Construction method and application of yarrowia lipolytica engineering bacteria for high yield of itaconic acid

Abstract

The invention discloses a construction method and application of yarrowia lipolytica engineering bacteria for high yield of itaconic acid, and the construction method comprises the following steps: step S1, by utilizing a CRISPR / Cas9 technology and combining a multi-copy insertion strategy, carrying out precise integration on a citrate lyase gene, a cis-aconitic acid synthetase gene and an itaconic acid synthetase gene, optimizing metabolic carbon flow and improving the yield of a target product; s2, constructing a dynamic regulation and control system, and regulating key gene expression by stages through an inductive promoter and a feedback regulation and control element to realize dynamic optimization of a metabolic pathway; s3, accurately positioning itaconic acid synthetase in mitochondria or peroxides through signal peptide, reducing generation of byproducts, enhancing DGA1 gene expression by utilizing a CRISPR activation technology, and improving the tolerance of the strain in a low-pH environment; and step S4, adding a xylose isomerase gene, a xylulose reductase gene and a fatty acyl-coenzyme A dehydrogenase gene, optimizing metabolic pathways of xylose and fatty acid, and realizing efficient conversion of multiple carbon sources.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a construction method and application of an engineering bacterium of Yarrowia lipolytica capable of producing high itaconic acid. Background Art

[0002] Itaconic acid is an important bio-based platform chemical, widely used in biodegradable plastics, pharmaceutical intermediates, adhesives and coatings. The industrial production of itaconic acid mainly adopts microbial fermentation. Among them, the traditional itaconic acid production strain, Aspergillus niger, has been widely used due to its high yield and mature process. However, this strain has many limitations in industrial production. for example: The fermentation process requires strict control of oxygen supply and pH conditions, and the production process consumes a lot of energy; Many by-products are generated: such as citric acid and malic acid, which results in low purity of the target product and increased separation costs; In recent years, Yarrowia lipolytica has become a non-model industrial strain with development potential due to its strong metabolic plasticity and convenient genetic engineering operation. The excellent performance of Yarrowia lipolytica in lipid metabolism and tolerance to various environmental conditions makes it an ideal candidate microorganism for itaconic acid production. However, there are still problems in using Yarrowia lipolytica for itaconic acid production: 1. The natural metabolic pathway of Yarrowia lipolytica cannot efficiently guide itaconic acid synthesis, and the carbon flow is dispersed in the by-product (such as citric acid and malic acid) production pathway, resulting in insufficient yield of the target product; 2. Industrial fermentation for itaconic acid production usually needs to be carried out under acidic conditions of pH 3.0 to 4.5. The cell membrane stability of existing strains is insufficient, which limits their application in industrial environments. 3. Industrial fermentation often requires the use of multiple carbon sources, but the metabolic adaptability of the existing Yarrowia lipolytica is limited and cannot achieve efficient conversion of multiple carbon sources.

[0003] Based on this, a method for constructing and applying an engineered strain of Yarrowia lipolytica that produces high itaconic acid is 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 that produces high itaconic acid, the construction method comprising the following steps: Step S1, selecting Yarrowia lipolytica as a host strain; Step S2, integrating the citrate lyase gene citL, the aconitate synthase gene ACO and the itaconate synthase gene CAD into the host strain genome by using CRISPR / Cas9 gene editing technology; Step S3, constructing a dynamic regulatory system, using the inductive promoter XPR2 to enhance the expression of citL and ACO in the early stage of metabolism, and activating the expression of CAD in the middle and late stages of metabolism through feedback regulatory elements; Step S4, localizing itaconate synthase to mitochondria or peroxisomes by fusing a mitochondrial targeting signal peptide or a peroxisome targeting signal peptide; Step S5, using CRISPR / Cas9 gene editing technology to target and knock out the citrate synthase gene and the malate dehydrogenase gene; Step S6, adding xylose isomerase gene XYL1, xylulose reductase gene XYL2 and fatty acyl-CoA dehydrogenase gene POX1, and realizing multi-carbon source metabolism optimization by dynamically regulating POX1 gene expression; Step S7, editing the promoter region of the lipid membrane protein gene DGA1 by CRISPR activation technology to increase its expression level, so as to enhance the lipid membrane function.

[0005] Further preferably, the dynamic regulation system includes an inductive promoter XPR2 and a feedback regulatory element, wherein the inductive promoter XPR2 is used to enhance the initial expression of the citrate lyase gene citL and the aconitate synthase gene ACO, and the feedback regulatory element controls the activity of the transactivator by detecting changes in the concentrations of aconitate and itaconic acid, thereby accurately activating the expression of the itaconic acid synthase gene CAD in the middle and late stages of metabolism; The inductive promoter XPR2 is used to enhance the expression of the citrate lyase gene citL and the aconitate synthase gene ACO in the early stage of metabolism, promoting the rapid accumulation of citric acid and aconitate, thereby laying a sufficient precursor foundation for itaconic acid synthesis. At the same time, the feedback regulatory element dynamically regulates the activity of the trans-activator by real-time monitoring of the concentration changes of aconitate and itaconic acid, so that the expression of the itaconate synthase gene CAD is accurately activated in the middle and late stages of metabolism, thereby optimizing the carbon flow of the metabolic pathway and avoiding the waste of precursors and the accumulation of by-products.

[0006] Further preferably, the mitochondrial targeting signal peptide is composed of the signal sequence MLS of Saccharomyces cerevisiae, and the peroxisome targeting signal peptide is composed of a sequence containing a termination signal PTS1; the mitochondrial targeting signal MLS is used to guide itaconate synthase into the mitochondrial matrix and directly co-localize with aconitic acid to improve local catalytic efficiency; the peroxisome targeting signal PTS1 locates itaconate synthase into the peroxisome through the peroxisome membrane transport system, thereby optimizing the target metabolic pathway; The mitochondrial targeting signal MLS guides itaconate synthase into the mitochondrial matrix and co-localizes with aconitic acid in the same subcellular location, thereby utilizing the local high concentration of aconitic acid and efficient coenzyme supply in the mitochondria to improve the catalytic efficiency. The peroxisome targeting signal PTS1 recognizes the peroxisome membrane transport system and transports itaconate synthase to the peroxisome, effectively isolating competitive metabolic reactions and reducing the generation of by-products. The synergistic effect of the two signal peptides optimizes the flow distribution of target metabolites and enhances the synthesis efficiency.

[0007] Further preferably, a dual gRNA editing tool based on the CRISPR / Cas9 system is used to edit the citrate synthase gene and the malate dehydrogenase gene; the dual gRNA targets the promoter region and the coding sequence of the citrate synthase gene and the malate dehydrogenase gene, respectively, and the editing efficiency is detected by PCR amplification and agarose gel electrophoresis, and the mutation site is verified by sequencing technology; Citrate synthase and malate dehydrogenase will cause the dispersion of carbon flow during metabolism and generate non-target products, thereby reducing the synthesis efficiency of itaconate. Through dual gRNA technology, the knockout of these genes significantly reduced the generation of by-products, making the carbon flow more concentrated on the target metabolic pathway. PCR amplification and agarose gel electrophoresis were used to detect the editing efficiency, and sequencing technology was used to verify the accuracy of the mutation site to ensure the reliability and genetic stability of the editing operation.

[0008] Further preferably, the multi-carbon source adaptability modification includes adding xylose isomerase gene XYL1, xylulose reductase gene XYL2 and fatty acyl-CoA dehydrogenase gene POX1; regulating the expression levels of XYL1 and XYL2 genes by a dynamic expression control module to optimize the xylose metabolic pathway; optimizing the fatty acid β-oxidation pathway by dynamically regulating the expression of POX1 gene, limiting the production of acetate and increasing the supply of acetyl-CoA; The xylose isomerase gene XYL1 and the xylulose reductase gene XYL2 were added to construct the xylose metabolic pathway. XYL1 converted xylose into xylulose, and XYL2 further converted xylulose into glycolysis intermediates, providing additional carbon flow for itaconic acid synthesis. The fatty acyl-CoA dehydrogenase gene POX1 optimized the fatty acid β-oxidation pathway through dynamic expression regulation, reduced the production of acetic acid products, and increased the supply of acetyl-CoA, thereby promoting the synthesis of target metabolites.

[0009] Further preferably, the promoter region of the lipid membrane protein gene DGA1 is modified by CRISPR activation technology; the modified promoter enhances the transcriptional activity of the DGA1 gene, and the triacylglycerol synthase encoded by the DGA1 gene regulates the lipid bilayer stability of the cell membrane; The promoter region of the lipid membrane protein gene DGA1 was modified through CRISPR activation technology to enhance the transcriptional activity of the DGA1 gene, thereby improving the stability and adaptability of the strain's cell membrane. The DGA1 gene encodes triglyceride synthase, which can increase the thickness and stability of the lipid bilayer. The modification of the promoter region significantly increased the expression level of DGA1, making the strain more tolerant in low pH environments and adapting to the requirements of acidic conditions in industrial fermentation. The enhancement of the lipid membrane effectively reduced the leakage of the cell membrane to the target product, thereby improving the efficiency of intracellular metabolic flux and the final yield of itaconic acid.

[0010] Further preferably, the itaconate synthase gene CAD adopts a multi-copy insertion strategy during the genome integration process, and the insertion strategy is based on multi-site homologous recombination technology to insert the CAD gene into a high expression site in the host strain genome; The high expression sites are obtained by transcriptomic analysis screening, and are characterized by genomic regions with high promoter activity and high ribosome binding efficiency; The multi-copy insertion is accomplished by CRISPR / Cas9 technology, wherein the homology arm length is 500 bp to 1000 bp, the insertion site is verified by detecting the gene copy number by quantitative PCR, and the stable expression of the CAD gene is verified by transcriptional level analysis.

[0011] In addition, the present invention also provides an application of a method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid yield, which is used for industrial-scale production of itaconic acid, and the application comprises the following steps: Step A1, culturing the engineered strain constructed by the method under optimized fermentation conditions, wherein the fermentation conditions include controlling the pH value at 3.0 to 4.5, the temperature at 28 to 32° C., and maintaining the dissolved oxygen level at more than 30%; Step A2, using glucose, xylose or waste oil as the main carbon source, with an initial carbon source concentration of 20 to 50 g / L; Step A3, using a batch or continuous fed-batch fermentation mode to maintain the metabolic activity of the strain by dynamically supplementing carbon and nitrogen sources; Step A4, separating itaconic acid by acid precipitation or ion exchange at the end of the fermentation, and obtaining a high-purity itaconic acid crystal product by spray drying; Step A5: using the obtained itaconic acid product for the production of bio-based plastics, biodegradable polymers and food additives.

[0012] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects: 1. The present invention integrates the citrate lyase gene (citL), aconitate synthase gene (ACO) and itaconate synthase gene (CAD) of Yarrowia lipolytica through CRISPR / Cas9 technology, and significantly improves the expression of the target gene by using a multi-copy insertion strategy; guides the insertion of the target gene into the high expression site of the genome through homologous recombination, optimizes the carbon flow distribution, reduces the generation of by-products, and ensures that the metabolic carbon flow is concentrated on the itaconate synthesis pathway, thereby greatly improving the yield and production efficiency of the target product; 2. The present invention realizes the phased optimization of gene expression by constructing a dynamic regulation system including an inductive promoter and feedback regulatory elements; the XPR2 promoter is used to enhance the expression of key enzymes in the early stage of metabolism and rapidly accumulate metabolic precursors; the feedback element dynamically activates the expression of the CAD gene by detecting the changes in the concentrations of aconitic acid and itaconic acid, accurately regulates the direction of carbon flow, and effectively improves the dynamic balance and efficiency of the target metabolic pathway.

[0013] 3. The present invention utilizes signal peptides to precisely direct itaconate synthase to mitochondria or peroxisomes, thereby achieving efficient reactions within specific subcellular organelles, avoiding competitive generation of by-products, and optimizing metabolic flux; at the same time, CRISPR activation technology is used to upregulate DGA1 gene expression, increase triglyceride synthase levels, and significantly improve cell membrane stability, thereby enhancing the tolerance and metabolic activity of the engineered strain under low pH conditions, and adapting to the needs of industrial production.

[0014] 4. The present invention optimizes xylose metabolism and fatty acid β-oxidation pathways by adding xylose isomerase gene, xylulose reductase gene and fatty acyl-CoA dehydrogenase gene respectively; the dynamic expression control module adjusts gene expression according to the external carbon source concentration to ensure efficient conversion of xylose into glycolysis intermediates, reduce the generation of acetic acid by-products in fatty acid metabolism, and increase the supply of acetyl-CoA, thereby realizing flexible and efficient utilization of multiple carbon sources. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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.

[0016] Figure 1 Schematic diagram of gene integration of the present invention. DETAILED DESCRIPTION

[0017] 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.

[0018] 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.

[0019] Example like Figure 1 As shown, a method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid in this embodiment comprises the following steps: Step S1, select Yarrowia lipolytica, which is commonly used in industrial applications, as the host strain. This strain has strong metabolic plasticity and environmental adaptability, especially in the field of lipid metabolism and organic acid production. In the breeding process, the metabolic efficiency of the strain is further improved by combining natural isolate screening with ultraviolet mutagenesis, while ensuring its growth stability under high sugar and low pH conditions. In the screening process, low pH resistance (pH 3.0 to 4.5), rapid proliferation ability and high genetic modification efficiency are clearly defined as the main criteria to ensure that it meets the needs of industrial production; Step S2, in order to efficiently express the citrate lyase gene citL, the aconitate synthase gene ACO and the itaconate synthase gene CAD, the CRISPR / Cas9 technology is combined with a multi-copy insertion strategy to achieve precise integration of the genes; A single plasmid containing the Cas9 protein coding sequence and the target gene gRNA sequence was constructed. The plasmid vector includes the following modules: Cas9 protein sequence: The expression of Cas9 is driven by a highly efficient promoter to generate double-strand breaks (DSBs) at the target genomic site; gRNA sequence: Design targeting highly expressed sites to guide Cas9 to cut the target region, and optimize the gRNA sequence through online tools to ensure high specificity matching with the target sequence and avoid off-target effects; Target gene expression module: Integrate the expression modules of citL, ACO and CAD genes, and design homology arms of 500 bp to 1000 bp on both sides of the genes to guide multi-site homologous recombination; Screening marker: antibiotic resistance gene, used for screening positive strains after transformation; The construction and assembly of plasmids use efficient DNA recombination technology to ensure the correct connection and functional integrity of all modules; Selection of high expression sites: The integration sites of target genes are determined by transcriptomic analysis; high-throughput sequencing of gene transcription data of host strains at different growth stages is used to screen genomic regions with high promoter activity and high ribosome binding efficiency, such as regions near ribosomal protein genes; these sites can provide strong transcription and translation efficiency, thereby supporting high-level expression of key enzymes; the selection of high expression sites is ensured to be compatible with the multi-copy insertion strategy through multiple rounds of prediction and experimental verification; Gene integration and multi-copy insertion: During gene integration, the CRISPR / Cas9 system is combined with multi-site homologous recombination technology; after the Cas9 protein generates DSB in the target genomic region, the homologous arms match the genomic sequence, guiding the precise insertion of the target gene into the high expression site; in order to further increase the expression of the CAD gene, a multi-copy insertion strategy is used to achieve multi-copy arrangement of the target gene through repetitive integration; multi-copy integration significantly enhances the transcription level of CAD while avoiding the problem of unstable insertion sites; After gene integration is completed, the integration results are verified by a variety of technologies: Fluorescence PCR detection: Use a PCR reaction system containing fluorescent dyes to amplify the extracted genomic DNA of the engineered strain; the fluorescent dye binds to the double-stranded DNA during the amplification process, and the intensity of its fluorescent signal increases with the increase in the amount of amplification. The insertion success rate of the target gene is judged by observing the intensity of the fluorescent signal. The real-time fluorescence signal intensity curve is used to determine whether the target gene has been inserted. Positive samples show a specific amplification curve, while non-integrated samples have no amplification signal or the amplification signal is significantly weakened; Quantitative PCR analysis: Extract genomic DNA from positive strains and perform qPCR amplification using specific primers for the target gene and the host's internal reference gene. Calculate the absolute copy number of the target gene by comparing the Ct values. Substitute the Ct value of the sample into the standard curve calculation formula to obtain the copy number of the target gene in each sample, and calculate the success rate of multi-copy insertion and integration efficiency. Expression level verification: The transcription levels of citL, ACO, and CAD genes were measured by quantitative PCR to ensure the stability and consistency of integrated gene expression; The total RNA of the positive strain was extracted and the residual DNA was removed by DNase I treatment to ensure that the detection signal came only from the transcription product of the target gene; the RNA was reverse transcribed into cDNA using reverse transcriptase; a specific and efficient reverse transcription kit was selected to ensure high-fidelity synthesis of the target gene cDNA; specific primers of the target gene (citL, ACO, CAD) and the internal reference gene (such as 18SrRNA or GAPDH) were used for qPCR amplification; the expression level of the target gene was evaluated by comparing the ratio of the relative expression of the target gene to the expression of the internal reference gene.

[0020] Step S3, the dynamic regulation system realizes time-sharing regulation of gene expression through the inductive promoter XPR2 and the feedback regulatory element; The dynamic regulation system consists of the inductive promoter XPR2 and feedback regulatory elements, which realize the temporal expression of genes according to the different needs of the metabolic process. By optimizing the function of the inductive promoter XPR2, the natural XPR2 promoter that is highly sensitive to metabolic signals is selected to construct its expression module connection with citL and ACO. At the same time, the transcription efficiency of the promoter is improved by integrating the enhancer sequence, so that it can significantly increase the expression level of citL and ACO in the early stage of metabolism, quickly accumulate citric acid and aconitic acid metabolism, and ensure the efficient utilization of the initial carbon flow of the metabolic pathway. Secondly, feedback regulatory elements are constructed to respond to fluctuations in the concentration of metabolic intermediates and dynamically adjust the expression intensity of CAD. The feedback regulatory elements are composed of signal molecule binding sites and trans-activating factors. By detecting changes in the concentrations of aconitic acid and itaconic acid, the activity of the trans-activating factors is regulated in real time. It is necessary to ensure the sensitivity of the signal binding elements to metabolites so that the expression module of CAD can be triggered when aconitic acid reaches an appropriate level, thereby efficiently directing the metabolic carbon flow to the synthesis pathway of itaconic acid, avoiding the waste of precursor substances, reducing the generation of by-products, and improving the overall efficiency of the metabolic system.

[0021] Step S4, by designing the mitochondrial targeting signal MLS and the peroxisome targeting signal PTS1, CAD is accurately guided to locate to a specific subcellular organelle, the by-product generation is reduced and the itaconate synthesis efficiency is optimized, and the nucleic acid sequence of the mitochondrial targeting signal MLS is extracted from Saccharomyces cerevisiae. The signal peptide can guide the target protein to cross the mitochondrial membrane and enter the matrix, ensuring its compatibility with the fusion expression module of the itaconate synthase gene CAD. At the same time, the peroxisome targeting sequence of the termination signal PTS1 is used to accurately guide the itaconate synthase into the peroxisome. The PTS1 signal peptide binds to the receptor protein on the peroxisome membrane to achieve efficient transport through the membrane receptor protein, and the itaconate synthase is ensured to function efficiently in a specific subcellular region through the optimized binding of the signal peptide; In the vector construction stage, the signal peptide is fused with the CAD gene to construct a plasmid vector, and the plasmid with the fusion gene is integrated into a specific site of the genome through the CRISPR / Cas9 gene editing tool, and the fluorescent marker protein is combined with the signal peptide for fusion expression; The distribution of the fusion protein was observed under a fluorescence microscope to visually assess whether itaconate synthase was accurately positioned in the mitochondrial matrix or peroxisome. Subcellular component separation technology was further used to extract the mitochondria and peroxisomes in the cells, and the concentrations of CAD protein in different components were measured. The extracted samples were tested by Western blot analysis to confirm the targeting accuracy and concentration distribution of the CAD protein, and the effect of positioning optimization was verified by metabolic flux analysis to observe whether the optimization of subcellular localization significantly improved the efficiency of itaconate production and reduced the generation of by-products.

[0022] Step S5, using CRISPR / Cas9 dual gRNA tools to target and knock out citrate synthase gene and malate dehydrogenase gene, reduce the generation of byproducts in the metabolic process of Yarrowia lipolytica and optimize the conversion of carbon flow to the target pathway of itaconate, through two gRNA sequences targeting citrate synthase gene (Citrate Synthase, CS) and malate dehydrogenase gene (Malate Dehydrogenase, MDH), respectively, the gRNA target of each gene covers its promoter region and coding sequence, to double ensure the destruction of functional regions, block gene expression, and use online tools to predict and optimize the specificity of gRNA with the target sequence, while avoiding off-target effects; When constructing a CRISPR vector, two gRNA sequences and the Cas9 protein coding sequence are co-loaded into a single plasmid, and a screening marker that is compatible with the host bacteria is added so that positive strains can be quickly screened out after the subsequent editing is completed. The vector uses an efficient promoter to drive the expression of Cas9 and gRNA, and efficiently induces gene editing in the host bacteria. After the vector is introduced into the host strain by electroporation, positive strains are screened in a selective culture medium. After the editing is completed, the knockout of the target gene is detected by PCR amplification technology, the DNA fragment of the editing region is amplified, and the size change of the PCR product is analyzed by agarose gel electrophoresis to preliminarily confirm whether gene editing has occurred. The genomic DNA of the positive strain is extracted for Sanger sequencing, and the base changes at the target gene editing site are analyzed to confirm whether the double-strand break guided by the gRNA is successfully repaired as the expected deletion or insertion mutation, and to exclude the possibility of off-target effects; After the editing verification was completed, metabolic flux analysis was used to evaluate the changes in the production of citric acid and malic acid in the strain. The unedited wild-type strain usually accumulates more citric acid and malic acid by-products, resulting in dispersed carbon flow; while the engineered strain that successfully knocked out the citrate synthase gene and malate dehydrogenase gene significantly reduced the accumulation of these by-products, making the carbon flow more concentrated on the metabolic pathway of itaconate.

[0023] Step S6, by adding xylose isomerase gene XYL1, xylulose reductase gene XYL2 and fatty acyl-CoA dehydrogenase gene POX1, the metabolic pathways of xylose and fatty acids are optimized respectively. Through the dynamic expression regulation module, it is ensured that the gene expression level can be adjusted in real time according to the external carbon source concentration, thereby improving the metabolic efficiency and reducing the generation of by-products; Xylose is an economical and readily available carbon source, but the conversion efficiency is low in the metabolic system of Yarrowia lipolytica, so the xylose isomerase gene XYL1 and the xylulose reductase gene XYL2 were added to improve the xylose metabolism capacity through dynamic expression regulation; XYL1 encodes xylose isomerase, which catalyzes the conversion of xylose into xylulose, while XYL2 encodes xylulose reductase, which further converts xylulose into intermediate metabolites that can enter the glycolytic pathway, thus enabling efficient metabolism of xylose; The dynamic expression control module regulates the expression levels of XYL1 and XYL2 genes through the carbon source sensing element. When the xylose concentration is high, the control module activates the high-level expression of XYL1 and XYL2 to quickly metabolize xylose. When the xylose concentration decreases, the dynamic expression control module automatically downregulates gene expression to avoid excessive enzyme accumulation and waste of resources. The XYL1 and XYL2 genes and their dynamic expression control module are assembled into a plasmid vector and integrated into the high expression site of the host bacteria through CRISPR / Cas9 technology. Fatty acids are another important carbon source, but they may produce acetic acid as a byproduct during metabolism, reducing carbon flow efficiency. Therefore, the fatty acyl-CoA dehydrogenase gene POX1 was added, and its expression level was optimized through a dynamic expression control module. POX1 encodes fatty acyl-CoA dehydrogenase, which is a key enzyme in fatty acid β-oxidation. It can promote the decomposition of fatty acids and reduce the production of acetate, while increasing the supply of acetyl-CoA, providing sufficient precursors for itaconic acid synthesis. The expression intensity of the POX1 gene is regulated by the regulatory element that senses the fatty acid concentration. When the fatty acid concentration is high, the expression of the POX1 gene is upregulated to accelerate the metabolic rate of fatty acids and generate sufficient acetyl-CoA to supply the itaconate synthesis pathway; when the fatty acid concentration decreases, the regulatory module downregulates the expression level of POX1 to avoid the accumulation of byproducts and metabolic waste; Construct a dynamic expression control module and POX1 gene fusion expression vector, and use CRISPR / Cas9 tools to integrate it into the host bacterial genome; The XYL1, XYL2 and POX1 genes were integrated into the high-efficiency expression sites of the host strain through CRISPR / Cas9 technology to ensure stable gene expression: Integration verification: Use fluorescent PCR to detect gene integration, amplify the DNA fragment of the insertion area, observe the fluorescent signal to determine the insertion success rate, and use quantitative PCR to determine the gene copy number to confirm whether the integration efficiency meets the design requirements; Transcription verification: By extracting total RNA from the engineered strain, quantitative PCR was used to analyze the transcription levels of XYL1, XYL2, and POX1 to ensure that the dynamic expression regulatory module regulates gene expression according to the external carbon source concentration; Metabolic efficiency verification: Xylose and fatty acid metabolism experiments were conducted to determine the xylose conversion rate, fatty acid β-oxidation efficiency and acetic acid production, respectively. High performance liquid chromatography HPLC was used to analyze the itaconic acid production and evaluate the overall metabolic performance.

[0024] Step S7, transforming the promoter region of the lipid membrane protein gene DGA1 by CRISPR activation technology to enhance the transcriptional activity of the DGA1 gene; the DGA1 gene encodes triacylglycerol synthase, and by increasing its expression level, the lipid bilayer stability of the cell membrane is regulated, the tolerance and metabolic activity of the strain in a low pH environment are enhanced, and a stable cell structure support is provided for efficient itaconic acid synthesis; CRISPR activation (CRISPRa) technology achieves transcriptional enhancement in the promoter region of target genes through dCas9 (deactivated Cas9 protein) combined with transcription activators; The plasmid vector for constructing the CRISPR activation system is as follows: Deactivated Cas9 protein (dCas9) is used as a gene targeting tool. Its ability to bind DNA is not affected, but its cutting function is lost. Transcription activators (VP64, p65 or RTA) are fused to the dCas9 protein to enhance the transcription of the DGA1 gene. By designing 2 to 3 gRNA sequences for the DGA1 gene promoter region, targeting key sites in the core promoter region, ensuring that dCas9-VP64 can effectively initiate gene transcription after binding, and using online tools to optimize the gRNA sequence to ensure high specificity and avoid off-target binding with other genomic regions; Add dCas9-VP64 and gRNA expression modules to the plasmid vector, add a strong promoter to drive the efficient expression of the system, and include a resistance marker in the vector for positive strain screening; By constructing a vector of the CRISPR activation system, dCas9-VP64 or other activating factors with enhanced transcription function are used to bind to the promoter region of the DGA1 gene to form a powerful transcription activation module, which then specifically binds to the DGA1 promoter region to start the transcription of the DGA1 gene, thereby increasing the expression level of triglyceride synthase. Unlike traditional gene editing methods, CRISPR activation technology does not destroy the DNA sequence, but increases its expression level by regulating the transcriptional activity of the gene; The constructed plasmid vector was introduced into the Yarrowia lipolytica host strain by electroporation. The transformed strain was first inoculated into a selective medium containing antibiotics to screen for positive transformants. Then, the genomic RNA of the positive strain was extracted and the transcription level of the DGA1 gene was analyzed by quantitative PCR. The positive strain should show the characteristics of significantly increased DGA1 transcription level. The following methods were used to verify the effect of CRISPR activation technology on improving strain membrane stability and acid resistance: The RNA of positive strains was extracted, and the transcription level of DGA1 was measured by qPCR, and the relative increase compared with the wild-type strain was calculated; The membrane permeability changes of the strains under pH 3.0 to 4.5 were measured by staining experiments. The cell membrane was labeled with a specific fluorescent dye, and the membrane integrity was evaluated by the change in fluorescence intensity. The cell membrane morphology and thickness of the modified strains were observed using an electron microscope and compared with those of the unmodified strains to confirm the enhanced effect of the membrane structure; The strains were cultured under low pH (3.0, 3.5, 4.0) conditions, and the growth rate, survival rate, and metabolite production of the strains were measured to evaluate the strains' ability to adapt to acidic environments.

[0025] 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.

[0026] 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. A method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid, characterized in that: The construction method comprises the following steps: Step S1, selecting Yarrowia lipolytica as a host strain; Step S2, integrating the citrate lyase gene citL, the aconitate synthase gene ACO and the itaconate synthase gene CAD into the host strain genome by using CRISPR / Cas9 gene editing technology; Step S3, constructing a dynamic regulatory system, using the inductive promoter XPR2 to enhance the expression of citL and ACO in the early stage of metabolism, and activating the expression of CAD in the middle and late stages of metabolism through feedback regulatory elements; Step S4, localizing itaconate synthase to mitochondria or peroxisomes by fusing a mitochondrial targeting signal peptide or a peroxisome targeting signal peptide; Step S5, using CRISPR / Cas9 gene editing technology to target and knock out the citrate synthase gene and the malate dehydrogenase gene; Step S6, adding xylose isomerase gene XYL1, xylulose reductase gene XYL2 and fatty acyl-CoA dehydrogenase gene POX1, and realizing multi-carbon source metabolism optimization by dynamically regulating POX1 gene expression; Step S7, editing the promoter region of the lipid membrane protein gene DGA1 by CRISPR activation technology to increase its expression level, so as to enhance the lipid membrane function.

2. The method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid according to claim 1, characterized in that: The dynamic regulation system includes an inductive promoter XPR2 and a feedback regulatory element. The inductive promoter XPR2 is used to enhance the initial expression of the citrate lyase gene citL and the aconitate synthase gene ACO. The feedback regulatory element controls the activity of the transactivator by detecting changes in the concentrations of aconitate and itaconic acid, thereby accurately activating the expression of the itaconic acid synthase gene CAD in the middle and late stages of metabolism. The inductive promoter XPR2 can enhance the expression of the citrate lyase gene citL and the aconitate synthase gene ACO according to the initial metabolic needs of the engineered strain.

3. The method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid according to claim 1, characterized in that: The mitochondrial targeting signal peptide is composed of the signal sequence MLS of Saccharomyces cerevisiae, and the peroxisome targeting signal peptide is composed of a sequence containing the termination signal PTS1; the mitochondrial targeting signal MLS is used to guide itaconate synthase into the mitochondrial matrix and directly co-localize with aconitic acid to improve local catalytic efficiency; the peroxisome targeting signal PTS1 locates itaconate synthase into the peroxisome through the peroxisome membrane transport system, thereby optimizing the target metabolic pathway.

4. The method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid according to claim 1, characterized in that: The citrate synthase gene and the malate dehydrogenase gene were edited using a dual gRNA editing tool based on the CRISPR / Cas9 system; the dual gRNAs targeted the promoter regions and coding sequences of the citrate synthase gene and the malate dehydrogenase gene, respectively, the editing efficiency was detected by PCR amplification and agarose gel electrophoresis, and the mutation site was verified by sequencing technology.

5. The method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid according to claim 1, characterized in that: The multi-carbon source adaptability modification includes adding a xylose isomerase gene XYL1, a xylulose reductase gene XYL2 and a fatty acyl-CoA dehydrogenase gene POX1; regulating the expression levels of the XYL1 and XYL2 genes by a dynamic expression control module to optimize the xylose metabolic pathway; and optimizing the fatty acid β-oxidation pathway by dynamically regulating the expression of the POX1 gene, thereby limiting acetic acid production and increasing acetyl-CoA supply.

6. The method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid according to claim 1, characterized in that: The promoter region of the lipid membrane protein gene DGA1 was modified by CRISPR activation technology; the modified promoter enhanced the transcriptional activity of the DGA1 gene, and the triglyceride synthase encoded by it regulates the lipid bilayer stability of the cell membrane.

7. The method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid according to claim 1, characterized in that: The itaconate synthase gene CAD adopts a multi-copy insertion strategy during the genome integration process. The insertion strategy is based on multi-site homologous recombination technology to insert the CAD gene into a high expression site in the host strain genome; The high expression sites are obtained by transcriptomic analysis screening, and are characterized by genomic regions with high promoter activity and high ribosome binding efficiency; The multi-copy insertion is accomplished by CRISPR / Cas9 technology, wherein the homology arm length is 500 bp to 1000 bp, the insertion site is verified by detecting the gene copy number by quantitative PCR, and the stable expression of the CAD gene is verified by transcriptional level analysis.

8. The use of the method for constructing an engineered strain of Yarrowia lipolytica that produces high itaconic acid according to any one of claims 1 to 7, characterized in that: For industrial-scale production of itaconic acid, the application comprises the following steps: Step A1, culturing the engineered strain constructed by the method under optimized fermentation conditions, wherein the fermentation conditions include controlling the pH value at 3.0 to 4.5, the temperature at 28 to 32° C., and maintaining the dissolved oxygen level at more than 30%; Step A2, using glucose, xylose or waste oil as the main carbon source, with an initial carbon source concentration of 20 to 50 g / L; Step A3, using a batch or continuous fed-batch fermentation mode to maintain the metabolic activity of the strain by dynamically supplementing carbon and nitrogen sources; Step A4, separating itaconic acid by acid precipitation or ion exchange at the end of the fermentation, and obtaining a high-purity itaconic acid crystal product by spray drying; Step A5: using the obtained itaconic acid product for the production of bio-based plastics, biodegradable polymers and food additives.

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