Method for constructing yarrowia lipolytica engineering bacteria and producing itaconic acid

Abstract

The invention discloses a method for constructing yarrowia lipolytica engineering bacteria and producing itaconic acid, which comprises the following steps: adopting an optimized heterologous expression strategy, utilizing mitochondrial targeting signal peptide to improve the positioning efficiency of a core enzyme Cis-acetic acid decarboxylase, enhancing the contact between the enzyme and a substrate and promoting the synthesis of itaconic acid, and then, utilizing mitochondrial targeting signal peptide to enhance the reaction between the enzyme and the substrate to promote the synthesis of itaconic acid. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of escherichia coli are added, cofactor circulation in mitochondria is regulated and controlled, the generation and regeneration efficiency of NADPH and NADH is improved, finally, a transmembrane transporter gene ldhT is modified, the amino acid sequence and the spiral structure of the transmembrane transporter gene ldhT are optimized, the excretion efficiency of itaconic acid is improved, and the yield of itaconic acid is increased. The negative influence of intracellular accumulation on the cells is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of Yarrowia lipolytica engineering bacteria, in particular to the construction of Yarrowia lipolytica engineering bacteria and a method for producing itaconic acid. Background Art

[0002] Itaconic acid is an important organic chemical raw material, widely used in medicine, food additives and polymer materials. Its green and biomanufacturing methods have environmental and economic advantages. As an industrial microorganism with strong natural adaptability, Yarrowia lipolytica has become the preferred host for itaconic acid production due to its wide utilization of carbon sources and tolerance to environmental stress. However, in the biological production of itaconic acid, the metabolic performance and fermentation efficiency of Yarrowia lipolytica are still limited. First, CAD, the key enzyme in itaconate synthesis, needs to be in full contact with cis-aconitic acid in mitochondria. The accuracy of its subcellular localization directly affects the catalytic efficiency. However, heterologous CAD enzymes are usually expressed in the cytoplasm and isolated from the mitochondria where the substrate is generated, resulting in low efficiency of the target metabolic pathway. Second, itaconic acid production depends on the participation of a large number of cofactors, but the cofactor production pathway of Yarrowia lipolytica is inefficient and cannot meet the needs of efficient enzymatic reactions; 3. The accumulation of cytosolic anaconazole can easily induce metabolic feedback inhibition and reduce the metabolic flux of the target pathway. At the same time, the acidic stress of the weak acid environment has an adverse effect on the metabolic stability and cell viability of Yarrowia lipolytica.

[0003] Based on this, the construction of Yarrowia lipolytica engineered bacteria and the method for producing itaconic acid are needed. Summary of the invention

[0004] To achieve the above object, the present invention provides the following scheme: a method for constructing an engineered strain of Yarrowia lipolytica and producing itaconic acid, comprising the following steps: Step S1: introducing a cis-aconiticacid decarboxylase CAD gene derived from Aspergillus into Yarrowia lipolytica by genetic engineering means, and adding a mitochondrial targeting signal peptide to target the CAD enzyme to the mitochondrial cavity, wherein the mitochondrial targeting signal peptide is an N-terminal sequence derived from the CoxIV protein of Saccharomyces cerevisiae, and a polypeptide sequence MGWSK is added thereto, and the CAD enzyme catalyzes cis-aconitic acid to generate itaconic acid in the mitochondria; Step S2: heterologously expressing the glucose-6-phosphate dehydrogenase zwf gene and the 6-phosphogluconate dehydrogenase gnd gene from Escherichia coli and regulating the expression of the mitochondrial NADH dehydrogenase gene to increase the generation and circulation levels of the cofactors NADPH and NADH, wherein the NADPH concentration is increased to 2 to 2.5 times the original metabolic level, thereby providing sufficient reducing power for the catalytic reaction of the CAD enzyme in step S1; Step S3: integrating the lactic acid transmembrane transporter gene ldhT from Lactobacillus into the Yarrowia lipolytica genome, and optimizing the key amino acid residues in the hydrophobic region of the transmembrane transporter to mutate the K86 site to A, so that the transmembrane transporter can adapt to the transmembrane transport requirements of itaconic acid; Step S4: using CRISPR Cas9 gene editing to knock out fatty acid synthesis-related genes FAS1 and FAS2, and citric acid diversion-related gene CIT1, to adjust the carbon flow metabolic pathway of Yarrowia lipolytica, and to allocate metabolic carbon flow to cis-aconitic acid production; Step S5: Under fermentation conditions with a low pH value of 3.5 to 4.5, the carbon source ratio is 60wt% to 70wt% of glucose, 20wt% to 30wt% of glycerol, and 5wt% to 15wt% of waste oil and fat based on the total carbon source, and a phosphate buffer system is used to adjust the pH value of the fermentation liquid, and the oxygen supply is adjusted by controlling the stirring rate and the gas flow rate to maintain the dissolved oxygen level at 20% to 30%.

[0005] Further preferably, the amino acid sequence of the mitochondrial targeting signal peptide in step S1 is an optimized N-terminal fragment of Saccharomyces cerevisiae CoxIV protein, and the optimized fragment contains 20 amino acids, wherein the valine at position 4 is replaced by leucine, and the serine at position 12 is replaced by alanine.

[0006] Further preferably, the CAD gene integration position in step S1 is located in the 26S ribosomal RNA operator region of the Yarrowia lipolytica genome, and the integration is inserted in the form of multiple copies, with each copy spaced 500 bases apart, and termination signal sequences derived from the Saccharomyces cerevisiae ENO1 gene are added upstream and downstream of the insertion region.

[0007] Further preferably, the generation of the cofactor in step S2 is enhanced by driving the expression of the zwf gene and the gnd gene through a dual promoter system, wherein the zwf gene front driver selects the PGK1 promoter of Saccharomyces cerevisiae, and the gnd gene front driver selects the GPD1 promoter with strong transcriptional activity, and a signal sequence is added to the 3' end of the gene.

[0008] Further preferably, the optimization of the lactate transmembrane transporter in step S3 includes site-directed mutation of the K86 site to enhance hydrophobicity, and adjusting the helical inclination of the transmembrane region by molecular dynamics simulation, the inclination angles are 15°, 20° and 25°, respectively, to form a larger molecular channel pore size, and the optimized transmembrane protein channel is suitable for the transport of small molecules with a molecular weight of 160 to 200 Daltons.

[0009] Further preferably, in step S4, the knockout of the fatty acid metabolism genes FAS1 and FAS2 and the shearing of the citric acid shunt gene CIT1 are completed by CRISPR gene editing, the editing target sites of the FAS1 and FAS2 genes are located at the start position of the coding region, the CIT1 gene shearing retains its first exon and deletes the second exon, and at the same time, the transcription level of the PPT2 gene related to fatty acid metabolism is inhibited.

[0010] Further preferably, in step S4, a heterologous sphingosine synthase derived from Agrobacterium is introduced to enhance the metabolic efficiency of cofactors in the cytoplasm, and the endogenous yeast aconitase gene is replaced by a plant-derived aconitase gene, the plant aconitase gene is selected from Arabidopsis thaliana, and its expression is regulated by the TEF1 promoter.

[0011] Further preferably, in step S5, the carbon source ratio control adopts a phased supply strategy, the glucose concentration is controlled at 40 g / L to 50 g / L in the initial fermentation stage, the glycerol concentration added in the middle fermentation stage is 15 g / L to 20 g / L, the waste oil addition time is set to 48 hours after fermentation, and the addition rate per hour is 5 g / L to 10 g / L.

[0012] Further preferably, in the step S5, the fermentation process adopts batch feeding, the feeding interval of each batch is 24 hours to 36 hours, the total feeding amount is controlled at 20% to 30% of the initial culture solution volume, and the pH of the culture solution at the end of fermentation is controlled at 4.0 to 4.2.

[0013] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects: 1. The present invention improves the localization efficiency of the CAD enzyme by adding a mitochondrial targeting signal peptide derived from the CoxIV protein of Saccharomyces cerevisiae and optimizing the signal peptide sequence; an optimized CoxIV signal peptide version is added to the front end of the expression sequence of the CAD gene, wherein the 4th valine is replaced by leucine and the 12th serine is replaced by alanine to enhance the stability of the signal peptide and the mitochondrial recognition efficiency, and the CAD gene is precisely integrated into the 26S ribosomal RNA control region of the Yarrowia lipolytica genome by using CRISPR-Cas9 to ensure efficient expression, and a transcription termination signal derived from the ENO1 gene is added upstream and downstream to reduce nonspecific transcription interference and improve expression stability.

[0014] 2. The present invention enhances the generation and circulation efficiency of cofactors through heterologous gene expression and regulation; by adding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of Escherichia coli, and using PGK1 and GPD1 dual promoters to drive the production of NADPH, a polyA signal sequence derived from Saccharomyces cerevisiae ACT1 is added to the 3' end of the gene to enhance the stability and expression of mRNA.

[0015] 3. The present invention adds the transmembrane transport protein gene ldhT derived from lactobacillus, optimizes its key amino acid sequence through molecular dynamics simulation, replaces the K86 site in the hydrophobic region with A, enhances the binding ability of itaconic acid to the transport protein, and at the same time, adjusts the inclination angle of the helical structure in the transmembrane region and optimizes it to 15°, 20° and 25°, expands the pore size of the molecular channel, integrates the optimized ldhT gene into the high expression site of the Yarrowia lipolytica genome, and adds termination signals upstream and downstream to reduce transcription interference, thereby ensuring the continuous and efficient expression of the efflux protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The present invention is a flow chart from the construction of Yarrowia lipolytica engineered bacteria to the production of itaconic acid. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Example like Figure 1 As shown, the construction of the Yarrowia lipolytica engineered bacteria and the method for producing itaconic acid in this embodiment include the following steps: Step S1: CAD gene introduction and mitochondrial targeting optimization In this step, the CAD gene is integrated into the 26S ribosomal RNA operator region of the Yarrowia lipolytica genome. During the integration process, the CAD gene is inserted in the form of multiple copies, with each copy spaced 500 bases apart, so that the transcription space between the copies is independent, and transcription interference caused by dense copies is avoided. In addition, termination signal sequences derived from the ENO1 gene of Saccharomyces cerevisiae are added to the upstream and downstream of the integrated gene. The termination signal sequence can effectively terminate the transcription process and avoid interference of non-specific transcription extension on downstream genes. Through the optimization of this step, the expression level of the CAD gene is increased, and its product CAD protein accounts for more than 20% of the total protein content of Yarrowia lipolytica, providing sufficient enzyme basis for the subsequent synthesis of itaconic acid; On the basis of achieving efficient expression, this step precisely locates the CAD enzyme into the mitochondrial cavity through mitochondrial targeting. Mitochondria are the main production site of cis-aconiticacid. Targeting the CAD enzyme into the mitochondrial cavity improves the spatial synergistic efficiency of the enzyme and the substrate and increases the catalytic rate. Specifically: A mitochondrial targeting signal peptide was added to the front end of the expression sequence of the CAD gene. The signal peptide was derived from the N-terminal sequence of the CoxIV protein of Saccharomyces cerevisiae, and the amino acid sequence of the signal peptide was further modified: the 4th valine was replaced by leucine, and the 12th serine was replaced by alanine to further optimize the targeting efficiency and stability of the signal peptide. The experimental objectives of this step S1 are to optimize the signal peptide sequence through gene editing, verify the effect of the optimized signal peptide on improving the mitochondrial targeting efficiency and localization stability in Yarrowia lipolytica, and evaluate its effect on the incidence of enzyme localization errors; Experimental Materials 1. Strain and vector construction Tested strains: Yarrowia lipolytica; Vector: pYL-CAD plasmid was constructed to express the CAD gene and contain the mitochondrial targeting signal peptide sequence. The optimized signal peptide sequence (optimized version) and the original signal peptide sequence (unoptimized version) were cloned into the plasmid respectively. The optimized version was named pYL-CAD-opt and the unoptimized version was named pYL-CAD-wt. Targeting signal peptide sequence optimization: Original sequence: derived from the N-terminus of CoxIV protein of Saccharomyces cerevisiae; Optimized sequence: valine at position 4 was replaced by leucine, and serine at position 12 was replaced by alanine.

[0021] 2. Gene Editing CRISPR-Cas9 was used to integrate the optimized and unoptimized versions of the CAD gene into the 26S ribosomal RNA operator region of the Yarrowia lipolytica genome, ensuring that the upstream and downstream of the insertion region carried the ENO1 termination signal sequence to improve transcriptional stability. Real-time fluorescence quantitative PCR was used to verify the insertion success rate of gene editing.

[0022] 3. Targeting efficiency test Green fluorescent protein GFP was fused to the C-terminus of the CAD gene to facilitate the observation of the mitochondrial localization of the CAD enzyme. The cells were cultured in YNB medium at 37°C for 48 hours, and the co-localization of GFP signal and mitochondrial-specific dye MitoTracker was observed using a confocal fluorescence microscope. Data analysis: Targeting efficiency was defined as the ratio of pixels where GFP signal colocalized with MitoTracker signal; 4. Positioning stability test method The strain was cultured under high-intensity transcription conditions and high glucose concentration of 50 g / L for 48 hours to observe the stability of the signal peptide and the changes in the co-localization ratio of GFP signal and MitoTracker signal during the culture period; Method for the incidence of enzyme localization errors: The GFP signal intensity in the cytoplasm was detected under the same conditions, and the proportion of CAD-GFP that was not targeted to mitochondria was calculated.

[0023] Experimental Results 1. Mitochondrial targeting efficiency Optimized signal peptide pYL-CAD-opt: The co-localization efficiency of GFP signal and MitoTracker signal was 85%±2%; Unoptimized signal peptide pYL-CAD-wt: The co-localization efficiency was 65%±3%; The optimized version was improved by about 20% compared with the unoptimized version; 2. Positioning stability Optimized signal peptide: under 48 hours of high transcription pressure, the co-localization efficiency dropped from 85% to 80%; non-optimized signal peptide: the co-localization efficiency dropped from 65% to 50%; The optimized signal peptide has improved positioning stability; 3. Incidence of enzyme localization errors Optimized signal peptide: GFP signal ratio in the cytoplasm is 10%±1%; Unoptimized signal peptide: The GFP signal ratio in the cytoplasm is 30%±2%; Optimization effect: The optimized version reduces the incidence of CAD enzyme positioning errors.

[0024] Experimental conclusion: The optimized signal peptide sequence achieves improved mitochondrial targeting efficiency through gene editing. Its positioning efficiency is about 20% higher than that of the original sequence, and the positioning stability is significantly better than that of the unoptimized version under long-term culture conditions. At the same time, the optimized signal peptide reduces the incidence of CAD enzyme positioning errors, laying a foundation for stable enzyme expression to achieve efficient itaconate synthesis. The results of this experiment verify the effectiveness of the step S1 optimization strategy and provide a guarantee for the enhancement of cofactor generation in step S2.

[0025] Step S2: Enhancement of cofactor production and regulation of the cycle After achieving efficient expression and mitochondrial targeting of the CAD gene in step S1, the generation and circulation regulation of cofactors NADPH and NADH are optimized. The catalytic reaction of the CAD enzyme requires NADPH and NADH as providers of reducing power, and their concentration levels directly affect the generation rate and metabolic efficiency of itaconic acid. The endogenous cofactor generation pathway of Yarrowia lipolytica is inefficient and cannot meet the needs of efficient metabolic pathways. The cofactor generation capacity is enhanced by heterologous gene expression. The invention uses the glucose-6-phosphate dehydrogenase zwf gene and the 6-phosphogluconate dehydrogenase gnd gene derived from Escherichia coli, the promoter before the zwf gene is the PGK1 promoter of Saccharomyces cerevisiae, and the promoter before the gnd gene is the GPD1 promoter with strong transcriptional activity, so as to enhance the transcription level of the enzyme and improve the generation capacity of NADPH, and add the polyA signal sequence derived from the ACT1 gene of Saccharomyces cerevisiae to the 3' end of the two genes, so as to prolong the half-life of mRNA and improve the stability and efficiency of gene expression; The experimental objectives of this step S2 are: to optimize the gene expression regulation in the cofactor production pathway, to verify the effect of the dual promoter system and the 3'-end polyA signal sequence on the efficiency of NADPH and NADH production, and to evaluate the improvement effect on the catalytic efficiency and overall metabolic stability of the CAD enzyme; Experimental Materials 1. Strain and vector construction: Tested strain: Yarrowia lipolytica; Vector construction: pYL-zwf and pYL-gnd vectors were constructed to express the glucose-6-phosphate dehydrogenase zwf gene and the 6-phosphogluconate dehydrogenase gnd gene, respectively. The Saccharomyces cerevisiae PGK1 promoter was inserted into the front end of the zwf gene, and the GPD1 promoter was inserted into the front end of the gnd gene to form a dual promoter system. The polyA signal sequence derived from the Saccharomyces cerevisiae ACT1 gene was added to the 3' end of the zwf and gnd genes to enhance the mRNA stability. 2. Gene integration and verification: Use CRISPR-Cas9 to integrate the constructed pYL-zwf and pYL-gnd into specific sites of the Yarrowia lipolytica genome respectively; detect the integration success rate of the gene by real-time fluorescence quantitative PCR, and measure the mRNA transcription level of the gene to evaluate the function of the polyA signal sequence; 3. Cofactor generation efficiency test: The optimized strain and the control strain (non-optimized Yarrowia lipolytica) were cultured in YNB medium containing 50 g / L glucose for 48 hours; the concentrations of intracellular NADPH and NADH were detected by enzyme-linked immunosorbent assay (ELISA), respectively; the production efficiency of NADPH and NADH was calculated by the ratio of the concentration of the optimized strain to that of the control strain; 4.CAD enzyme catalytic efficiency test: Enzyme activity determination: After the CAD enzyme was extracted from the culture fluid of the optimized strain and the control strain, its enzyme activity for converting cis-aconiticacid into itaconic acid was determined by spectrometry; Itaconic acid production determination: High performance liquid chromatography (HPLC) was used to analyze the itaconic acid concentration in the culture broth to evaluate the actual effect of CAD enzyme catalytic efficiency; 5. Overall metabolic stability test: During the culture process, the biomass OD600 value, glucose consumption rate and by-product generation of the strain were measured; the fermentation was continued for 72 hours to observe the metabolic stability of the strain.

[0026] Experimental Results 1.NADPH and NADH generation efficiency: Optimized strain: NADPH concentration was 42.5±2.3μM, NADH concentration was 38.6±1.8μM; Control strain: NADPH concentration was 18.7 ± 1.2 μM, NADH concentration was 15.4 ± 1.0 μM; Improvement: The cofactor production efficiency of the optimized strain was increased to 2.3 times that of the wild-type strain.

[0027] 2. CAD enzyme catalytic efficiency: The CAD enzyme activity of the optimized strain was 8.2 ± 0.4 μmol / min / mg protein, while that of the control strain was 3.5 ± 0.2 μmol / min / mg protein; The itaconic acid production of the optimized strain reached 48 g / L, while that of the control strain was 22 g / L; Improvement effect: CAD enzyme catalytic efficiency and itaconic acid production rate increased by 2.3 times and 2.2 times respectively; 3. Overall metabolic stability: Biomass: The OD600 value of the optimized strain was stable at 36±2, while that of the control strain was 28±2; Byproduct production: The citric acid production of the optimized strain was 3.2 g / L, while that of the control strain was 6.8 g / L; Metabolic balance: The optimized strain showed higher metabolic stability and 52% less by-product production.

[0028] Experimental conclusion: Through the combination of the dual promoter system and the 3'-end polyA signal sequence, the production efficiency of NADPH and NADH in Yarrowia lipolytica is enhanced, and the cofactor production is increased to 2.3 times that of the wild-type strain. The catalytic activity of the CAD enzyme is effectively improved through optimization, and its enzyme activity is increased by 2.3 times. The production rate of itaconic acid is increased by 2.2 times. The optimized cofactor cycle improves the overall metabolic stability of the strain, reduces the generation of by-products, and improves the production efficiency of the target product.

[0029] Step S3: Optimization of transmembrane transporters and itaconate efflux After optimizing the cofactor generation cycle and enhancing the catalytic efficiency of the CAD enzyme in step S2, the generation rate of itaconic acid is improved. However, as a weak acid, excessive accumulation of itaconic acid in the cell will have a negative impact on the metabolic balance of Yarrowia lipolytica. This step uses transmembrane transporter optimization to achieve efficient efflux of itaconic acid by introducing and modifying the lactic acid transmembrane transporter gene derived from Lactobacillus. Specifically, the amino acid sequence of the lactate transmembrane transporter was optimized, and the key amino acid K86 site in the hydrophobic region of the transporter was replaced with A through site-directed mutagenesis to enhance hydrophobicity and improve the binding ability of the transporter with hydrophobic molecules. At the same time, molecular dynamics simulation was used to refine the helical structure of the transmembrane region and adjust the inclination angles of the three main helices: optimizing them to 15°, 20° and 25°, expanding the pore size of the transmembrane channel, so that it can adapt to the transportation needs of small molecules with a molecular weight of 160 to 200 Daltons. In this step S1, Experimental objectives: To verify the improvement effect of the optimized lactate transmembrane transporter on itaconic acid transmembrane transport efficiency, intracellular acidity relief and strain metabolic balance through experiments; Experimental Materials 1. Strain and vector construction: Tested strain: Yarrowia lipolytica; Vector construction: pYL-ldhT vector was used to construct the unoptimized pYL-ldhT-wt and optimized pYL-ldhT-opt lactate transporter gene expression vectors; The unoptimized version directly uses the original ldhT gene sequence of Lactobacillus; the optimized version replaces the K86 site with A through site-directed mutagenesis and adjusts the helical inclination of the transmembrane region to 15°, 20°, and 25°; Gene integration: CRISPR-Cas9 was used to integrate pYL-ldhT-opt and pYL-ldhT-wt into the high expression sites of the Yarrowia lipolytica genome, and termination signal sequences derived from the ENO1 gene of Saccharomyces cerevisiae were added upstream and downstream of the integration region to ensure transcriptional stability; 2. Experimental groups: Control group: wild-type Yarrowia lipolytica (no ldhT expression); Unoptimized group: expressing the original lactate transmembrane transporter (pYL-ldhT-wt); Optimization group: expression of optimized lactate transmembrane transporter (pYL-ldhT-opt); 3. Transmembrane transport efficiency detection Culture conditions: YNB medium (supplemented with 50 g / L glucose and 20 g / L glycerol) was used and cultured at 37°C in a shaking incubator for 48 hours. Samples were taken regularly during the culture process to measure the intracellular and extracellular itaconic acid concentrations. Detection method: High performance liquid chromatography (HPLC) was used to measure the intracellular and extracellular itaconic acid concentrations; Calculation of transmembrane transport efficiency: ; 4. Intracellular acidity relief effect Detection method: Use fluorescent pH probe (pHrodoGreen) to measure intracellular pH value, and compare it 48 hours after fermentation. Analyze the negative correlation between the decrease of intracellular pH value and the acid relief effect; 5. Metabolic balance and biomass detection Detection indicators: biomass, measured by OD600; by-product production, measured by HPLC; continuous culture for 72 hours, regular sampling.

[0030] Experimental Results 1. Transmembrane transport efficiency: Control group: transmembrane efficiency was 15%±2%; Unoptimized group: transmembrane efficiency was 28%±3%; Optimized group: transmembrane efficiency was 68%±4%; Improvement effect: The transmembrane efficiency of the optimized lactate transmembrane transporter increased by 40 percentage points compared with the unoptimized version and by 53 percentage points compared with the control group. 2. Intracellular acidity relief effect: Control group: intracellular pH value decreased to 4.8±0.1; Unoptimized group: intracellular pH was 5.3 ± 0.2; Optimization group: intracellular pH was 6.1±0.1; Improvement effect: The optimized transporter alleviated the intracellular acidic environment, and the pH drop was further reduced by 30% compared with the non-optimized group; 3. Metabolic balance and biomass Biomass: Control group: OD600 value was 28±2; Unoptimized group: OD600 value was 34±2; Optimization group: OD600 value was 42±2; 4. Amount of by-products generated: Control group: byproduct generation was 6.5 g / L; Unoptimized group: byproduct generation was 4.2 g / L; Optimized group: byproduct generation was 2.7 g / L; Improvement effect: The optimized version reduces the amount of by-products generated and the biomass increases by 50% compared with the control group.

[0031] The above experiments verified that the optimized lactate transmembrane transporter pYL-ldhT-opt improved the transmembrane transport efficiency of itaconic acid in Yarrowia lipolytica. Its transmembrane efficiency was 40 percentage points higher than that of the non-optimized group, reaching 68%. The intracellular pH value of the optimized group was maintained at 6.1, and the decline was nearly 30% less than that of the control group, effectively solving the problem of metabolic feedback inhibition caused by itaconic acid accumulation, reducing the amount of by-products generated and increasing the biomass of the strain.

[0032] Step S4: Optimization of carbon flow allocation and metabolic network reconstruction After improving the transmembrane efflux efficiency of itaconic acid in step S3, this step solves the problem of insufficient precursors for itaconic acid synthesis by optimizing carbon flow distribution and reconstructing the metabolic network. This step adopts a multi-level metabolic network optimization strategy, specifically: CRISPR-Cas9 gene editing was used to precisely knock out genes related to fatty acid synthesis and citric acid diversion to reduce non-target allocation of carbon flow, as follows: On the one hand, the fatty acid synthesis-related genes FAS1 and FAS2 were knocked out: these two genes encode important subunits of fatty acid synthase, and their knockout can reduce the flow of acetyl-CoA to fatty acids. The editing target is located at the start position of the gene coding region, inhibiting fatty acid anabolism from the root; On the other hand, the CIT1 gene encodes citrate synthase, whose function is to combine acetyl-CoA and oxaloacetate to produce citric acid. In order to maintain the basal metabolic balance of the cell and reduce the excessive production of citric acid, a precise shearing strategy is used to retain the first exon of CIT1 and delete the second exon to achieve partial inhibition of citric acid synthesis. qPCR and protein electrophoresis are used to verify that the expression level of CIT1 after shearing is reduced to 40% of the original, but the basal metabolism can still proceed normally.

[0033] In this step, the utilization efficiency of the cofactor NADPH is improved by adding heterologous sphingosine synthetase; In this step, the endogenous aconitase gene of Yarrowia lipolytica is replaced with a plant-derived aconitase gene to enhance the efficiency of converting citric acid into cis-aconitase acid. The plant aconitase gene is regulated by the strong promoter TEF1, and its expression level is increased by 50% compared with the endogenous gene, thereby accelerating the generation rate of cis-aconitase acid.

[0034] In this step S4, 1. Experimental objectives: Verify the effects of gene knockout and splicing strategies on target metabolic pathways; Determine the proportion of cis-aconiticacid in carbon flow distribution after optimization; Evaluate the functionality of heterologous enzymes and plant aconitase gene replacements; 2. Experimental Methods Strain grouping: Control group: wild-type Yarrowia lipolytica; Optimized group 1: knockout of only FAS1 and FAS2; Optimized group 2: knockout of FAS1, FAS2 and cleavage of CIT1; Optimization group 3: adding sphingosine synthase and plant aconitase genes; Carbon flow distribution determination: using [U-13C] glucose isotope labeling method combined with gas chromatography-mass spectrometry GC-MS to analyze the distribution ratio of acetyl-CoA and citrate in different metabolic pathways; Metabolic efficiency determination: The production of cis-aconiticacid and itaconic acid was determined by high performance liquid chromatography (HPLC).

[0035] 3. Experimental results Carbon flow distribution ratio: Control group: cis-aconiticacid accounted for 40% ± 2% of the total carbon flow allocation; Optimized group 1: the cis-aconiticacid distribution ratio increased to 52%±3%; Optimized group 2: the allocation ratio was further increased to 58%±2%; Optimization group 3: The allocation ratio reached 65%±3%, achieving improvement; Target metabolite production: The amount of cis-aconiticacid produced: the optimized group 3 reached 18.6 g / L, and the control group was 8.5 g / L; Itaconic acid production: 64 g / L in the optimized group 3 and 22 g / L in the control group; Amount of by-products generated: The citric acid production in the optimized group 3 was reduced by 70% compared with that in the control group.

[0036] The metabolic flow distribution of Yarrowia lipolytica was optimized by precisely knocking out the fatty acid synthesis-related genes FAS1 and FAS2, cutting the citric acid shunt gene CIT1, and adding heterologous sphingosine synthase and plant aconitase genes. In the optimized strain, the distribution ratio of cis-aconiticacid in the carbon flow increased from 40% to more than 65%, the production of the target metabolite itaconic acid reached 64g / L, and the production of by-products was reduced.

[0037] Step S5: Fermentation process optimization Through steps S1-S4, the metabolic pathway of Yarrowia lipolytica is optimized. Since itaconic acid production needs to be carried out in an acidic environment, this step further improves the fermentation process for itaconic acid production. Specifically: By controlling the pH value of fermentation between 3.5 and 4.5 and using a phosphate buffer system to maintain acid-base balance, pH fluctuations caused by itaconic acid accumulation can be avoided; According to the carbon source demand at different fermentation stages, a phased supply strategy is adopted. In the initial fermentation period of 0 to 24 hours, glucose is used as the main carbon source, and the concentration is controlled at 40g / L to 50g / L to quickly increase the bacterial biomass. In the middle period of 24 to 48 hours, glycerol is gradually added to 15g / L to 20g / L to balance the metabolic flow and reduce the generation of non-target products. After 48 hours in the later stage, waste oil is added as a supplementary carbon source at a rate of 5g / L to 10g / L per hour. The utilization of waste oil not only reduces production costs, but also improves the carbon source allocation efficiency and promotes the continuous generation of itaconic acid. A batch feeding strategy is adopted, with each batch feeding interval of 24 to 36 hours, and the total feeding amount is controlled at 20% to 30% of the initial culture solution volume. Such feeding can accurately meet the nutritional needs of the bacteria, while avoiding metabolic disorders caused by excessive carbon source. By adjusting the stirring rate and gas flow, the fermentation dissolved oxygen level is stabilized at 20% to 30%, ensuring the metabolic activity of the bacteria throughout the fermentation process.

[0038] In this step S5, Experimental objectives: To experimentally verify the fermentation process conditions of optimizing pH control, dynamic carbon source supply and batch feeding strategy, and evaluate their effects on itaconic acid production efficiency, by-product generation and bacterial metabolic stability; Experimental Materials and Methods 1. Experimental strains Using genetically engineered Yarrowia lipolytica, including optimized CAD enzyme expression, cofactor cycle enhancement and metabolic flux reconstruction, steps S1-S4 are completed; 2. Fermentation conditions setting The fermentation environment was set to an acidic condition of 3.5 to 4.5, and the pH value was adjusted using a phosphate buffer system. The pH values ​​at the fermentation endpoint were set to 4.0, 4.2, and 4.5, respectively, and the production efficiency of itaconic acid under different conditions was compared. Dynamic carbon source supply strategy: Initial stage (0 to 24 hours): 40 to 50 g / L glucose; Mid-term (24 to 48 hours): 15 to 20 g / L glycerol; Later stage (after 48 hours): waste oil is supplemented at 5 to 10 g / L per hour; Feeding: The feeding interval is set to 24 to 36 hours, and the total amount is 20% to 30% of the initial culture volume; 3. Experimental Grouping Control group: traditional process, fixed carbon source supply, no pH optimization; Experimental group: optimized process, dynamic carbon source supply, pH optimization and batch feeding; 4. Detection Methods Itaconic acid production: The itaconic acid concentration in the fermentation broth was determined using high performance liquid chromatography (HPLC). Unit carbon source efficiency: calculated by the ratio of the amount of itaconic acid generated to the amount of carbon source consumed; Byproduct formation: The concentration of citric acid byproduct was determined using HPLC; Bacterial biomass: The bacterial density was determined by OD600 value; Detection of contaminating bacteria: After fermentation, the growth of contaminating bacteria was evaluated by plate spreading method.

[0039] Experimental Results 1. Effect of pH Control End point pH value and itaconic acid generation efficiency: pH 4.0: The unit carbon source efficiency reaches 0.62g / g, and the total output is 65g / L; pH 4.2: unit carbon source efficiency is 0.60 g / g, total output is 63 g / L; pH 4.5: The unit carbon source efficiency dropped to 0.54 g / g, and the total yield was 58 g / L; Inhibition of contaminating bacteria: No growth of contaminating bacteria was detected in the pH range of 4.0 to 4.2, while the growth rate of contaminating bacteria in the pH 4.5 group reached 5%.

[0040] Conclusion: The pH value at the end point of fermentation was optimized to 4.0 to 4.2, which helped to maximize the production of itaconic acid and effectively inhibit contaminating bacteria.

[0041] 2. Impact of dynamic carbon source supply Effect of different carbon sources on itaconic acid production: Single carbon source supply (control group): itaconic acid production was 38 g / L, and byproduct citric acid production was 8.5 g / L; Dynamic carbon source supply (experimental group): itaconic acid production increased to 65g / L, and citric acid production decreased to 2.5g / L; Contribution of glycerol and waste oils: Glycerol supply reduced citric acid production and improved mid-term metabolic efficiency, while the addition of waste oil prolonged late-term metabolic activity and increased final itaconic acid production; Conclusion: The dynamic carbon source supply strategy optimized the carbon flow distribution, improved the efficiency of itaconic acid production and reduced the formation of by-products.

[0042] 3. Impact of feeding strategy Feed interval and itaconic acid production: Feed interval 24 hours: total production 65g / L; Feed interval 36 hours: total production 62 g / L; Without feed: total yield is 45g / L; Bacterial biomass: OD600 value of the feeding group was stable at 40±2, and that of the control group was 30±2; Conclusion: Batch feeding can effectively maintain bacterial activity and increase itaconic acid production.

[0043] 4. Overall fermentation performance Itaconic acid generation efficiency: Optimized process group: unit carbon source efficiency is 0.58 to 0.62 g / g, total output is 65 g / L; Control group: unit carbon source efficiency was 0.42 g / g, total yield was 38 g / L; By-product generation: The citric acid generation in the optimized process group was reduced by more than 70%, and the experimental group was stabilized below 2.5g / L; the citric acid generation in the control group was 8.5g / L; Inhibition of contaminating bacteria: No contaminating bacteria were detected in the optimization group, and the contamination rate in the control group was 8%.

[0044] This step experiment verified the effect of optimizing the fermentation process. Under the optimized conditions, the pH value at the fermentation endpoint was maintained in the range of 4.0 to 4.2 to maximize the production of itaconic acid, while effectively inhibiting the growth of contaminating bacteria to ensure the stability and controllability of the fermentation. The phased carbon source supply strategy improved the carbon source utilization efficiency and reduced the production of non-target metabolites. Batch feeding ensured the stability of bacterial biomass and metabolic efficiency, so that the unit carbon source production efficiency of itaconic acid reached 0.62 g / g and the total output reached 65 g / L.

[0045] In this embodiment, step S1 improves the catalytic efficiency by optimizing CAD gene expression and mitochondrial targeting; step S2 ensures sufficient energy supply for metabolic reactions by enhancing the generation and circulation of NADPH and NADH; step S3 optimizes transmembrane transporters to improve the efflux efficiency of itaconic acid while maintaining the stability of the intracellular environment; step S4 reconstructs the carbon flow distribution path to concentrate more precursor substances on itaconic acid production; step S5 optimizes fermentation conditions by precise pH control, dynamic carbon source supply and feeding strategy to improve the production efficiency of the target product and the stability of the fermentation process.

[0046] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0047] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Construction of an engineered strain of Yarrowia lipolytica and a method for producing itaconic acid, characterized in that: The following steps are involved: Step S1: introducing a cis-aconiticacid decarboxylase CAD gene derived from Aspergillus into Yarrowia lipolytica by genetic engineering means, and adding a mitochondrial targeting signal peptide to target the CAD enzyme to the mitochondrial cavity, wherein the mitochondrial targeting signal peptide is an N-terminal sequence derived from the CoxIV protein of Saccharomyces cerevisiae, and a polypeptide sequence MGWSK is added thereto, and the CAD enzyme catalyzes cis-aconitic acid to generate itaconic acid in the mitochondria; Step S2: heterologously expressing the glucose-6-phosphate dehydrogenase zwf gene and the 6-phosphogluconate dehydrogenase gnd gene from Escherichia coli and regulating the expression of the mitochondrial NADH dehydrogenase gene to increase the generation and circulation levels of the cofactors NADPH and NADH, wherein the NADPH concentration is increased to 2 to 2.5 times the original metabolic level, thereby providing sufficient reducing power for the catalytic reaction of the CAD enzyme in step S1; Step S3: integrating the lactic acid transmembrane transporter gene ldhT from Lactobacillus into the Yarrowia lipolytica genome, and optimizing the key amino acid residues in the hydrophobic region of the transmembrane transporter to mutate the K86 site to A, so that the transmembrane transporter can adapt to the transmembrane transport requirements of itaconic acid; Step S4: using CRISPR Cas9 gene editing to knock out fatty acid synthesis-related genes FAS1 and FAS2, and citric acid shunt-related gene CIT1, to adjust the carbon flow metabolic pathway of Yarrowia lipolytica, and to allocate metabolic carbon flow to cis-aconiticacid production; Step S5: Under fermentation conditions with a low pH value of 3.5 to 4.5, the carbon source ratio is 60wt% to 70wt% of glucose, 20wt% to 30wt% of glycerol, and 5wt% to 15wt% of waste oil and fat based on the total carbon source, and a phosphate buffer system is used to adjust the pH value of the fermentation liquid, and the oxygen supply is adjusted by controlling the stirring rate and the gas flow rate to maintain the dissolved oxygen level at 20% to 30%.

2. The method for constructing an engineered strain of Yarrowia lipolytica and producing itaconic acid according to claim 1, characterized in that: The amino acid sequence of the mitochondrial targeting signal peptide in step S1 is the optimized N-terminal fragment of the Saccharomyces cerevisiae CoxIV protein, and the optimized fragment contains 20 amino acids, wherein the 4th valine is replaced by leucine, and the 12th serine is replaced by alanine.

3. The method for constructing an engineered strain of Yarrowia lipolytica and producing itaconic acid according to claim 1, characterized in that: In step S1, the CAD gene integration position is located in the 26S ribosomal RNA operator region of the Yarrowia lipolytica genome, and the integration is inserted in the form of multiple copies, with each copy spaced 500 bases apart, and termination signal sequences derived from the Saccharomyces cerevisiae ENO1 gene are added upstream and downstream of the insertion region.

4. The method for constructing an engineered strain of Yarrowia lipolytica and producing itaconic acid according to claim 1, characterized in that: The generation of the cofactor in step S2 is enhanced by driving the expression of the zwf gene and the gnd gene through a dual promoter system, wherein the zwf gene front driver uses the PGK1 promoter of Saccharomyces cerevisiae, and the gnd gene front driver uses the GPD1 promoter with strong transcriptional activity, and a signal sequence is added to the 3' end of the gene.

5. The method for constructing an engineered strain of Yarrowia lipolytica and producing itaconic acid according to claim 1, characterized in that: The optimization of the lactate transmembrane transporter in step S3 includes site-directed mutation of the K86 site to enhance hydrophobicity, and adjusting the helical inclination of the transmembrane region by molecular dynamics simulation, the inclination angles being 15°, 20° and 25°, respectively, to form a larger molecular channel pore size. The optimized transmembrane protein channel is suitable for the transport of small molecules with a molecular weight of 160 to 200 Daltons.

6. The method for constructing an engineered strain of Yarrowia lipolytica and producing itaconic acid according to claim 1, characterized in that: In step S4, the knockout of fatty acid metabolism genes FAS1 and FAS2 and the shearing of citric acid shunt gene CIT1 are completed by CRISPR gene editing, the editing target sites of FAS1 and FAS2 genes are located at the start position of the coding region, the shearing of CIT1 gene retains its first exon and deletes the second exon, and at the same time inhibits the transcription level of PPT2 gene related to fatty acid metabolism.

7. The method for constructing an engineered strain of Yarrowia lipolytica and producing itaconic acid according to claim 1, characterized in that: In step S4, a heterologous sphingosine synthase derived from Agrobacterium is introduced to enhance the metabolic efficiency of cofactors in the cytoplasm, and an endogenous yeast aconitase gene is replaced by a plant-derived aconitase gene. The plant aconitase gene is selected from Arabidopsis thaliana, and its expression is regulated by the TEF1 promoter.

8. The method for constructing an engineered strain of Yarrowia lipolytica and producing itaconic acid according to claim 1, characterized in that: In step S5, the carbon source ratio control adopts a phased supply strategy, the glucose concentration is controlled at 40g / L to 50g / L in the early stage of fermentation, the glycerol concentration added in the middle stage of fermentation is 15g / L to 20g / L, the addition time of waste oil is set to 48 hours after fermentation, and the addition rate per hour is 5g / L to 10g / L.

9. The method for constructing an engineered strain of Yarrowia lipolytica and producing itaconic acid according to claim 1, characterized in that: In step S5, the fermentation process is fed in batches, with the interval between each batch of feeding being 24 to 36 hours, the total amount of feeding being controlled at 20% to 30% of the initial culture solution volume, and the pH of the culture solution at the end of fermentation being controlled at 4.0 to 4.2.

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