Method for constructing engineered yarrowia lipolytica strain and producing itaconic acid

Abstract

Disclosed in the present invention is a method for constructing an engineered Yarrowia lipolytica strain and producing itaconic acid, which method comprises the following steps: improving the localization efficiency of a core enzyme, i.e., Cis-aconitic acid decarboxylase by using a mitochondrial targeting signal peptide according to an optimized heterologous expression strategy, thereby enhancing the contact between the enzyme and a substrate and promoting the synthesis of itaconic acid; secondly, improving the generation and regeneration efficiency of NADPH and NADH by adding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from Escherichia coli and regulating a cofactor cycle in mitochondria; and finally, modifying a transmembrane transporter gene ldhT, and optimizing the amino acid sequence and helical structure thereof, thereby increasing the efflux efficiency of itaconic acid and reducing the negative impact of intracellular accumulation on cells.

Description

Construction of engineered Yersinia lipophila and methods for producing itaconic acid Technical Field

[0001] This invention relates to the field of engineered Yersinia lipolytica, and in particular to the construction of engineered Yersinia lipolytica and a method for producing itaconic acid. Background Technology

[0002] Itaconic acid is an important organic chemical raw material, widely used in pharmaceuticals, food additives, and polymer materials. Its green and bio-manufacturing methods have environmental and economic advantages. Yersinia lipolytica, as a naturally adaptable industrial microorganism, is a preferred host for itaconic acid production due to its wide utilization of carbon sources and tolerance to environmental stress. However, the metabolic performance and fermentation efficiency of Yersinia lipolytica still have limitations in the bio-production of itaconic acid.

[0003] I. The key enzyme CAD in itaconic acid 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 are isolated from the mitochondria where the substrate is produced, resulting in low efficiency of the target metabolic pathway.

[0004] II. Itaconic acid production depends on the participation of a large number of cofactors, but the cofactor production pathway of Yersinia lipolyticis is inefficient and cannot meet the requirements of efficient enzymatic reactions.

[0005] Third, the accumulation of intracellular citric acid can easily trigger metabolic feedback inhibition, reducing the metabolic flux of the target pathway. At the same time, the acidic stress of the weakly acidic environment has an adverse effect on the metabolic stability and cell viability of Yersinia lipolytica.

[0006] Therefore, it is necessary to construct engineered Yersinia lipophila strains and develop methods for producing itaconic acid. Summary of the Invention

[0007] To achieve the above objectives, the present invention provides the following solution: a method for constructing engineered *Yarrowia lipophila* strains and producing itaconic acid, comprising the following steps:

[0008] Step S1: The cis-aconitic acid decarboxylase CAD gene from Aspergillus spp. was introduced into Yersinia lipolytica through genetic engineering, and a mitochondrial targeting signal peptide was added to target the CAD enzyme to the mitochondrial cavity. The mitochondrial targeting signal peptide is the N-terminal sequence of the Cox IV protein from Saccharomyces cerevisiae, and the polypeptide sequence MGWSK (SEQ ID NO: 1) was added to it. The CAD enzyme catalyzes the conversion of cis-aconitic acid to itaconic acid in the mitochondria.

[0009] Step S2: By heterologously expressing the glucose-6-phosphate dehydrogenase zwf gene and the 6-phosphate gluconate dehydrogenase gnd gene from E. coli, and regulating the expression level of the mitochondrial NADH dehydrogenase gene, the production and circulation levels of cofactors NADPH and NADH are increased. The NADPH concentration is increased to 2 to 2.5 times the original metabolic level, providing sufficient reducing power for the catalytic reaction of CAD enzyme in step S1.

[0010] Step S3: Integrate the lactic acid transmembrane transporter gene ldhT from Lactobacillus into the genome of Yersinia lipophila, and optimize the key amino acid residues in the hydrophobic region of the transmembrane transporter by mutating the K86 site to A, so that the transmembrane transporter can adapt to the transmembrane transport requirements of itaconic acid.

[0011] Step S4: Knock out fatty acid synthesis-related genes FAS1 and FAS2 using CRISPR Cas9 gene editing, and / or cleave citric acid diversion-related gene CIT1, and / or adjust the carbon flux metabolic pathway of Yersinia lipolytica to allocate metabolic carbon flux to cis-aconitic acid production.

[0012] Step S5: Under low pH conditions of 3.5 to 4.5, use a carbon source ratio of 60 wt% to 70 wt% of glucose, 20 wt% to 30 wt% of glycerol, and 5 wt% to 15 wt% of waste oil. Use a phosphate buffer system to adjust the pH of the fermentation broth and adjust the oxygen supply by controlling the stirring rate and gas flow rate to maintain the dissolved oxygen level at 20% to 30%.

[0013] More preferably, in step S1, the mitochondrial targeting signal peptide is selected from the N-terminal 20 amino acids of the Saccharomyces cerevisiae COX IV protein, and the N-terminal positions 1-20 of its amino acid sequence are used as the mitochondrial targeting fragment.

[0014] More preferably, the brewer's yeast COX IV protein includes the amino acid sequence shown in NCBI accession number NP_011328.1.

[0015] More preferably, the mitochondrial-targeting signal peptide includes an optimized amino acid sequence, wherein the optimized amino acid sequence is based on the N-terminal 20 amino acids of the Saccharomyces cerevisiae COX IV protein, with valine at position 4 replaced by leucine and serine at position 12 replaced by alanine.

[0016] More preferably, in step S1, the CAD gene integration site is located in the 26S ribosomal RNA control region of the Yersinia lipolyticis genome, and the integration is carried out in the form of multiple copies, with each copy spaced 500 bases apart. Termination signal sequences from the ENO1 gene of Saccharomyces cerevisiae are added upstream and downstream of the insertion region, respectively.

[0017] Preferably, the protein encoded by the CAD gene includes the amino acid sequence shown in NCBI accession number BAG49047.1, and the CAD gene includes the nucleotide sequence shown in NCBI accession number AB326105.1.

[0018] Preferably, the protein sequence encoded by the Saccharomyces cerevisiae ENO1 gene includes the amino acid sequence shown in UniProt accession number: P00924, and the ENO1 gene includes the nucleotide sequence shown in NCBI accession number: NM_001181383.3.

[0019] Preferably, the termination signal sequence derived from the Saccharomyces cerevisiae ENO1 gene is located at the 3′ end of NM_001181383.3.

[0020] More preferably, in step S2, the enhancement of cofactor generation drives the expression of the zwf gene and the gnd gene through a dual promoter system, wherein the pre-promoter of the zwf gene is the PGK1 promoter of Saccharomyces cerevisiae, and the pre-promoter of the gnd gene is the GPD1 promoter with strong transcriptional activity, and a signal sequence is added to the 3′ end of the gene.

[0021] Preferably, the signal sequence is a polyA signal sequence derived from the ACT1 gene of Saccharomyces cerevisiae.

[0022] Preferably, the protein encoded by the zwf gene includes the amino acid sequence shown in NCBI accession number NP_417461.1, and the zwf gene includes the nucleotide sequence shown in NCBI accession number NC_000913.3 (gene interval 1911265-1913184).

[0023] Preferably, the protein encoded by the gnd gene includes the amino acid sequence shown in NCBI accession number NP_417457.1, and the gnd gene includes the nucleotide sequence shown in NCBI accession number NC_000913.3 (gene interval 1909399-1911261).

[0024] Preferably, the PGK1-encoded protein includes the amino acid sequence shown in NCBI accession number NP_009976.1, and the GPD1-encoded protein includes the amino acid sequence shown in NCBI accession number NP_009948.1.

[0025] Preferably, the polyA signal sequence comprises the 3′ nucleotide sequence of the Saccharomyces cerevisiae ACT1 gene (corresponding protein NCBI accession number: NP_116614.1).

[0026] In one specific implementation, the zwf and gnd genes are integrated into the leu2 and ura3 sites, respectively.

[0027] Preferably, the leu2 site corresponds to a safe harbor site in the Yarrowialipolytica genome, and its sequence is shown in NCBI accession number XM_503734.1;

[0028] The ura3 site corresponds to a safe harbor site in the Yarrowialipolytica genome, and its sequence is shown in NCBI accession number XM_503865.1.

[0029] Further preferably, the optimization of the lactate transmembrane transporter in step S3 includes site-directed mutagenesis at the K86 site to enhance hydrophobicity, and adjustment of the helical tilt angle of the transmembrane region through molecular dynamics simulations, with tilt angles of 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.

[0030] More preferably, the optimization of the lactate transmembrane transporter includes replacing the K86 site with A.

[0031] More preferably, in step S4, the knockout of fatty acid metabolism genes FAS1 and FAS2 and the cleavage of citrate shunt gene CIT1 are accomplished through CRISPR gene editing. The editing target sites of FAS1 and FAS2 genes are located at the initiation position of the coding region. The CIT1 gene cleavage retains its first exon and deletes the second exon, while inhibiting the transcription level of PPT2 gene, which is related to fatty acid metabolism.

[0032] In a further preferred embodiment, in step S4, a heterologous sphingosine synthase derived from Agrobacterium is introduced to enhance the metabolic efficiency of cofactors in the cytoplasm, and a plant-derived aconitase gene is used to replace the yeast endogenous aconitase gene. The plant aconitase gene is selected from Arabidopsis thaliana, and its expression is regulated by the TEF1 promoter.

[0033] Preferably, the FAS1 gene contains the nucleotide sequence shown at positions 486068 to 488035 of NC_006045.2; and the FAS2 gene contains the nucleotide sequence shown at positions 848340 to 848579 of NC_006045.2.

[0034] Preferably, the sequence of the citric acid diversion gene CIT1 contains the amino acid sequence shown in NCBI accession number XP_503380.1.

[0035] Preferably, the sphingosine synthase sequence comprises the amino acid sequence shown in NCBI accession number WP_007405449.1, the protein encoded by the plant aconitase gene comprises the amino acid sequence shown in NCBI accession number NP_178634.2, and the protein encoded by the endogenous aconitase gene comprises the amino acid sequence shown in NCBI accession number XP_503960.1.

[0036] Preferably, the TEF1-encoded protein contains the amino acid sequence shown in NCBI accession number XP_503490.1.

[0037] More preferably, in step S5, the carbon source ratio is controlled using a phased supply strategy. In the early stage of fermentation, the glucose concentration is controlled at 40 g / L to 50 g / L, and in the middle stage of fermentation, the glycerol concentration is added at 15 g / L to 20 g / L. The waste oil is added after 48 hours of fermentation, and the addition rate is 5 g / L to 10 g / L per hour.

[0038] More preferably, in step S5, the fermentation process is carried out in batches with an interval of 24 to 36 hours between each batch, the total amount of feed is controlled at 20% to 30% of the initial culture medium volume, and the pH of the culture medium at the end of fermentation is controlled at 4.0 to 4.2.

[0039] According to specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0040] I. This 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 version of the CoxIV signal peptide is added to the front end of the expression sequence of the CAD gene, wherein the valine at position 4 is replaced with leucine and the serine at position 12 is replaced with alanine to enhance the stability of the signal peptide and the mitochondrial recognition efficiency. Furthermore, the CAD gene is precisely integrated into the 26S ribosomal RNA control region of the Yersinia lipolyticis genome using CRISPR-Cas9 to ensure efficient expression. Transcription termination signals derived from the ENO1 gene are added upstream and downstream to reduce non-specific transcriptional interference and improve expression stability.

[0041] II. This invention enhances the generation and cycling efficiency of cofactors through heterologous gene expression and regulation; by adding glucose-6-phosphate dehydrogenase and 6-phosphate gluconate dehydrogenase from Escherichia coli, and using PGK1 and GPD1 dual promoters respectively to drive the NADPH generation capacity, a polyA signal sequence from Saccharomyces cerevisiae ACT1 is added to the 3′ end of the gene to enhance the stability and expression level of mRNA.

[0042] Third, this invention incorporates the transmembrane transporter gene ldhT from Lactobacillus and optimizes its key amino acid sequence through molecular dynamics simulations. The K86 site in the hydrophobic region is replaced with A to enhance the binding ability of itaconic acid to the transporter. At the same time, the helical structure tilt angle of the transmembrane region is adjusted to 15°, 20° and 25° to expand the molecular channel pore size. By integrating the optimized ldhT gene into the high expression site of the Yersinia lipolytica genome and adding termination signals upstream and downstream to reduce transcriptional interference, the continuous and efficient expression of the efflux protein is ensured. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 is a flowchart of the process from the construction of engineered Yersinia lipophila to the production of itaconic acid in this invention. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] Example 1: From the construction of engineered Yersinia lipophila to the production method of itaconic acid

[0048] As shown in Figure 1, the method for constructing the engineered Yersinia lipolytica strain and producing itaconic acid in this embodiment includes the following steps:

[0049] (1) Step S1: Introduction of CAD gene and mitochondrial targeting optimization

[0050] This step involves integrating the (cis-aconitic acid decarboxylase)CAD gene into the 26S ribosomal RNA operator region of the *Yersinia lipolytica* genome. The CAD gene originates from the genus *Aspergillus terreus*, and its corresponding encoded protein amino acid sequence is shown in NCBI accession number BAG49047.1; the corresponding gene sequence is shown in NCBI accession number AB326105.1. The CAD gene was integrated into the 26S ribosomal RNA operator region of the *Yersinia lipolytica* genome via CRISPR-Cas9 editing. The rDNA region was inserted in a multiple-copy manner, with each copy spaced 500 bases apart. Transcription termination signal sequences from the Saccharomyces cerevisiae ENO1 gene were added upstream and downstream of the inserted fragment, respectively. The ENO1 gene corresponds to the amino acid sequence shown in UniProt accession number: P00924. The ENO1 gene is the nucleotide sequence shown in NCBI accession number: NM_001181383.3. The transcription termination signal sequence is located at the 3′ end of the gene nucleotide sequence to enhance transcriptional stability. The amino acid sequence of the signal peptide was further modified: the 4th amino acid, valine (Val), was replaced with leucine (Leu), and the 12th amino acid, serine (Ser), was replaced with alanine (Ala), resulting in an optimized amino acid sequence.

[0051] By optimizing the fusion expression of the signal peptide and CAD protein, the CAD enzyme is directed to the mitochondrial cavity, ensuring its spatial synergy with the substrate cis-aconitic acid and improving catalytic efficiency.

[0052] Experimental materials

[0053] 1. Construction of strains and vectors

[0054] Test strain: Yersinia lipophila (accession number: ATCC MYA-2613), which is a commonly used engineered chassis strain with genetic stability and strong metabolic regulation ability;

[0055] For example, CN104711201A, "A Yarrowia lipolytica and its application", was published on 2022-09-16. The article uses "Yarrowia lipolytica" as the subject, as evidence that strains of this genus are prior art.

[0056] Vector: pYL-CAD plasmid was constructed to express the CAD gene (derived from Aspergillus) and contain the mitochondrial targeting signal peptide sequence. The polypeptide sequence MGWSK (SEQ ID NO: 1) was added to it. 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.

[0057] Targeted signal peptide sequence optimization:

[0058] Original sequence: derived from the N-terminal 20 amino acids of the Saccharomyces cerevisiae Cox IV protein, the amino acid sequence of which is shown in NCBI accession number: NP_011328.1;

[0059] Optimized sequence: valine at position 4 was replaced with leucine, and serine at position 12 was replaced with alanine.

[0060] 2. Gene Editing

[0061] Using CRISPR-Cas9, optimized and unoptimized versions of the CAD gene, i.e., the gene encoding the CAD fusion protein (containing CAD, mitochondrial targeting signal peptide sequences (optimized and unoptimized), and the added polypeptide sequence MGWSK (SEQ ID NO: 1)), were integrated into the 26S rDNA region of the Yarrowia lipolytica genome. The target sequence of the 26S rDNA region was obtained by screening using the online tool CRISPOR / Benchling, and sites with GC content of 40%-60% and high off-target scores near the PAM sequence "NGG" were selected.

[0062] The gRNA target sequence selected in this embodiment is 5′-GCTGATCGGACCGTTAGGAC-3′ (SEQ ID NO: 2);

[0063] Its 3′ PAM sequence is "TGG". To avoid off-target effects, it is necessary to perform a genome-wide alignment to confirm that there are no high homology potential off-target sites ≥15bp.

[0064] The Cas9 protein and gRNA work together to create a double-strand break in the 26S rDNA region. Subsequently, the CAD gene is integrated through the homologous arms of the donor DNA (800 bp upstream and downstream). The transcription termination signal sequence of the ENO 1 gene (derived from Saccharomyces cerevisiae) is added upstream and downstream of the integrated fragment to enhance mRNA stability and prevent transcription from extending to adjacent genes.

[0065] 3. Targeting efficiency test

[0066] Green fluorescent protein (GFP) was fused to the C-terminus of the CAD gene to facilitate 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 the GFP signal and the mitochondrial-specific dye Mito Tracker was observed using a confocal fluorescence microscope.

[0067] Data analysis: Targeting efficiency is defined as the ratio of pixels co-localized by GFP signal and Mito Tracker signal.

[0068] 4. Positioning Stability Test Method

[0069] The strain was cultured under high-intensity transcription conditions for 48 consecutive hours at a high glucose concentration of 50 g / L. The stability of the signal peptide and the change in the colocalization ratio of GFP signal and Mito Tracker signal during the culture period were observed.

[0070] 5. Enzyme localization error rate test method

[0071] Enzyme localization error rate method: Under the same conditions, detect the GFP signal intensity in the cytoplasm and calculate the proportion of CAD-GFP that is not targeted to mitochondria.

[0072] Experimental results

[0073] 1. Mitochondrial targeting efficiency

[0074] The optimized signal peptide pYL-CAD-opt achieved a co-localization efficiency of 85% ± 2% for the GFP signal and the MitoTracker signal; the unoptimized signal peptide pYL-CAD-wt achieved a co-localization efficiency of 65% ± 3%; the optimized version represents an improvement of approximately 20% over the unoptimized version.

[0075] 2. Positioning stability

[0076] The optimized signal peptide showed a decrease in co-localization efficiency from 85% to 80% under 48 hours of high transcriptional pressure; the unoptimized signal peptide showed a decrease in co-localization efficiency from 65% to 50%.

[0077] The optimized signal peptide exhibits improved localization stability.

[0078] 3. Enzyme localization error rate

[0079] Optimized signal peptide: The proportion of GFP signal in the cytoplasm is 10% ± 1%;

[0080] Unoptimized signal peptide: The proportion of GFP signal in the cytoplasm is 30% ± 2%;

[0081] Optimization effect: The optimized version reduces the incidence of CAD enzyme positioning errors.

[0082] Experimental conclusions: The optimized signal peptide sequence improved mitochondrial targeting efficiency through gene editing, with a localization efficiency approximately 20% higher than the original sequence. Its localization stability was also significantly better than the unoptimized version under long-term culture conditions. At the same time, the optimized signal peptide reduced the incidence of CAD enzyme localization errors, laying a stable enzyme expression foundation for efficient itaconic acid synthesis. The results of this experiment verify the effectiveness of the optimization strategy in step S1 and provide a guarantee for the enhancement of cofactor generation in step S2.

[0083] In this step, the CAD gene is inserted in multiple copies, with each copy spaced 500 bases apart, ensuring the transcriptional space is independent between copies and avoiding transcriptional interference caused by copy density. Furthermore, termination signal sequences are added upstream and downstream of the integrated gene to effectively terminate the transcription process and prevent non-specific transcriptional elongation from interfering with downstream genes. Through this optimization 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 Yersinia lipolytica, providing a sufficient enzymatic basis for the subsequent synthesis of itaconic acid.

[0084] Building upon efficient expression, this step precisely targets the CAD enzyme to the mitochondrial cavity via mitochondrial targeting. Mitochondria are the main site of cis-aconitic acid production. Targeting the CAD enzyme to the mitochondrial cavity improves the spatial coordination efficiency between the enzyme and the substrate, thereby enhancing the catalytic rate.

[0085] (2) Step S2: Enhancement and cyclic regulation of cofactor generation

[0086] This step involves introducing the glucose-6-phosphate dehydrogenase zwf gene (the amino acid sequence of the encoded protein is shown in NCBI accession number: NP_417461.1) and the 6-phosphate gluconate dehydrogenase gnd gene (the amino acid sequence of the encoded protein is shown in NCBI accession number: NP_417457.1) from *Escherichia coli* (K-12 MG1655) to design expression regulatory elements.

[0087] The promoter preceding the zwf gene is the PGK1 promoter from *Saccharomyces cerevisiae*, and the upstream promoter of the zwf gene is the PGK1 promoter from *Saccharomyces cerevisiae* (NCBI accession number for the protein encoded by PGK1: NP_009976.1). The promoter preceding the gnd gene is the GPD1 promoter with strong transcriptional activity, and the upstream promoter of the gnd gene is the GPD1 promoter from *Saccharomyces cerevisiae* (NCBI accession number for the protein encoded by GPD1: NP_009948.1). A polyA signal sequence from the ACT1 gene from *Saccharomyces cerevisiae* (NCBI accession number for the protein encoded by ACT1: NP_116614.1) is added to the 3′ end of both the zwf and gnd genes. The polyA signal sequence originates from the 3′ untranslated region of the *Saccharomyces cerevisiae* ACT1 gene. The ACT1 mRNA sequence has the NCBI accession number: NM_001179927.1. This is to enhance mRNA stability and transcriptional levels.

[0088] We optimized gene expression regulation in the cofactor generation pathway, verified the effects of the dual promoter system and the 3′ polyA signaling sequence on improving the efficiency of NADPH and NADH generation, and evaluated their effects on improving the catalytic efficiency and overall metabolic stability of CAD enzymes.

[0089] Experimental materials

[0090] 1. Construction of strains and vectors:

[0091] Test strain: Yarrowia lipolytica (accession number: ATCC MYA-2613), which is a commonly used engineered chassis strain with genetic stability and strong metabolic regulation ability.

[0092] Vector construction: pYL-zwf and pYL-gnd vectors were constructed to express the glucose-6-phosphate dehydrogenase zwf gene and the 6-phosphate gluconate dehydrogenase gnd gene, respectively. The PGK1 promoter of Saccharomyces cerevisiae 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 from the Saccharomyces cerevisiae ACT1 gene was added to the 3′ end of the zwf and gnd genes, respectively, to enhance mRNA stability.

[0093] 2. Gene integration and validation: The pYL-zwf and pYL-gnd vectors were integrated into the leu2 and ura3 sites, respectively, using CRISPR-Cas9.

[0094] The leu2 site corresponds to a safe harbor site in the Yarrowialipolytica genome, and its sequence is shown in NCBI accession number XM_503734.1;

[0095] The ura3 site corresponds to a safe harbor site in the Yarrowialipolytica genome, and its sequence is shown in NCBI accession number XM_503865.1;

[0096] These two gene loci are "safe harbor" sites for genome integration commonly used by this host. Their integration does not affect the host's core metabolism or growth performance and can ensure the long-term genetic stability of exogenous genes. The integration success rate of the genes was detected by real-time quantitative PCR, and the mRNA transcription level of the genes was measured to assess the function of the poly A signal sequence.

[0097] 3. Cofactor generation efficiency test:

[0098] The optimized strain and the control strain (unoptimized Yersinia lipolyticis) were cultured in YNB medium containing 50 g / L glucose for 48 hours. The concentrations of NADPH and NADH in the cells were detected by enzyme-linked immunosorbent assay (ELISA). The production efficiency of NADPH and NADH was calculated by the ratio of the concentrations of the optimized strain to the control strain.

[0099] 4. CAD enzyme catalytic efficiency test:

[0100] Enzyme activity assay: CAD enzyme was extracted from the culture media of the optimized strain and the control strain, and its enzyme activity for the conversion of cis-aconiticacid to itaconic acid was determined by spectrophotometry.

[0101] Itaconic acid yield determination: The concentration of itaconic acid in the culture medium was analyzed by high performance liquid chromatography (HPLC) to assess the actual impact on the catalytic efficiency of CAD enzyme.

[0102] 5. Overall metabolic stability test:

[0103] During the culture process, the biomass OD600 value, glucose consumption rate and by-product generation of the strain were measured; the metabolic stability of the strain was observed after 72 hours of continuous fermentation.

[0104] Experimental results

[0105] 1. NADPH and NADH generation efficiency:

[0106] Optimized strains: NADPH concentration of 42.5±2.3μM, NADH concentration of 38.6±1.8μM;

[0107] Control strains: NADPH concentration was 18.7±1.2 μM, and NADH concentration was 15.4±1.0 μM;

[0108] Improvement: The cofactor generation efficiency of the optimized strain was increased to 2.3 times that of the wild-type strain.

[0109] 2. CAD enzyme catalytic efficiency:

[0110] The optimized strain had a CAD enzyme activity of 8.2 ± 0.4 μmol / min / mg protein, while the control strain had an activity of 3.5 ± 0.2 μmol / min / mg protein.

[0111] The optimized strain produced 48 g / L of itaconic acid, while the control strain produced 22 g / L.

[0112] Improved efficacy: The catalytic efficiency of CAD enzyme and the rate of itaconic acid production increased by 2.3 times and 2.2 times, respectively.

[0113] 3. Overall metabolic stability:

[0114] Biomass: The OD600 value of the optimized strain was stable at 36±2, while that of the control strain was 28±2;

[0115] Byproduct generation: The citric acid production of the optimized strain was 3.2 g / L, while that of the control strain was 6.8 g / L;

[0116] Metabolic balance: The optimized strains exhibited higher metabolic stability and a 52% reduction in byproduct production.

[0117] Experimental conclusions: By combining the dual promoter system and the 3′ polyA signal sequence, the production efficiency of NADPH and NADH in Yersinia lipolytica was enhanced, and the amount of cofactors produced was increased to 2.3 times that of the wild-type strain. Optimization effectively improved the catalytic activity of CAD enzyme, increasing its enzyme activity by 2.3 times and the itaconic acid production rate by 2.2 times. The optimized cofactor cycle improved the overall metabolic stability of the strain, reduced by-product generation, and improved the production efficiency of the target product.

[0118] Step S2 optimized the generation and cycling regulation of cofactors NADPH and NADH. The catalytic reaction of 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 Yeast lipolyticis has low efficiency and cannot meet the needs of efficient metabolic pathways. The cofactor generation capacity was enhanced by heterologous gene expression.

[0119] The selection of promoters enhances the transcriptional level of enzymes, thereby increasing the NADPH production capacity. The addition of a poly A signal sequence from the Saccharomyces cerevisiae ACT1 gene to the 3′ end of both genes prolongs the half-life of mRNA, thus improving the stability and efficiency of gene expression.

[0120] (3) Step S3: Optimization of transmembrane transport proteins and itaconic acid efflux

[0121] This step involves targeted optimization of the amino acid sequence of the lactate transmembrane transporter. By using site-directed mutagenesis, the key amino acid K86 in the hydrophobic region of the transporter is replaced with A to enhance hydrophobicity and improve the transporter's ability to bind to hydrophobic molecules. Simultaneously, molecular dynamics simulations are used to refine the helical structure of the transmembrane region, and the tilt angles of the three main helices are adjusted to 15°, 20°, and 25° respectively, thereby expanding the pore size of the transmembrane channel to accommodate the transport requirements of small molecules with molecular weights ranging from 160 to 200 Daltons.

[0122] Experimental objective: To verify the improved effects of the optimized lactate transmembrane transporter on itaconic acid transmembrane transport efficiency, intracellular acidity mitigation, and bacterial metabolic balance through experiments;

[0123] Experimental materials

[0124] 1. Construction of strains and vectors:

[0125] Tested strain: Yarrowia lipolytica (ATCC MYA-2613);

[0126] Vector construction: The unoptimized version of pYL-ldhT-wt and the optimized version of pYL-ldhT-opt lactate transporter gene expression vectors were constructed using the pYL-ldhT vector;

[0127] 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 tilt angle of the transmembrane region to 15°, 20° and 25°.

[0128] The tertiary structure of the ldhT protein was predicted using SWISS-MODEL, and a 100ns kinetic simulation was performed in the POPC lipid bilayer environment using the CHARMM36 force field in GROMACS software. The natural tilt angle was obtained by measuring the angle between the transmembrane α-helix backbone vector and the lipid bilayer normal vector.

[0129] Gene integration: pYL-ldhT-opt and pYL-ldhT-wt were integrated into the high expression sites leu2 and ura3 of the Yeast lipolyticis genome using CRISPR-Cas9. Termination signal sequences from the ENO1 gene of Saccharomyces cerevisiae were added upstream and downstream of the integration region to ensure transcriptional stability.

[0130] When using CRISPR-Cas9 for site-specific integration, a gRNA sequence 5'-GATGACCTTGACGATCTGGA-3′ (SEQ ID NO: 3) was designed for the leu2 site, with its 3′ end adjacent to the PAM sequence AGG; a gRNA sequence 5′-CTGATGTTGACGACTCGTGA-3′ (SEQ ID NO: 4) was designed for the ura3 site, with its 3′ end adjacent to the PAM sequence TGG, to generate double-strand breaks at the corresponding gene sites and achieve homologous integration of exogenous fragments.

[0131] 2. Experimental Groups:

[0132] Control group: Wild-type Yersinia lipolyticis (without ldhT expression);

[0133] Unoptimized group: Expresses primitive lactate transmembrane transporter (pYL-ldhT-wt);

[0134] Optimized group: Expresses an optimized version of the lactate transmembrane transporter (pYL-ldhT-opt).

[0135] 3. Transmembrane transport efficiency testing

[0136] Culture conditions: YNB medium (with added glucose 50 g / L and glycerol 20 g / L) was used, and the culture was carried out at 37°C in a shaker for 48 hours. During the culture, samples were taken at regular intervals to determine the intracellular and extracellular zeaxanthin concentrations.

[0137] Detection method: Intracellular and extracellular chiconic acid concentrations were determined using high performance liquid chromatography (HPLC).

[0138] Transmembrane transport efficiency calculation:

[0139] 4. Relief effect on intracellular acidity

[0140] Detection method: Intracellular pH was measured using a fluorescent pH probe (pHrodoGreen), and the results were compared 48 hours into the later stage of fermentation; the degree of decrease in intracellular pH and the degree of acidity mitigation were analyzed.

[0141] 5. Metabolic balance and biomass detection

[0142] Detection indicators: Biomass, determined by OD600; Byproduct generation, determined by HPLC; Continuous culture for 72 hours, with sampling at regular intervals.

[0143] Experimental results

[0144] 1. Transmembrane transport efficiency:

[0145] Control group: Transmembrane efficiency was 15% ± 2%;

[0146] Unoptimized group: Transmembrane efficiency was 28% ± 3%;

[0147] Optimized group: Transmembrane efficiency was 68% ± 4%;

[0148] Improved effect: The transmembrane efficiency of the optimized lactate transmembrane transporter was increased by 40 percentage points compared with the unoptimized version and by 53 percentage points compared with the control group.

[0149] 2. Relief effect on intracellular acidity:

[0150] Control group: Intracellular pH decreased to 4.8±0.1;

[0151] Unoptimized group: Intracellular pH was 5.3 ± 0.2;

[0152] Optimized group: Intracellular pH was 6.1 ± 0.1;

[0153] Improvement effect: The optimized transporter protein alleviated the intracellular acidic environment, and the pH value decreased by 30% less than that of the unoptimized group.

[0154] 3. Metabolic balance and biomass

[0155] Biomass:

[0156] Control group: OD600 value was 28±2;

[0157] Unoptimized group: OD600 value is 34±2;

[0158] Optimized group: OD600 value is 42±2.

[0159] 4. Byproduct generation:

[0160] Control group: Byproduct formation amount was 6.5 g / L;

[0161] Unoptimized group: Byproduct formation amount was 4.2 g / L;

[0162] Optimized group: Byproduct generation amount was 2.7 g / L;

[0163] Improved results: The optimized version reduces the amount of byproducts generated, and increases biomass by 50% compared to the control group.

[0164] The above experiments verified that the optimized lactate transmembrane transporter pYL-ldhT-opt improved the itaconic acid transmembrane transport efficiency of Yersinia lipolytica, with a transmembrane efficiency 40 percentage points higher than that of the unoptimized group, reaching 68%. The intracellular pH of the optimized group was maintained at 6.1, with a decrease of nearly 30% compared with the control group. This effectively solved the problem of metabolic feedback inhibition caused by itaconic acid accumulation, reduced the amount of by-products generated, and increased the biomass of the strain.

[0165] After optimizing the cofactor generation cycle and enhancing the catalytic efficiency of CAD enzyme in step S2, the production rate of itaconic acid was improved. However, as a weak acid, excessive accumulation of itaconic acid in the cell can negatively affect the metabolic balance of Yersinia lipolyticis. This step uses transmembrane transport protein optimization. By introducing and modifying the lactic acid transmembrane transport protein gene derived from Lactobacillus, efficient efflux of itaconic acid was achieved.

[0166] (4) Step S4: Optimization of carbon flux allocation and reconstruction of metabolic network

[0167] 1. Experimental objective:

[0168] To verify the impact of gene knockout and splicing strategies on target metabolic pathways;

[0169] The proportion of cis-aconiticacid in carbon flux partitioning was determined after optimization.

[0170] To assess the functionality of heterologous enzymes and plant aconitase gene substitutions.

[0171] 2. Experimental Methods

[0172] Strains are grouped as follows:

[0173] Control group: Wild-type Yersinia lipolyticis;

[0174] Optimization Group 1: Only FAS1 and FAS2 were knocked out, derived from Yarrowia lipolytica PO1f, ATCC MYA-2613.

[0175] Corresponding gene to FAS1:

[0176] The FAS1 gene includes the nucleotide sequence located at positions 486068-488035 on the Y. lipolytica reference genome NC_006045.2;

[0177] The FAS2 gene includes the nucleotide sequence located at positions 848340-848579 on the Y. lipolytica reference genome NC_006045.2;

[0178] Optimization Group 2: Knock out FAS1 and FAS2 and cleave CIT1 (cleavage retains its first exon and deletes the second exon). The protein sequence encoded by the citrate shunt gene CIT1 includes the amino acid sequence shown in NCBI accession number XP_503380.1.

[0179] Optimization Group 3: Added sphingosine synthase from the soil bacterium *Sphingomonaspaucimobilis*, whose encoded protein sequence includes the amino acid sequence shown in NCBI accession number WP_007405449.1, and aconitase3 from *Arabidopsis thaliana*, whose encoded protein sequence includes the amino acid sequence shown in NCBI accession number NP_178634.2. The *Arabidopsis thaliana* aconitase gene was used to replace the endogenous aconitase gene *YALI0E14949g* from *Yarrowia lipolytica*, whose encoded protein sequence includes the amino acid sequence shown in NCBI accession number XP_503960.1.

[0180] Carbon flow partition determination: The partition ratio of acetyl-CoA and citrate in different metabolic pathways was analyzed using [U-13C] glucose isotope labeling combined with gas chromatography-mass spectrometry (GC-MS).

[0181] Metabolic efficiency determination: The production of cis-aconiticacid and itaconic acid was determined by high performance liquid chromatography (HPLC).

[0182] 3. Experimental Results

[0183] Carbon stream distribution ratio:

[0184] Control group: cis-aconiticacid accounted for 40% ± 2% of the total carbon flux distribution;

[0185] Optimization Group 1: The cis-aconiticacid allocation ratio was increased to 52% ± 3%;

[0186] Optimization Group 2: The allocation ratio is further increased to 58% ± 2%;

[0187] Optimization Group 3: The allocation ratio reached 65% ± 3%, achieving an improvement;

[0188] Target metabolite production amount:

[0189] Cis-aconiticacid production: Optimized group 3 reached 18.6 g / L, while the control group was 8.5 g / L;

[0190] Itaconic acid production: 64 g / L in optimized group 3, compared to 22 g / L in the control group;

[0191] Byproduct generation:

[0192] The amount of citric acid produced in optimized group 3 was reduced by 70% compared with the control group.

[0193] By precisely knocking out fatty acid synthesis-related genes FAS1 and FAS2, cleaving the citric acid diversion gene CIT1, and adding heterosphingosine synthase and plant aconitase genes, the metabolic flux allocation of *Yarrowia lipolytica* was optimized. In the optimized strain, the proportion of cis-aconiticacid in the carbon flux increased from 40% to over 65%, the production of the target metabolite itaconic acid reached 64 g / L, and the production of byproducts was reduced.

[0194] After improving the transmembrane efflux efficiency of itaconic acid in step S3, this step addresses the shortage of itaconic acid synthesis precursors through carbon flow allocation optimization and metabolic network reconstruction. This step employs a multi-level metabolic network optimization strategy. This step uses CRISPR-Cas9 gene editing to precisely knock out genes related to fatty acid synthesis and citrate diversion, reducing non-target carbon flow allocation. On one hand, it knocks out fatty acid synthesis-related genes FAS1 and FAS2: these two genes encode important subunits of fatty acid synthases, and their knockout reduces the translocation of acetyl-CoA to fatty acids. The editing target is located at the initiation position of the gene coding region, inhibiting fatty acid synthesis metabolism at its source.

[0195] On the other hand, the CIT1 gene encodes citrate synthase, which combines acetyl-CoA and oxaloacetate to produce citrate. To maintain cellular basal metabolic balance and reduce excessive citrate production, a precise cleavage strategy was employed to retain the first exon of CIT1 and delete the second exon, thus partially inhibiting citrate synthesis. qPCR and protein electrophoresis confirmed that the expression level of the cleaved CIT1 decreased to 40% of its original level, but basal metabolism could still proceed normally.

[0196] This step improves the utilization efficiency of the cofactor NADPH by adding heterosphingosine synthase.

[0197] This step involves replacing the endogenous aconitase gene in Yersinia lipolytica with a plant-derived aconitase gene to enhance the efficiency of citric acid conversion to cis-aconiticacid. The plant aconitase gene is regulated by the strong promoter TEF1, and its expression level is 50% higher than that of the endogenous gene, thus accelerating the production rate of cis-aconiticacid.

[0198] Step S5: Fermentation process optimization

[0199] The metabolic pathways of Yersinia lipolytica were optimized through steps S1-S4. Since itaconic acid production requires an acidic environment, this step further refined the fermentation process for itaconic acid production. Specifically:

[0200] 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.

[0201] To address the carbon source requirements at different fermentation stages, a phased supply strategy was adopted. In the initial stage (0-24 hours), glucose was used as the main carbon source, with a concentration controlled at 40-50 g / L to rapidly increase cell biomass. In the middle stage (24-48 hours), glycerol was gradually added to 15-20 g / L to balance metabolic flux and reduce the generation of non-target products. In the later stage (48 hours), waste oil was added as a supplementary carbon source at a rate of 5-10 g / L per hour. The utilization of waste oil not only reduced production costs but also improved carbon source allocation efficiency and promoted the continuous generation of itaconic acid.

[0202] By employing a batch feeding strategy, with each batch fed 24 to 36 hours apart, and the total feeding volume controlled at 20% to 30% of the initial culture liquid volume, the nutritional needs of the microorganisms can be precisely met, while avoiding metabolic disorders caused by excessive carbon sources. Furthermore, by adjusting the stirring rate and gas flow rate, the dissolved oxygen level during fermentation is stabilized at 20% to 30%, ensuring the metabolic activity of the microorganisms throughout the fermentation process.

[0203] Experimental objective: To verify the effects of optimized pH control, dynamic carbon source supply, and batch feeding strategies on itaconic acid production efficiency, byproduct formation, and cell metabolic stability through experiments.

[0204] Experimental Materials and Methods

[0205] 1. Experimental strain

[0206] Using genetically engineered Yersinia lipase, including optimized CAD enzyme expression, enhanced cofactor cycling, and metabolic flux reconstruction, steps S1-S4 were completed.

[0207] 2. Fermentation conditions settings

[0208] 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 itaconic acid production efficiency under different conditions was compared.

[0209] Dynamic carbon source supply strategy:

[0210] Initial stage (0 to 24 hours): 40 to 50 g / L glucose;

[0211] Intermediate stage (24 to 48 hours): 15 to 20 g / L glycerin;

[0212] Later stage (after 48 hours): Replenish waste oil at a rate of 5 to 10 g / L per hour;

[0213] Feeding: The feeding interval is set at 24 to 36 hours, and the total amount is 20% to 30% of the initial culture medium volume;

[0214] 3. Experimental Grouping

[0215] Control group: Traditional process, fixed carbon source supply, no pH optimization;

[0216] Experimental group: Optimized process, dynamic carbon source supply, pH optimization, and batch feeding;

[0217] 4. Detection Method

[0218] Itaconic acid production: The concentration of itaconic acid in the fermentation broth was determined by high performance liquid chromatography (HPLC).

[0219] Unit carbon source efficiency: calculated as the ratio of the amount of itaconic acid generated to the amount of carbon source consumed;

[0220] Byproduct formation: The concentration of citric acid byproducts was determined using HPLC;

[0221] Bacterial biomass: Bacterial density was determined by OD600 value;

[0222] Contamination bacteria detection: After fermentation, the growth of contaminating bacteria was assessed by plate coating method.

[0223] Experimental results

[0224] 1. The effect of pH regulation

[0225] Endpoint pH and itaconic acid formation efficiency:

[0226] pH 4.0: The unit carbon source efficiency reaches 0.62 g / g, and the total yield is 65 g / L;

[0227] pH 4.2: The unit carbon source efficiency is 0.60 g / g, and the total yield is 63 g / L;

[0228] pH 4.5: The unit carbon source efficiency decreased to 0.54 g / g, and the total yield was 58 g / L;

[0229] Inhibition of contaminating bacteria:

[0230] No contaminant growth was detected in the pH range of 4.0 to 4.2, while the contaminant growth rate reached 5% in the pH 4.5 group.

[0231] Conclusion: Optimizing the pH value at the fermentation endpoint to 4.0 to 4.2 helps to maximize itaconic acid production and effectively inhibit contaminating bacteria.

[0232] 2. Impact of dynamic carbon source supply

[0233] The effect of staged carbon sources on itaconic acid formation:

[0234] Single carbon source supply (control group): itaconic acid production was 38 g / L, and citric acid production as a byproduct was 8.5 g / L;

[0235] Dynamic carbon source supply (experimental group): Itaconic acid production increased to 65 g / L, while citric acid production decreased to 2.5 g / L;

[0236] The contributions of glycerin and waste oils:

[0237] Glycerol supply reduces citric acid production and improves mid-term metabolic efficiency, while the addition of waste oil prolongs late-term metabolic activity and increases the final itaconic acid yield.

[0238] Conclusion: The dynamic carbon source supply strategy optimizes carbon flow distribution, improves itaconic acid formation efficiency, and reduces byproduct formation.

[0239] 3. Impact of Feeding Strategy

[0240] Feeding interval and itaconic acid formation:

[0241] Feeding interval 24 hours: Total output is 65g / L;

[0242] Feeding interval 36 hours: Total output is 62g / L;

[0243] No refeeding: Total output is 45g / L;

[0244] Microbial biomass: The OD600 value of the fed group was stable at 40±2, while that of the control group was 30±2;

[0245] Conclusion: Batch feeding effectively maintained cell activity and increased itaconic acid production.

[0246] 4. Overall fermentation performance

[0247] Itaconic acid formation efficiency:

[0248] Optimized process group: unit carbon source efficiency is 0.58 to 0.62 g / g, total yield is 65 g / L;

[0249] Control group: unit carbon source efficiency was 0.42 g / g, total yield was 38 g / L;

[0250] Byproduct formation: The citric acid formation in the optimized process group was reduced by more than 70%, and the experimental group was stable at below 2.5 g / L; the citric acid formation in the control group was 8.5 g / L.

[0251] Inhibition of contaminating bacteria: No contaminating bacteria were detected in the optimized group, while the contamination rate in the control group was 8%.

[0252] This step of the experiment verifies the effect of optimizing the fermentation process. Under optimized conditions, the final pH value of fermentation is 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 and ensuring the stability and controllability of fermentation. The staged carbon source supply strategy improves the carbon source utilization efficiency and reduces the production of non-target metabolites. The batch feeding ensures the stability of cell biomass and metabolic efficiency, so that the unit carbon source production efficiency of itaconic acid reaches 0.62g / g and the total yield reaches 65g / L.

[0253] In this embodiment, step S1 improves catalytic efficiency by optimizing CAD gene expression and mitochondrial targeting; step S2 ensures sufficient energy supply for metabolic reactions by enhancing NADPH and NADH generation and cycling; step S3 improves itaconic acid efflux efficiency by optimizing transmembrane transport proteins while maintaining intracellular environmental stability; step S4 reconstructs carbon flow distribution pathways to concentrate more precursor substances for itaconic acid generation; and step S5 optimizes fermentation conditions through precise pH control, dynamic carbon source supply, and fed-batch strategies to improve the generation efficiency of the target product and the stability of the fermentation process.

[0254] The technical features of the above embodiments can be combined in any way. For the sake of brevity, 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.

[0255] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for constructing engineered *Yarrowia lipophila* strains and producing itaconic acid, characterized in that, Includes the following steps: Step S1: The cis-aconitic acid decarboxylase CAD gene from Aspergillus was introduced into Yersinia lipolytica through genetic engineering, and a mitochondrial targeting signal peptide was added to target the CAD enzyme to the mitochondrial cavity. The mitochondrial targeting signal peptide is the N-terminal sequence of the Cox IV protein from Saccharomyces cerevisiae, and the polypeptide sequence MGWSK (SEQ ID NO: 1) was added to it. Step S2: Heterologous expression of the glucose-6-phosphate dehydrogenase zwf gene and the 6-phosphate gluconate dehydrogenase gnd gene derived from Escherichia coli; Step S3: Integrate the lactic acid transmembrane transporter gene ldhT from Lactobacillus into the genome of Yersinia lipophila, and mutate the K86 site to A by optimizing the key amino acid residues in the hydrophobic region of the transmembrane transporter. Step S4: Knock out fatty acid synthesis-related genes FAS1 and FAS2 using CRISPR Cas9 gene editing, and / or cleave citric acid diversion-related gene CIT1, and / or adjust the carbon flux metabolic pathway of Yersinia lipolytica to allocate metabolic carbon flux to cis-aconitic acid production. Step S5: Under low pH conditions of 3.5 to 4.5, use a carbon source ratio of 60 wt% to 70 wt% of glucose, 20 wt% to 30 wt% of glycerol, and 5 wt% to 15 wt% of waste oil. Use a phosphate buffer system to adjust the pH of the fermentation broth and adjust the oxygen supply by controlling the stirring rate and gas flow rate to maintain the dissolved oxygen level at 20% to 30%.

2. The method for constructing the engineered *Yersinia lipophila* strain and producing itaconic acid according to claim 1, characterized in that, In step S1, the amino acid sequence of the mitochondrial targeting signal peptide is an optimized N-terminal fragment of the Saccharomyces cerevisiae CoxIV protein. The optimized fragment contains 20 amino acids, in which valine at position 4 is replaced with leucine and serine at position 12 is replaced with alanine.

3. The method for constructing the engineered *Yersinia lipophila* strain and producing itaconic acid according to claim 1, characterized in that, In step S1, the CAD gene is integrated into the 26S ribosomal RNA control region of the Yersinia lipolyticis genome. Integration is performed using a multi-copy insertion method, with each copy spaced 500 bases apart. Termination signal sequences from the ENO1 gene of Saccharomyces cerevisiae are added upstream and downstream of the insertion region, respectively.

4. The method for constructing the engineered *Yersinia lipophila* strain and producing itaconic acid according to claim 1, characterized in that, In step S2, the enhancement of cofactor generation drives the expression of the zwf and gnd genes through a dual promoter system. The zwf gene pre-promoter is the PGK1 promoter of Saccharomyces cerevisiae, and the gnd gene pre-promoter is the GPD1 promoter with strong transcriptional activity. At the same time, a signal sequence is added to the 3′ end of the gene.

5. The method for constructing the engineered *Yersinia lipophila* strain and producing itaconic acid according to claim 1, characterized in that, The optimization of the lactate transmembrane transporter in step S3 includes site-directed mutagenesis at the K86 site to enhance hydrophobicity, and adjustment of the helical tilt angle of the transmembrane region through molecular dynamics simulations. The tilt angles are 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 the engineered *Yersinia lipophila* strain 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 cleavage of the citrate shunt gene CIT1 are accomplished through CRISPR gene editing. The editing target sites of FAS1 and FAS2 genes are located at the initiation position of the coding region. The CIT1 gene cleavage retains its first exon and deletes the second exon, while inhibiting the transcription level of the PPT2 gene, which is related to fatty acid metabolism.

7. The method for constructing the engineered *Yersinia lipophila* strain 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. The endogenous yeast aconitase gene is replaced with a plant-derived aconitase gene selected from Arabidopsis thaliana, and its expression is regulated by the TEF1 promoter.

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

9. The method for constructing the engineered *Yersinia lipophila* strain according to any one of claims 1-8 and for producing itaconic acid, characterized in that, In step S5, the fermentation process is carried out in batches with an interval of 24 to 36 hours between each batch. The total amount of feed is controlled at 20% to 30% of the initial culture medium volume, and the pH of the culture medium at the end of fermentation is controlled at 4.0 to 4.2.

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