor PdhR preferentially binds to pyruvate and detaches from the modified promoter, and the downstream gene resumes normal transcriptional expression. The results showed that PdhR can bind to the promoter P TDH3-2 The results are shown in Figure 2. Figure 2 As shown, PdhR is expressed on three pdhO The inhibition folds of the insertion sites were 5.7, 22.0, and 8.5, respectively. Site No. 2 (-128) was selected for subsequent investigation and evaluation.

[0040] Amplify the weak promoter P by existing methods PRM and P AGA , expression cassettes with different promoter strengths were obtained by overlapping extension PCR. PRM / P AGA -NLS-pdhR-PEST-T ADH1 The expression cassettes with different promoter strengths were constructed into the recombinant plasmid pRS416-GFP2 to obtain the recombinant plasmid pRS416-PPdhR-GFP2 (pdhR promoter is P PRM ) and pRS416-APdhR-GFP2 (pdhR promoter is P AGA ), were transformed into Saccharomyces cerevisiae BY4741. The results are as follows Figure 3 As shown, under weak PdhR expression, the pyruvate-activated gene circuit is turned on, and the two promoters (P AGA 、P PRM ) The dynamic multiples are 3.7 times and 3.4 times respectively.

[0041] (2) Construction of pyruvate-repressible gene circuit The XylR binding sequence (xylO) was inserted into the constitutive promoter PTEF1 -105 site of the synthesized pyruvate-repressible promoter P TEF1-1 The nucleotide sequences of XylR and xylO are shown in SEQ ID NO.6 and SEQ ID NO.7 respectively. The expression cassette of reporter gene mCherry was obtained by overlapping extension PCR. TEF1-1 -mcherry-T CYC1 The expression cassette was constructed into the vector pRS416 to obtain the recombinant plasmid pRS416-mCherry1.

[0042] The nuclear localization tag NLS was fused to the N-terminus of the xylose transcription factor XylR, and the protein degradation tag CLN2 was fused to the C-terminus to synthesize the codon-optimized xylose transcription factor NLS-xylR-CLN2 containing the nuclear localization signal peptide and the degradation tag. The nucleotide sequence of CLN2 is shown in SEQ ID NO. 8. The expression cassette of XylR was obtained by overlap extension PCR. TDH3-2 -NLS-xylR-CLN2-T CYC1 The expression cassette was constructed into the recombinant plasmid pRS416-mCherry1 to obtain the recombinant plasmid pRS416-XylR-mCherry1. PRM -NLS-pdhR-PEST-T ADH1 , constructed into the vector pRS416-XylR-mCherry1, and obtained the pyruvate-repressed recombinant plasmid pRS416-PdhR-XylR-mCherry1, which was then transformed into Saccharomyces cerevisiae BY4741 to obtain a pyruvate-repressed gene circuit. Pyruvate can activate the expression of XylR and further inhibit the promoter P TEF1-1 The inhibition factor of the pyruvate-repressible gene circuit was tested using different concentrations of glucose. The results showed that under the condition of a glucose concentration of 10g / L, the inhibition factor of the pyruvate-repressible gene circuit was 3.1 times.

[0043] (3) Construction and orthogonality verification of a bifunctional pyruvate-responsive gene circuit Amplified expression cassette P TDH3-2 -gfp-T CYC1 The expression cassette was constructed into the recombinant plasmid pRS416-PdhR-XylR-mCherry1 to obtain the recombinant plasmid pRS416-PdhR-XylR-mCherry1-GFP2, which was then transformed into Saccharomyces cerevisiae BY4741. Figure 4 The Saccharomyces cerevisiae cells shown contain both a pyruvate-activated gene circuit and a pyruvate-repressed gene circuit. Using glucose as an activator, the performance of the bifunctional pyruvate-responsive gene circuit was tested using different concentrations of glucose. Figure 5 As shown, glucose activated GFP expression while simultaneously repressing mCherry expression. Under 10 g / L glucose conditions, the activation factor was 3.6-fold and the repression factor was 3.0-fold, similar to the performance of the two gene circuits tested separately. These results demonstrate that the constructed pyruvate-activated and repressed gene circuits have good orthogonality and can be used simultaneously to regulate the metabolic network of Saccharomyces cerevisiae.

[0044] Example 3: Construction of a Dynamically Regulated Malic Acid Metabolic Pathway This example takes malic acid synthesis as an example, and uses the growth-defective pyruvate decarboxylase (pdc)-deficient Saccharomyces cerevisiae TAM as the research object. The TAM strain was genetically modified to construct different malic acid-synthesizing strains TAM1, TAM2, and TAM3.

[0045] like Figure 7 As shown, the control strain TAM1 contains a malate synthesis pathway based on pyc2, mdh3, and mae1 genes, the control strain TAM2 adds a Pdc1 complementation module based on TAM1, and the control strain TAM3 introduces the bifunctional pyruvate response gene circuit constructed in Example 2 as a regulatory module based on TAM2. Figure 6 shown.

[0046] The specific construction method is as follows: Amplify constitutive promoters using the Saccharomyces cerevisiae genome as a template P TPL1 , pyruvate carboxylase gene pyc2 (Genbank: 850733) and the malate dehydrogenase gene without the C-terminal peroxisome targeting tag SKL mdh3ΔSKL (SEQ ID NO.9). A codon-optimized malate transporter mutant gene from fission yeast was synthesized in our company. mae1 F253A (SEQ ID NO. 10), terminator T RPP1BT and T ENO2 The expression cassette was obtained by assembling multiple fragments through fusion PCR overlap extension. P TDH3-2 -pyc2-T RPP1BT 、 P TPL1- mdh3-T ENO2 and P TEF1 -mae1-T ADH1, and were successively constructed into the centromere expression vector pRS416 through one-step cloning, finally obtaining the recombinant plasmid pRS416-M containing the malic acid synthesis pathway, which was then transformed into the cerevisiae TAM to construct the malic acid-synthesizing strain TAM1 as a control strain.

[0047] Amplification of the pyruvate decarboxylase gene using the Saccharomyces cerevisiae genome as a template pdc1 (Genbank: 852519), amplify the pyruvate-repressible promoter using existing methods P TEF1-1 and terminator T CYC1 , fusion PCR overlap extension assembly multiple fragments to obtain expression cassette P TEF1-1 -pdc1-T CYC1 , was constructed into the vector pRS416-M by one-step cloning to obtain the recombinant plasmid pRS416-M-Pdc, which was then transformed into Saccharomyces cerevisiae TAM to construct the malic acid synthesis strain TAM2 containing the malic acid synthesis pathway and Pdc1 backfill module as a control strain.

[0048] Furthermore, the recombinant plasmid pRS416-PdhR-XylR-mCherry1-GFP2 in Example 2 was used as a template to amplify the transcription factor expression cassette. P TDH3-2 -xylR-T CYC1t -P PRM -pdhR-T ADH1 , cloned into plasmid pRS416-M-Pdc in one step to obtain plasmid pRS416-M-Pdc-XylR-PdhR, which was transformed into strain TAM to construct the regulated malate synthesis strain TAM3 as an experimental strain, containing the malate synthesis pathway, Pdc1 replenishment module and regulatory module.

[0049] The above recombinant plasmids were constructed in Escherichia coli DH10b competent cells and screened using LB-Amp plates. The pRS416-based recombinant plasmids were electroporated into Saccharomyces cerevisiae TAM competent cells and screened using SC-Ura plates. The cells were cultured at 30°C for 72 hours, and single colonies were selected for colony PCR verification. The genomes were then extracted and sequenced.

[0050] Example 4: Shake flask fermentation performance of strain TAM1 The strain TAM1 was isolated at an initial OD 6000.1 was inoculated into fermentation medium with 100 g / L glucose as the sole carbon source, and changes in parameters such as OD, glucose, pyruvate, ethanol, and malate were monitored. Fermentation medium: 3 g / L potassium dihydrogen phosphate, 6.6 g / L potassium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 1 g / L urea, trace elements, and vitamin solution.

[0051] like Figure 8 As shown, the growth of strain TAM1 was slow, and the OD 600 The maximum specific growth rate μ of strain TAM1 was max 0.13 h -1 , slightly lower than the 0.2 h reported in the literature -1 This is due to differences in the culture medium and fermentation environment. Due to the complete knockout of pdc, this strain completely eliminated ethanol accumulation and instead accumulated significant amounts of pyruvate. Pyruvate concentrations reached as high as 26 g / L on day 10. After glucose depletion, some pyruvate was reabsorbed and utilized. Strain TAM1 achieved a final malate production of 2.90 g / L.

[0052] Example 5: Comparison of shake flask fermentation performance between strains TAM2 and TAM3 Strains TAM2 and TAM3 were isolated at the same initial OD 600 0.1 was inoculated into the fermentation medium, and combined with the fermentation strategy regulated by glucose, 5 g / L glucose was fed at the beginning of the fermentation until OD 600 After reaching 15, the remaining glucose was replenished at one time, and the total glucose amount was 100 g / L. The OD 600 The fermentation performance of the control strain TAM2 and the experimental strain TAM3 was systematically evaluated. Figure 9 As shown in A, due to the complementation of Pdc1, the growth of strains TAM2 and TAM3 was restored and the OD 600 The growth situation is close to the maximum specific growth rate μ max 0.32h -1 , much larger than strain TAM1, the final OD 600 Close to TAM1. The byproduct ethanol production is as follows Figure 9 As shown in Figure B, the ethanol accumulation of strain TAM3 decreased. This is because the expression of Pdc1 was inhibited to a certain extent in the late fermentation period, and the ethanol synthesis flux decreased. Figure 9 As shown in Figure C, strain TAM3 accumulates a small amount of pyruvate during fermentation, reaching 6 g / L on day 5, which is then reabsorbed and utilized. This is due to a decrease in ethanol metabolic flux, which leads to the partial accumulation of pyruvate, providing more precursors for the synthesis of the product malate.

[0053] Malic acid production Figure 9 As shown in Figure D, strains TAM2 and TAM3 produced malic acid yields of 1.50 g / L and 1.95 g / L, respectively. Under pyruvate regulation, strain TAM3 increased malic acid yield by 30%. In the late fermentation phase, strain TAM3 significantly increased malic acid yield compared to strain TAM2. This is due to the inhibition of Pdc1 expression in the late fermentation phase, which effectively weakens the ethanol synthesis flux and provides more pyruvate precursors for malic acid synthesis. Under these fermentation conditions, although the final yield of strain TAM3 was lower than that of strain TAM1, due to the recovery of cell growth, the fermentation cycle was shortened to 6 days, and the production intensity increased to 0.33 g / (L·d), exceeding the 0.30 g / (L·d) of strain TAM1. This suggests that strain TAM3 can produce malic acid in a shorter fermentation time and with higher efficiency, shortening the production cycle and improving economic benefits. In summary, compared with the static transformation strategy of completely knocking out pdc, the dynamic regulation strategy of growth replenishment based on pyruvate response allows the restoration of TAM growth in Saccharomyces cerevisiae while improving production intensity and shortening the fermentation cycle.

[0054] Example 6: Fermentation parameter optimization Considering that there is a certain amount of leaky expression in the repressive pyruvate gene circuit, shutting down pdc expression when the strain growth density is the highest is not the optimal shut-down time. Therefore, this example further studied the effect of different glucose addition timings on the dynamic regulation of malic acid synthesis. Six experimental groups were set up, each at OD 600 The remaining glucose is replenished when the values are 0, 5, 7, 10, 13, and 15. The results are as follows Figure 10 As shown in Figure A, malic acid production was lowest at 1.2 g / L when 100 g / L glucose was fed once at the beginning of fermentation. Compared to feeding at maximum growth density, feeding within an OD range of 7-10 allowed the strain to obtain a more adequate carbon source for a longer period of time, increasing malic acid production to 2.23 g / L.

[0055] Based on the physiological characteristics of Saccharomyces cerevisiae, increasing the dissolved oxygen level can weaken ethanol metabolism to a certain extent. At the shake flask level, 50 mL of culture medium was loaded in different volumes of shake flasks for fermentation to provide different dissolved oxygen levels. Figure 10As shown in Figure B, ethanol production decreased significantly with increasing dissolved oxygen levels, while strain growth density increased. Malic acid production reached a maximum of 2.50 g / L at a shake flask volume of 300 mL. Adequate oxygen supply enabled cells to perform aerobic respiration more efficiently, increasing biomass. Simultaneously, it inhibited the activity of enzymes involved in ethanol fermentation, directing more carbon flux toward cell growth and malic acid synthesis, synergistically increasing malic acid production while further reducing ethanol production and boosting malic acid production intensity to 0.42 g / (L·d).

Claims

1. A method for redirecting ethanol metabolism in Saccharomyces cerevisiae mediated by a pyruvate gene circuit, characterized in that: include: Constructing a pyruvate-responsive gene circuit in the host strain Saccharomyces cerevisiae to obtain modified Saccharomyces cerevisiae; The host bacteria is Saccharomyces cerevisiae in which cell growth-related ethanol metabolism genes are knocked out; the pyruvate-responsive gene circuit comprises the cell growth-related ethanol metabolism genes, genes related to the product synthesis pathway, the transcription factor PdhR, a pyruvate-activated promoter, the xylose transcription repressor XylR, and a pyruvate-repressible promoter; The pyruvate-activated promoter is a constitutive promoter into which the PdhR binding sequence pdhO is inserted; the ketoacid-repressible promoter is a constitutive promoter into which the XylR binding sequence xylO is inserted; the N-termini of the PdhR and XylR are fused with nuclear localization signal peptides respectively; The PdhR inhibits or restores the expression of genes related to the product synthesis pathway by binding to pdhO or pyruvate, and the XylR restores or inhibits the expression of ethanol metabolism genes related to cell growth by binding to xylO or pyruvate; The modified brewer's yeast is fermented and cultured. During the fermentation culture, the initial glucose concentration of the fermentation medium is set, and the remaining glucose is replenished at one time when the fermentation culture cell density reaches a preset range.

2. The method according to claim 1, characterized in that The host bacteria is pyruvate decarboxylase-deficient Saccharomyces cerevisiae; The cell growth-related ethanol metabolism gene is pyruvate decarboxylase.

3. The method according to claim 2, characterized in that The host strain is Saccharomyces cerevisiae TAM.

4. The method according to claim 1, wherein The nucleotide sequence of the nuclear localization signal peptide is shown in SEQ ID NO.

2.

5. The method according to claim 1, wherein The C-terminal fusion protein degradation tags of the transcription factor PdhR and the xylose transcription repressor XylR.

6. The method according to claim 5, wherein The nucleotide sequence of the protein degradation tag fused to the C-terminus of the transcription factor PdhR is shown in SEQ ID NO.5, and the nucleotide sequence of the protein degradation tag fused to the C-terminus of the xylose transcription repressor XylR is shown in SEQ ID NO.

8.

7. The method according to claim 1, characterized in that The constitutive promoter of pdhO inserted into the pyruvate-activated gene circuit is P TDH3 The constitutive promoter inserted into the xylO in the pyruvate repressible gene circuit is P TEF1 .

8. The method according to claim 7, characterized in that The PdhO inserts into P TDH3 -150 site, -128 site or -5 site of the promoter; The XylO is inserted into the P TEF1 The -105 site of the promoter.

9. The method according to claim 1, characterized in that The PdhR expression promoter is selected from P PRM promoter; The expression promoter of the xylose transcription repressor XylR is a pyruvate-activated promoter.

10. The method according to claim 1, characterized in that The initial glucose concentration is ≤10 g / L.

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