Method for constructing engineered yarrowia lipolytica strain with high itaconic acid production and use thereof

Abstract

Provided are a method for constructing an engineered Yarrowia lipolytica strain with high itaconic acid production and the use thereof. The method comprises: step S1, precisely integrating a citL gene, ACO gene, and CAD gene by using CRISPR / Cas9 technology in combination with a multi-copy insertion strategy, and optimizing a metabolic carbon flow, thereby improving the yield of a target product; step S2, constructing a dynamic regulation system, and regulating the expression of a key gene in stages by means of an inducible promoter and a feedback regulatory element, thereby achieving the dynamic optimization of a metabolic pathway; step S3, precisely localizing an itaconic acid synthase in mitochondria or peroxisomes by means of a signal peptide to reduce the production of by-products, and also enhancing the expression of a DGA1 gene by using CRISPR activation technology to improve the tolerance of a strain to a low-pH environment; and step S4, optimizing xylose and fatty acid metabolic pathways by means of adding a xylose isomerase gene, a xylulose reductase gene, and a fatty acyl-CoA dehydrogenase gene, thereby achieving the efficient conversion of multiple carbon sources.

Description

Construction Method and Application of a High-Itaconic Acid-Producing Yersinia lipophilic acid Engineered Strain Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for constructing and applying an engineered strain of Yersinia lipophila that produces high itaconic acid. Background Technology

[0002] Itaconic acid is an important bio-based platform chemical widely used in biodegradable plastics, pharmaceutical intermediates, adhesives, and coatings. Industrial production of itaconic acid primarily employs microbial fermentation. Among these methods, the traditional itaconic acid production strain, *Aspergillus niger*, is widely used due to its high yield and mature process; however, this strain faces several limitations in industrial production.

[0003] for example:

[0004] The fermentation process requires strict control of oxygen supply and pH conditions, and the production process is energy-intensive.

[0005] The generation of many byproducts, such as citric acid and malic acid, leads to low purity of the target product and increased separation costs.

[0006] In recent years, *Yersinia lipolytica* has become a promising non-model industrial strain due to its strong metabolic plasticity and ease of genetic engineering. Its excellent performance in lipid metabolism and tolerance to various environmental conditions makes it an ideal candidate microorganism for itaconic acid production. However, problems still exist in using *Yersinia lipolytica* for itaconic acid production:

[0007] 1. The natural metabolic pathway of Yersinia lipolytica cannot efficiently guide itaconic acid synthesis, and the carbon flow is dispersed in the byproduct (such as citric acid and malic acid) generation pathway, resulting in insufficient yield of the target product.

[0008] 2. Industrial fermentation for itaconic acid production typically requires acidic conditions at pH 3.0 to 4.5. Existing strains have insufficient cell membrane stability, which limits their application in industrial environments.

[0009] 3. Industrial fermentation often requires the use of multiple carbon sources, but the existing Yeast lipolyticis has limited metabolic adaptability and cannot achieve efficient conversion of multiple carbon sources.

[0010] Therefore, there is a need for a method for constructing and applying an engineered strain of Yersinia lipolytica that produces high levels of itaconic acid. Summary of the Invention

[0011] To achieve the above objectives, the present invention provides the following solution: a method for constructing a high-itaconic acid-producing engineered Yersinia lipolytica strain, the construction method comprising the following steps:

[0012] Step S1: Select Yersinia lipophila as the host strain;

[0013] Step S2: The citrate lyase gene citL, the cis-aconitate synthase gene ACO, and the itaconic acid synthase gene CAD are integrated into the host strain genome using CRISPR / Cas9 gene editing technology.

[0014] Step S3: Construct a dynamic regulatory system, using the inductive promoter XPR2 to enhance the expression of citL and ACO in the early stages of metabolism, and using feedback regulatory elements to activate the expression of CAD in the middle and late stages of metabolism.

[0015] Step S4: By fusing mitochondrial-targeting signal peptides and / or peroxisome-targeting signal peptides, itaconic acid synthase is localized to mitochondria and / or peroxisomes;

[0016] Step S5: Target and knock out the citrate synthase gene and malate dehydrogenase gene using CRISPR / Cas9 gene editing technology;

[0017] Step S6: Add xylose isomerase gene XYL1, xylulose reductase gene XYL2 and / or fatty acyl-CoA dehydrogenase gene POX1, and optimize multi-carbon source metabolism by dynamically regulating POX1 gene expression;

[0018] Step S7: Edit the promoter region of the lipid membrane protein gene DGA1 using CRISPR activation technology to increase its expression level and enhance lipid membrane function.

[0019] Preferably, step S1 further includes screening for natural isolates and ultraviolet mutagenesis of the strain.

[0020] In one specific embodiment, a 254nm ultraviolet lamp was used, with the lamp surface approximately 30cm away from the sample. An irradiation gradient of 20–100s was set, and conditions corresponding to a survival rate of 2%–5% were selected as mutagenesis parameters. After irradiation, the sample was incubated at 30°C in the dark for approximately 1 hour. Single clones were obtained by plate separation and inoculated into a medium with a pH of 3.5 (3.0–4.5 is permissible) and a carbon source of 100–200 g / L for 48 hours. Stable growth (OD) was then screened. 600 A strain that is ≥6.0 and can still proliferate under pH 3.0 conditions;

[0021] Genetic modification efficiency was evaluated using a standard electroporation / CRISPR integration assay, and lines with an integration positivity rate of ≥50% were selected.

[0022] Preferably, in step S2, the amino acid sequence of the citrate lyase gene citL includes the amino acid sequence shown in NCBI accession number XP_501064.1;

[0023] The aconitine synthase gene ACO includes the sequence shown in positions 1198468 to 1201161 of NCBI accession number NC_090773.1;

[0024] The itaconic acid synthase gene CAD includes the sequence shown in positions 79382 to 80910 of NCBI accession number NT_165939.1.

[0025] Preferably, step S2 includes integrating the citL, ACO, and CAD genes into the F-17 neutral integration site.

[0026] Further preferably, the citL, ACO, and / or CAD genes are expressed in multiple copies.

[0027] More preferably, the dynamic regulation system in step S3 includes a sensitive promoter XPR2 and a feedback regulatory element. The sensitive promoter XPR2 is used to enhance the initial expression of the citrate lyase gene citL and the cis-aconitate synthase gene ACO. The feedback regulatory element controls the activity of the trans-activator by detecting changes in the concentrations of cis-aconitate and itaconic acid, thereby precisely activating the expression of the itaconic acid synthase gene CAD in the later stages of metabolism.

[0028] The inductive promoter XPR2 enhances the expression of the citrate lyase gene citL and the aconitate synthase gene ACO in the early stages of metabolism, promoting the rapid accumulation of citrate and aconitate, thus laying a sufficient precursor foundation for itaconic acid synthesis. At the same time, the feedback regulatory element dynamically regulates the activity of the transactivator by monitoring the concentration changes of aconitate and itaconic acid in real time, so that the expression of the itaconic acid synthase gene CAD is precisely activated in the mid-to-late stages of metabolism, thereby optimizing the carbon flux of the metabolic pathway and avoiding the waste of precursors and the accumulation of byproducts.

[0029] Preferably, the sequence of the inductive promoter XPR2 includes the 5′ upstream control region of the sequence shown in NCBI accession number XM_500524.1.

[0030] Preferably, while retaining its core transcription initiation region, a functional fragment of UAS1 located in the upstream enhancement region of wild-type XPR2 is introduced to the 5′ end as an enhancer to improve the recruitment efficiency of RNA polymerase; at the same time, repetitive sequences with known repressive effects are deleted in the UAS2 region proximal to the XPR2 promoter to reduce the inhibition sensitivity to carbon and nitrogen sources; and the spatial position of carbon and nitrogen source response-related binding sites is further adjusted to make their distance from the enhancer region and the core promoter more coordinated.

[0031] Preferably, the feedback regulation element includes a signal molecule binding site and an inverse activator; more preferably, the signal molecule binding site is a PIRO promoter region binding site and the inverse activator is a RipR activator.

[0032] More preferably, the PIRO sequence includes nucleotides 2794581 to 2794732 of NCBI accession number NC_003197.2;

[0033] The RipR activator is derived from Salmonella enterica and encodes a LysR-type transcriptional regulator.

[0034] More preferably, in step S4, the mitochondrial targeting signal peptide is composed of the signal sequence MLS of Saccharomyces cerevisiae, and the perosome targeting signal peptide is composed of a sequence containing the termination signal PTS1; the mitochondrial targeting signal MLS is used to guide itaconic acid synthase into the mitochondrial matrix and directly co-localize with cis-aconitine to improve local catalytic efficiency; the perosome targeting signal PTS1 localizes itaconic acid synthase to the perosome through the perosome membrane transport system, optimizing the itaconic acid metabolic pathway;

[0035] The mitochondrial targeting signal MLS guides itaconic acid synthase into the mitochondrial matrix, where it co-localizes with cis-aconitine in the same subcellular location. This allows it to utilize the high local concentration of cis-aconitine and efficient coenzyme supply within the mitochondria, thereby enhancing catalytic efficiency. The perosome targeting signal PTS1 targets itaconic acid synthase to the perosome by recognizing the perosome membrane transport system, effectively isolating it from competitive metabolic reactions and reducing byproduct formation. The synergistic effect of the two signal peptides optimizes the flow distribution of target metabolites and enhances synthesis efficiency.

[0036] Preferably, the MLS signal peptide is derived from the cytochrome oxidase subunit IV (COX4) precursor protein of Saccharomyces cerevisiae; its encoded protein includes the amino acid sequence shown in NCBI accession number: NP_009313.1.

[0037] The PTS1 signal peptide is derived from the N-terminal signal sequence of the peroxisome labeling protein thiolase;

[0038] The sequence of the PTS1 signal peptide is the typical perosome tripeptide termination sequence SKL of S. cerevisiae.

[0039] The peroxisome marker protein thiolase includes the amino acid sequence shown in NCBI accession number NP_013581.1.

[0040] More preferably, step S5 uses a dual gRNA editing tool based on the CRISPR / Cas9 system to edit the citrate synthase gene and the malate dehydrogenase gene; the dual gRNAs target the promoter region and 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.

[0041] Citrate synthase and malate dehydrogenase can cause carbon flux dispersion and generate non-target products during metabolism, thereby reducing the efficiency of itaconic acid synthesis. By using dual gRNA technology, the knockout of these genes significantly reduces the generation of byproducts and makes the carbon flux more concentrated on the target metabolic pathway. PCR amplification and agarose gel electrophoresis are used to detect the editing efficiency, and sequencing technology is used to verify the accuracy of the mutation site, ensuring the reliability and genetic stability of the editing operation.

[0042] Preferably, the dual gRNA for knocking out the citrate synthase gene CIT1 includes the sequence shown in SEQ ID NO: 1-2.

[0043] Preferably, the dual gRNA for knocking out the malate dehydrogenase gene MDH2 includes the sequences shown in 3-4.

[0044] Preferably, the CIT1 gene in the citrate synthase gene includes the sequence shown in positions 100604 to 101983 of NCBI accession number NC_090774.1; the CIT2 gene in the citrate synthase gene encodes a protein including the sequence shown in NCBI accession number XP_503380.1.

[0045] Preferably, the malate dehydrogenase gene includes the NCBI accession number NC_090773.1, positions 2067644 to 2068657, and the NCBI accession number NC_090774.1, positions 1720717 to 1721457.

[0046] Further preferably, the multi-carbon source adaptation modification in step S6 includes the addition of xylose isomerase gene XYL1, xylulose reductase gene XYL2, and fatty acyl-CoA dehydrogenase gene POX1; the expression levels of XYL1 and XYL2 genes are regulated by a dynamic expression regulation module to optimize the xylose metabolic pathway; and the fatty acid β-oxidation pathway is optimized by dynamically regulating the expression of POX1 gene, thereby reducing the generation of acetic acid byproducts and increasing the supply of acetyl-CoA.

[0047] The xylose isomerase gene XYL1 and xylulose reductase gene XYL2 were added to construct the xylose metabolic pathway. XYL1 converts xylose into xylulose, and XYL2 further converts xylulose into glycolysis intermediates, providing additional carbon flux for itaconic acid synthesis. The fatty acid acyl-CoA dehydrogenase gene POX1 optimizes the fatty acid β-oxidation pathway through dynamic expression regulation, reduces the generation of acetic acid products, and increases the supply of acetyl-CoA, thereby promoting the synthesis of target metabolites.

[0048] Preferably, the amino acid sequence encoded by the xylose isomerase gene XYL1 includes the amino acid sequence shown in NCBI accession number: NP_001307591.1;

[0049] The amino acid sequence encoded by the xylulose reductase gene XYL2 includes the amino acid sequence shown in NCBI accession number ADQ89194.1.

[0050] The fatty acyl-CoA dehydrogenase gene POX1 includes positions 3891200 to 3893269 of NCBI accession number NC_090774.1.

[0051] More preferably, step S7 includes modifying the promoter region of the lipid membrane protein gene DGA1 using CRISPR activation technology; the modified promoter enhances the transcriptional activity of the DGA1 gene, and the triglyceride synthase it encodes regulates the stability of the lipid bilayer of the cell membrane.

[0052] By modifying the promoter region of the lipid membrane protein gene DGA1 using CRISPR activation technology, the transcriptional activity of the DGA1 gene is enhanced, 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 improves the expression level of DGA1, making the strain more tolerant to low pH environments and adapting to the acidic conditions required in industrial fermentation. The enhancement of the lipid membrane effectively reduces leakage of the target product from the cell membrane, improves the efficiency of intracellular metabolic flux, and increases the final yield of itaconic acid.

[0053] Preferably, the DGA1 gene includes the sequence shown in positions 3880991 to 3882535 of NCBI accession number NC_090774.1.

[0054] Preferably, the modification includes introducing a fusion protein of dCas9 protein and a transcription activator. More preferably, the transcription activator includes, but is not limited to, VP64, p65, or RTA. More preferably, the itaconic acid synthase gene CAD employs a multi-copy insertion strategy during genome integration. This insertion strategy is based on multi-site homologous recombination technology, inserting the CAD gene into a highly expressed site in the host strain genome.

[0055] The highly expressed sites were identified through transcriptomic analysis and are characterized by genomic regions with high promoter activity and high ribosome binding efficiency.

[0056] The multiple copy insertion was performed using CRISPR / Cas9 technology, with homologous arms ranging from 500bp to 1000bp in length. The insertion site was verified by quantitative PCR to detect the gene copy number, and the stable expression of the CAD gene was verified by transcriptional analysis.

[0057] In addition, the present invention also provides a method for constructing a high-itaconic acid-producing *Yersinia lipolytica* engineered strain, and the application of the obtained *Yersinia lipolytica* engineered strain for industrial-scale production of itaconic acid, the application comprising the following steps:

[0058] Step A1: Cultivate the engineered strain constructed by the method under optimized fermentation conditions, including pH controlled at 3.0 to 4.5, temperature controlled at 28 to 32°C, and dissolved oxygen level maintained above 30%.

[0059] Step A2: Use glucose, xylose, or waste oil as the main carbon source, with an initial carbon source concentration of 20 to 50 g / L;

[0060] Step A3: Use batch or continuous fed-batch fermentation mode to maintain the metabolic activity of the strain by dynamically supplementing carbon and nitrogen sources;

[0061] Step A4: At the end of fermentation, itaconic acid is separated by acid precipitation or ion exchange, and high-purity itaconic acid crystals are obtained by spray drying.

[0062] Step A5: Apply the obtained itaconic acid product to the production of bio-based plastics, biodegradable polymers, and food additives.

[0063] According to specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0064] I. This invention integrates the citrate lyase gene (citL), cis-aconitate synthase gene (ACO), and itaconic acid synthase gene (CAD) of *Yarrowia lipolytica* using CRISPR / Cas9 technology, and utilizes a multi-copy insertion strategy to increase the expression level of the target genes. By guiding the target genes to insert into high-expression sites in the genome through homologous recombination, carbon flow allocation is optimized, byproduct generation is reduced, and metabolic carbon flow is ensured to be concentrated in the itaconic acid synthesis pathway, thereby significantly improving the yield and production efficiency of the target products.

[0065] II. This invention achieves phased optimization of gene expression by constructing a dynamic regulation system containing a sensory promoter and a feedback regulatory element. 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 CAD gene expression by detecting changes in the concentrations of cis-aconitine and itaconic acid, precisely regulating the direction of carbon flow and effectively improving the dynamic balance and efficiency of the target metabolic pathway.

[0066] Third, this invention utilizes a signal peptide to precisely direct itaconic acid synthase to mitochondria or peroxisomes, achieving efficient reactions within specific subcellular organelles, avoiding competitive generation of byproducts, and optimizing metabolic flux. Simultaneously, CRISPR activation technology is used to upregulate DGA1 gene expression, increase triglyceride synthase levels, and enhance cell membrane stability, thereby enhancing the tolerance and metabolic activity of engineered strains under low pH conditions and adapting to the needs of industrial production.

[0067] IV. This 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 regulation module adjusts gene expression according to the concentration of external carbon sources to ensure efficient conversion of xylose into glycolysis intermediates, reduce the generation of acetic acid byproducts in fatty acid metabolism, and increase the supply of acetyl-CoA, thereby achieving flexible and efficient utilization of multiple carbon sources. Attached Figure Description

[0068] 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.

[0069] Figure 1 is a flowchart of the construction method steps of the present invention. Detailed Implementation

[0070] 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.

[0071] 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.

[0072] Example 1: A method for constructing a high-itaconic acid-producing engineered Yersinia lipolytica strain.

[0073] As shown in Figure 1, this embodiment describes a method for constructing a high-itaconic acid-producing Yersinia lipolytica engineered strain, the method comprising the following steps:

[0074] Step S1: Select *Yersinia lipolyticis* W29 (accession number: ATCC 20460 / CLIB89, e.g., CN118006479B "A *Yersinia lipolyticis* strain utilizing formic acid and its preparation method", published on 2024-06-14, using "*Yersinia lipolyticis* W29" as evidence that this genus strain is prior art) as the host strain. During the selection process, natural isolate screening and ultraviolet mutagenesis were combined to improve metabolic efficiency and enhance growth stability under high sugar and low pH conditions. *Yersinia lipolyticis* was subjected to ultraviolet mutagenesis treatment.

[0075] A 254nm UV lamp was used, with the lamp surface approximately 30cm away from the sample. An irradiation gradient of 20–100s was set, and conditions corresponding to a survival rate of 2%–5% were selected as mutagenesis parameters. After irradiation, the sample was incubated at 30℃ in the dark for approximately 1 hour. Single clones were obtained by plate separation and inoculated into YNB medium with a pH 3.5 buffer (3.0–4.5 is permissible). The carbon source was 100–200 g / L glucose, and the culture was carried out for 48 hours. Samples with stable growth (OD) were screened. 600 A strain that is ≥6.0 and can still proliferate under pH 3.0 conditions;

[0076] The genetic modification efficiency was evaluated by standard electroporation / CRISPR integration assay. Lines with an integration positivity rate of ≥50% were selected. The derived lines obtained through the above screening were designated as W29*, which served as the starting host for subsequent engineering modification and fermentation applications in steps S2 to S7.

[0077] The comparative sample used was unmutated W29.

[0078] Step S2: The derived strain W29* obtained from the screening and modification in step S1 is subjected to gene integration and expression enhancement treatment. The following sources are selected for key gene modules related to itaconic acid synthesis:

[0079] The citrate lyase gene citL is derived from Yarrowialipolytica and corresponds to the large subunit ACL1 of ATP-citrate lyase. Its preferred amino acid sequence is the one shown in NCBI accession number XP_501064.1.

[0080] The aconitase synthase gene ACO is derived from Yarrowia lipolytica and corresponds to aconitase ACO1 (NCBI accession number: NC_090773.1, positions 1198468 to 1201161).

[0081] The itaconic acid synthase gene CAD is derived from Aspergillus terreus and corresponds to cadA (NCBI accession number: NT_165939.1, positions 79382 to 80910).

[0082] The CRISPR / Cas9 gene editing system, combined with a multi-copy insertion strategy, was used to construct and achieve stable and efficient expression of the above genes. The single plasmid vector used includes the following functional modules:

[0083] Cas9 expression module: The expression of Cas9 protein is driven by the TEF1 strong promoter (NCBI accession number: NC_006068.1, positions 558003 to 558802), which causes double-strand breaks at predetermined genomic targets.

[0084] gRNA module: gRNAs targeting highly expressed integration sites were designed (NCBI accession number: NC006074.1, positions 2431501 to 2431522), and optimized through online alignment and off-target analysis to ensure specificity of recognition and accuracy of editing;

[0085] Target gene module: contains the coding sequences of citL, ACO and CAD genes, with 500-1000bp homologous arms at both ends to mediate homologous recombination;

[0086] Screening marker module: Introduces antibiotic resistance genes (HygR or LEU2) to facilitate the screening of positive transformants.

[0087] Regarding the selection of high expression sites, combined with transcriptome sequencing analysis of the host strain under different carbon sources and pH conditions, and referring to the literature (Liu et al., Microbial Biotechnology, 2022), it was finally determined that the target gene should be integrated into the F-17 neutral integration site. This site is a region of high transcriptional activity in Y. lipolytica that has been experimentally verified, and has good genetic safety and stability, making it suitable for multi-copy gene integration.

[0088] During gene integration, the Cas9 protein undergoes a double-strand break at the F-17 site, and the recombinant fragment carrying the homologous arm is inserted into the host genome through homologous recombination. Among them, the CAD gene performs multi-copy repetitive integration, further improving its transcription and translation efficiency and ensuring that more metabolic carbon flows are directed to the itaconic acid synthesis pathway.

[0089] Step S3: The dynamic regulation system achieves time-sequential regulation of gene expression through the sensing promoter XPR2 and feedback regulatory elements;

[0090] The promoter sequence includes the sensitive promoter XPR2, whose nucleotide sequence is derived from Yarrowia lipolytica, preferably the 5′ upstream regulatory region of the sequence shown in NCBI accession number XM_500524.1;

[0091] The dynamic regulatory system consists of the sensory promoter XPR2 and feedback regulatory elements, enabling the temporal expression of genes according to different metabolic processes. Functional optimization is achieved by targeting the sensory promoter XPR2.

[0092] While retaining its core transcription initiation region, a functional fragment of UAS1 located upstream of the wild-type XPR2 enhancer region was introduced to the 5′ end as an enhancer to improve the recruitment efficiency of RNA polymerase. At the same time, repetitive sequences with known repressive effects were deleted from the UAS2 region proximal to the XPR2 promoter to reduce the inhibition sensitivity to carbon and nitrogen sources. Furthermore, the spatial position of carbon and nitrogen source response-related binding sites was adjusted to make them more coordinated with the distances from the enhancer region and the core promoter.

[0093] Through the above optimizations, the XPR2 promoter can enhance the transcriptional levels of citL and ACO in the early stages of metabolism, promote the rapid accumulation of citrate and cis-aconitine, and thus ensure the initial utilization efficiency of carbon flux.

[0094] Regarding the regulation of CAD, a feedback regulatory element was constructed to respond to fluctuations in the concentration of metabolic intermediates and dynamically adjust the expression intensity of CAD. The feedback regulatory element consists of a signal molecule binding site and a trans-activator. Preferably, the binding site of the PIRO promoter region derived from Salmonella enterica (the sequence of PIRO is shown in NCBI accession number: NC_003197.2, positions 2794581 to 2794732) and the activator RipR, derived from Salmonella enterica, which encodes a LysR-type transcriptional regulator, are used to activate the PIRO promoter region in response to the itaconate signal. By detecting changes in the concentrations of cis-aconate and itaconic acid, the activity of the trans-activator is regulated in real time. Thus, when cis-aconate reaches an appropriate level, the expression module of CAD is triggered, efficiently guiding the metabolic carbon flow to the itaconic acid synthesis pathway, avoiding the waste of precursor substances, reducing the generation of byproducts, and improving the overall efficiency of the metabolic system.

[0095] Step S4: By designing the mitochondrial targeting signal MLS and the peroxisome targeting signal PTS1, the CAD is precisely guided to locate specific subcellular organelles, reducing byproduct generation and optimizing itaconic acid synthesis efficiency.

[0096] The mitochondrial targeting signal peptide MLS is derived from the cytochrome oxidase subunit IV (COX4) precursor protein of Saccharomyces cerevisiae. The COX4 precursor protein includes the amino acid sequence shown in NCBI accession number: NP_009313.1. This signal can effectively guide the target protein across the mitochondrial membrane into the matrix.

[0097] The PTS1 signal peptide is the typical perosome tripeptide termination sequence SKL of S. cerevisiae, which is derived from the perosome marker protein thiolase (NCBI accession number: NP_013581.1). It achieves efficient transport of CAD protein to the perosome by specifically binding to the perosome membrane receptor Pex5p.

[0098] In the vector construction stage, the MLS or PTS1 signal peptide is fused with the CAD gene to form a fusion expression module, a plasmid vector is constructed, and the plasmid with the fusion gene is integrated into a specific site in the genome using CRISPR / Cas9 gene editing tools.

[0099] This application uses the CRISPR / Cas9 gene editing tool derived from the Cas9 protein of Streptococcus pyogenes (NCBI accession number: NP_269215.1).

[0100] Step S5: Target and knock out the citrate synthase gene and malate dehydrogenase gene using the CRISPR / Cas9 dual gRNA tool to reduce the generation of byproducts in the metabolism of Yersinia lipolytica and optimize the conversion of carbon to the target pathway, itaconic acid.

[0101] The dual gRNAs used to knock out the citrate synthase gene CIT1 include:

[0102] gRNA-CIT1-1: 5′-GACTTCGACATCGGTGACGA-3′ (SEQ ID NO: 1) (PAM: AGG);

[0103] gRNA-CIT1-2: 5′-CTGACGATGACCTTGGTCAT-3′ (SEQ ID NO: 2) (PAM: TGG);

[0104] The dual gRNAs used to knock out the malate dehydrogenase gene MDH2 include:

[0105] gRNA-MDH2-1: 5′-GGTGATCGACTTCGACATCA-3′ (SEQ ID NO: 3) (PAM: CGG);

[0106] gRNA-MDH2-2: 5'-CTGACCTTGATGACGTCGAT-3' (SEQ ID NO: 4) (PAM: AGG).

[0107] The citrate synthase genes correspond to CIT1 (NCBI accession number: NC_090774.1, positions 100604 to 101983) and CIT2 (corresponding protein NCBI accession number: XP_503380.1), both of which encode isozymes of citrate synthase.

[0108] The malate dehydrogenase genes correspond to MDH1 (NCBI accession number: NC_090773.1, positions 2067644 to 2068657) and MDH2 (NCBI accession number: NC_090774.1, positions 1720717 to 1721457);

[0109] Each gene's gRNA target site covers its promoter region and coding sequence respectively, to ensure the destruction of the functional region and block gene expression. All gRNA sequences are predicted by online tools and optimized by off-target analysis to ensure the specificity and accuracy of the editing.

[0110] When constructing the CRISPR vector, two gRNA sequences and the Cas9 protein coding sequence are loaded into a single plasmid, and a selection marker adapted to the host bacteria is added to quickly screen positive strains after subsequent editing. The vector uses an efficient promoter to drive the expression of Cas9 and gRNA. After the vector is introduced into the host strain by electroporation, positive strains are screened in a selective medium.

[0111] After editing and validation, metabolic flux analysis was used to assess changes in the production of citric acid and malic acid in the strains. Unedited wild-type strains typically accumulate more citric acid and malic acid byproducts, resulting in dispersed carbon flux. In contrast, engineered strains that successfully knocked out the citric acid synthase gene and the malic acid dehydrogenase gene reduced the accumulation of these byproducts, making the carbon flux more concentrated in the itaconic acid metabolic pathway.

[0112] Step S6: By adding xylose isomerase gene XYL1, xylulose reductase gene XYL2, and fatty acid acyl-CoA dehydrogenase gene POX1, the metabolic pathways of xylose and fatty acids are optimized respectively.

[0113] Xylose isomerase gene XYL1 (NCBI accession number: NP_001307591.1, amino acid sequence shown);

[0114] The xylulose reductase gene XYL2 is derived from Pichiastipitis (also known as Scheffersomycesstipitis), and the encoded protein is preferably the amino acid sequence shown in NCBI accession number: ADQ89194.1;

[0115] The dynamic expression regulation module ensures that gene expression levels can be adjusted in real time according to the concentration of external carbon sources, thereby improving metabolic efficiency and reducing byproduct generation.

[0116] The dynamic expression regulation module, composed of the PIRO promoter region and its activator RipR, uses a carbon source sensing element to regulate the expression levels of XYL1 and XYL2 genes. At high xylose concentrations, the regulation module activates high-level expression of XYL1 and XYL2, rapidly metabolizing xylose. When xylose concentration decreases, the dynamic expression regulation module automatically downregulates gene expression to avoid excessive enzyme accumulation and resource waste. The XYL1 and XYL2 genes and their dynamic expression regulation module are assembled into a plasmid vector and integrated into the host bacterium's high-expression site, namely the F-17 neutral integration site, using CRISPR / Cas9 technology.

[0117] Fatty acids are another important carbon source, but they may produce acetic acid byproducts during metabolism, which reduces carbon flow efficiency. Therefore, the fatty acid acyl-CoA dehydrogenase gene POX1 was added, and its expression level was optimized through a dynamic expression regulation module.

[0118] The fatty acyl-CoA dehydrogenase gene POX1, source: Yarrowia lipolytica (NCBI accession number: NC_090774.1, positions 3891200 to 3893269);

[0119] POX1 encodes fatty acid acyl-CoA dehydrogenase, a key enzyme in fatty acid β-oxidation. It promotes fatty acid breakdown and reduces acetic acid production, while increasing the supply of acetyl-CoA, providing sufficient precursors for itaconic acid synthesis.

[0120] The expression intensity of the POX1 gene is regulated by a regulatory element that senses fatty acid concentration. When the fatty acid concentration is high, the expression of the POX1 gene is upregulated to accelerate the metabolism of fatty acids and generate sufficient acetyl-CoA to supply the itaconic acid 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.

[0121] Step S7: Modify the promoter region of the lipid membrane protein gene DGA1 using CRISPR activation technology to enhance the transcriptional activity of the DGA1 gene; the DGA1 gene encodes triglyceride synthase, and by increasing its expression level, it regulates the stability of the lipid bilayer of the cell membrane, enhances the strain's tolerance and metabolic activity in low pH environments, and provides stable cellular structural support for efficient itaconic acid synthesis.

[0122] The DGA1 gene encodes triglyceride synthase, which corresponds to positions 3880991 to 3882535 in Yarrowia lipolytica NCBI accession number NC_090774.1.

[0123] CRISPR activation (CRISPRa) technology enhances transcription by binding dCas9 (deactivated Cas9 protein) to transcription activators in the promoter region of a target gene.

[0124] The plasmid vectors for constructing the CRISPR activation system are as follows:

[0125] Deactivated Cas9 protein (dCas9) was used as a gene localization tool. Its ability to bind DNA was not affected, but it lost its cleavage function. A transcription activator (VP64, p65 or RTA) was fused to the dCas9 protein to enhance the transcription of the DGA1 gene.

[0126] Two to three gRNA sequences were designed targeting the DGA1 gene promoter region, targeting the core regulatory region 200-400 bp upstream of the transcription start site, to ensure that dCas9-VP64 (where Cas9: NCBI accession number: NP_269215.1) can effectively initiate gene transcription after binding. Online tools were used to optimize the gRNA sequences to ensure high specificity and avoid off-target binding to other genomic regions.

[0127] Add the dCas9-VP64 expression module, gRNA expression module, and selection marker module to the plasmid vector;

[0128] The dCas9-VP64 expression module is driven by a strong promoter to achieve high-level expression.

[0129] The gRNA expression module carries multiple optimized gRNA sequences;

[0130] The screening marker module allows setting the HygR resistance gene to screen for positive transformants.

[0131] The constructed plasmid vector was introduced into the host strain of Yersinia lipolytica via electroporation. The transformed strain was then inoculated into a selective medium containing antibiotics, and positive transformants were screened. Genomic RNA was then extracted from the positive strains, and the transcriptional level of the DGA1 gene was analyzed by quantitative PCR. Positive strains should exhibit an increase in the transcriptional level of DGA1.

[0132] Comparative Example 1

[0133] Unmodified Yeast Rice W29 (ATCC 20460) was selected as the control strain.

[0134] Culture conditions and detection methods: The culture conditions are consistent with those in the examples.

[0135] Comparative Example 2:

[0136] In W29, only the citL and ACO genes are integrated, without CAD multiple copy insertion, dynamic regulation, or subcellular localization.

[0137] Culture conditions and detection methods: The culture conditions are consistent with those in the examples.

[0138] Comparative Example 3:

[0139] Only the itaconic acid synthase gene CAD was integrated into the genome of Yersinia lipophila W29, without carbon flow optimization of citL and ACO, or the introduction of dynamic regulatory modules or subcellular localization signals.

[0140] Culture conditions and detection methods: consistent with the examples.

[0141] Experimental Example 1:

[0142] Construction and verification of high-yield itaconic acid-producing Yersinia spp.

[0143] (I) Experimental Methods:

[0144] (1) The strain and culture conditions are as follows:

[0145] Engineered strain: The engineered Yersinia lipophila strain constructed as described in Examples S1 to S7 (denoted as W29-IC*); Control strain: The intermediate strain of W29 and citL+ACO in the comparative example.

[0146] Culture medium: YNB (150 g / L glucose, 5 g / L ammonium sulfate nitrogen source, pH 3.5, buffered with 1 mol / L HCl); Culture conditions: 30℃, 200 rpm shake flask culture; Aeration rate of 1 vvm and stirring at 500 rpm were used in the fermenter scale-up experiment.

[0147] (2) Gene expression detection is as follows:

[0148] Total RNA was extracted and reverse transcribed using the PrimeScript RT kit; real-time qPCR: SYBR Green Master Mix, internal control gene ACT1; detection instrument: Bio-Rad CFX96.

[0149] (3) Metabolite detection results are as follows:

[0150] HPLC: Aminex HPX-87H (300×7.8mm), mobile phase 5mmol / L H2SO4, flow rate 0.6mL / min, column temperature 65℃, detector RID.

[0151] (4) The membrane stability and acid resistance were tested as follows:

[0152] Membrane permeability: PI (propidium iodide) staining, fluorescence intensity measured by flow cytometry (BD Accuri C6).

[0153] Electron microscope: JEOL JEM-2100 for observing film thickness and structure.

[0154] Acid resistance test: The cells were cultured for 48 hours at pH 3.0, 3.5 and 4.0, and the survival rate and acid production were measured.

[0155] II. Comparison Table of Experimental Results

[0156] Table 1: Performance of different strains in itaconic acid synthesis

[0157] As shown in Table 1, strain W29-IC* in the example can reach 45.6 g / L under shake-flask conditions for 72 h, which is about 3 times higher than wild-type comparative example 1; the yield can reach 68.4 g / L in a 5L fermenter, with a yield of 0.42 g / g (glucose), which is better than all comparative examples.

[0158] The comparative strains generally accumulated high levels of citric acid and malic acid (4.2-8.5 g / L and 3.5-6.1 g / L, respectively), while the byproducts of the example strains were only 1.2 g / L and 0.8 g / L, indicating that the carbon flow was effectively concentrated in the itaconic acid synthesis pathway.

[0159] Under pH 3.0 conditions, the membrane integrity retention rate of the example strain reached 85% and the survival rate was 78%, while that of the comparative strain was only 25%-40%, demonstrating that CRISPRa activation of DGA1 enhances membrane structural stability and acid environment adaptability.

[0160] Comparative analysis:

[0161] While Comparative Example 2 shows some improvement, it lacks CAD multi-copy and control optimization, resulting in limited effectiveness.

[0162] Although Comparative Example 3 can increase itaconic acid formation, byproducts still accumulate significantly due to carbon flow dispersion.

[0163] Conclusion: The engineered strain of this invention exhibits stable growth and metabolic performance under low pH and high sugar conditions, with increased acid production and significantly reduced byproducts, verifying the effectiveness and superiority of the method of this invention.

[0164] 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.

[0165] 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 a high-itaconic acid-producing engineered Yersinia lipolyticis strain, characterized in that, The construction method includes the following steps: Step S1: Select Yersinia lipophila as the host strain; Step S2: The citrate lyase gene citL, the cis-aconitate synthase gene ACO, and the itaconic acid synthase gene CAD are integrated into the host strain genome using CRISPR / Cas9 gene editing technology. Step S3: Construct a dynamic regulatory system, using the inductive promoter XPR2 to enhance the expression of citL and ACO in the early stages of metabolism, and using feedback regulatory elements to activate the expression of CAD in the middle and late stages of metabolism. Step S4: By fusing mitochondrial-targeting signal peptides and / or peroxisome-targeting signal peptides, itaconic acid synthase is localized to mitochondria and / or peroxisomes; Step S5: Target and knock out the citrate synthase gene and malate dehydrogenase gene using CRISPR / Cas9 gene editing technology; Step S6: Add xylose isomerase gene XYL1, xylulose reductase gene XYL2 and / or fatty acyl-CoA dehydrogenase gene POX1, and optimize multi-carbon source metabolism by dynamically regulating POX1 gene expression; Step S7: Edit the promoter region of the lipid membrane protein gene DGA1 using CRISPR activation technology to enhance lipid membrane function.

2. The method for constructing a high-itaconic acid-producing engineered Yersinia lipolytica strain according to claim 1, characterized in that, The dynamic regulation system includes a sensing promoter XPR2 and a feedback regulatory element. The sensing promoter XPR2 is used to enhance the initial expression of the citrate lyase gene citL and the cis-aconitate synthase gene ACO. The feedback regulatory element controls the activity of the trans-activator by detecting changes in the concentrations of cis-aconitate and itaconic acid, thereby precisely activating the expression of the itaconic acid synthase gene CAD in the later stages of metabolism.

3. The method for constructing a high-itaconic acid-producing engineered Yersinia lipolytica strain according to claim 1, characterized in that, The mitochondrial targeting signal peptide is composed of the signal sequence MLS of Saccharomyces cerevisiae, and the perosome targeting signal peptide is composed of a sequence containing the termination signal PTS1. The mitochondrial targeting signal MLS is used to guide itaconic acid synthase into the mitochondrial matrix and directly co-localize it with cis-aconitine to improve local catalytic efficiency. The perosome targeting signal PTS1 localizes itaconic acid synthase to the perosome through the perosome membrane transport system, thereby optimizing the itaconic acid metabolic pathway.

4. The method for constructing a high-itaconic acid-producing engineered Yersinia lipolyticis strain according to claim 1, characterized in that, The citrate synthase gene and malate dehydrogenase gene were edited using a dual gRNA editing tool based on the CRISPR / Cas9 system; the dual gRNAs targeted the promoter region and coding sequence of the citrate synthase gene and malate dehydrogenase gene, respectively.

5. The method for constructing a high-itaconic acid-producing engineered Yersinia lipolytica strain according to claim 1, characterized in that, The multi-carbon source adaptation modification includes the addition of xylose isomerase gene XYL1, xylulose reductase gene XYL2, and fatty acyl-CoA dehydrogenase gene POX1; the expression levels of XYL1 and XYL2 genes are regulated by a dynamic expression regulation module to optimize the xylose metabolic pathway; and the fatty acid β-oxidation pathway is optimized by dynamically regulating the expression of POX1 gene, thereby reducing the generation of acetic acid byproducts and increasing the supply of acetyl-CoA.

6. The method for constructing a high-itaconic acid-producing engineered Yersinia lipolytica strain according to claim 1, characterized in that, The promoter region of the lipid membrane protein gene DGA1 was modified using CRISPR activation technology; the modified promoter enhanced the transcriptional activity of the DGA1 gene, and the triglyceride synthase it encodes regulates the stability of the lipid bilayer of the cell membrane.

7. The method for constructing a high-itaconic acid-producing engineered *Yarrowia lipolyticis* strain according to any one of claims 1-6, characterized in that, The itaconic acid synthase gene CAD employs a multi-copy insertion strategy during genome integration. This insertion strategy is based on multi-site homologous recombination technology, inserting the CAD gene into a highly expressed site in the host strain genome. The highly expressed sites were identified through transcriptomic analysis and are characterized by genomic regions with high promoter activity and high ribosome binding efficiency. The multiple copy insertion was performed using CRISPR / Cas9 technology, with homologous arms ranging from 500bp to 1000bp in length. The insertion site was verified by quantitative PCR to detect the gene copy number, and the stable expression of the CAD gene was verified by transcriptional analysis.

8. The application of the *Yersinia lipolytica* engineered strain obtained by the method for constructing a high-itaconic acid-producing *Yersinia lipolytica* strain according to any one of claims 1-7, characterized in that, For industrial-scale production of itaconic acid, the application includes the following steps: Step A1: Cultivate the engineered strain constructed by the method under optimized fermentation conditions, including pH controlled at 3.0 to 4.5, temperature controlled at 28 to 32°C, and dissolved oxygen level maintained above 30%. Step A2: Use glucose, xylose, or waste oil as the main carbon source, with an initial carbon source concentration of 20 to 50 g / L; Step A3: Use batch or continuous fed-batch fermentation mode to maintain the metabolic activity of the strain by dynamically supplementing carbon and nitrogen sources; Step A4: At the end of fermentation, itaconic acid is separated by acid precipitation or ion exchange, and high-purity itaconic acid crystals are obtained by spray drying. Step A5: Apply the obtained itaconic acid product to the production of bio-based plastics, biodegradable polymers, and food additives.

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