Recombinant yarrowia lipolytica strain with high itaconic acid yield as well as construction method and application of recombinant yarrowia lipolytica strain
By constructing a recombinant lipophilic yeast strain that produces high levels of itaconic acid, problems such as poor coordination of heterologous metabolic pathways, limited transmembrane transport, insufficient dynamic regulation, and carbon source inhibition were solved, achieving efficient synthesis and stable expression of itaconic acid and improving fermentation efficiency and yield.
Patent Information
- Application Number
- CN202511656048.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-12
- Publication Date
- 2026-02-13
AI Technical Summary
In existing technologies, poor coordination between heterologous metabolic pathways and the host metabolic network leads to the accumulation of intermediate metabolites; restricted transmembrane transport of aconitine results in the product remaining in the mitochondria; immobilized expression systems lack dynamic regulation capabilities, making it difficult to balance the needs of growth and acid production stages; insufficient reducing power and coenzyme regeneration limit high-throughput synthesis efficiency; and traditional single carbon source processes exhibit significant inhibitory effects and short fermentation stages.
A recombinant lipophilic yeast strain producing high itaconic acid was constructed. By inserting the optimized cis-aconitine decarboxylase gene cadA and fusing it with a mitochondrial targeting signal peptide, key enzyme genes were knocked out, NADH oxidase was introduced, and citrate synthase and cis-aconitine hydratase were overexpressed. Dynamic regulatory elements and multi-site gene integration were used to optimize carbon source utilization and fermentation process, thereby improving metabolic flux and transport efficiency.
It significantly improved the yield and conversion rate of itaconic acid, achieving efficient synthesis and stable expression of itaconic acid. It solved the problems of intermediate metabolite accumulation, limited transmembrane transport, insufficient dynamic regulation ability and carbon source inhibition, thus improving the efficiency and yield of the fermentation stage.
Smart Images
Figure CN121518293A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of strain cultivation, more particularly to a recombinant Yarrowia lipolytica strain with high yield of itaconic acid and a construction method and application thereof. BACKGROUND
[0002] As an important renewable platform chemical, itaconic acid is widely used in the fields of degradable plastics, medicines and fine chemicals. Early production of itaconic acid mainly relied on Aspergillus niger fermentation system, although the yield was high, but the strain was susceptible to infection, poor in substrate adaptability and complex in process control. Subsequently, researchers shifted their focus to yeast chassis, especially Yarrowia lipolytica, which has the advantages of strong lipid metabolism, wide carbon source utilization, mature genetic manipulation, etc., making it an ideal host for new generation of itaconic acid biological manufacturing. In recent years, with the development of synthetic biology and metabolic engineering, many breakthroughs have been made in heterologous cadA expression, mitochondrial transport pathway optimization and carbon flow redirection, and the yield of itaconic acid has been significantly improved.
[0003] However, the existing technology still has multiple bottlenecks: first, the heterologous metabolic pathway and the host metabolic network have poor coordination, causing accumulation of intermediate metabolites; second, the transmembrane transport of cis-aconitic acid is limited, and the product is retained in the mitochondria; third, the fixed expression system lacks dynamic regulation ability, making it difficult to balance the needs of the growth and acid production stages; fourth, the reducing power and coenzyme regeneration are insufficient, limiting the high-throughput synthesis efficiency; fifth, the traditional single carbon source process has obvious inhibition effect and short fermentation stage. Based on this, the present application systematically improves and strengthens the above deficiencies, and constructs a recombinant Yarrowia lipolytica strain with high stability, high conversion efficiency and industrialization potential for high-yield itaconic acid. SUMMARY
[0004] The present application aims to provide a recombinant Yarrowia lipolytica strain with high yield of itaconic acid and a construction method and application thereof, to solve the problems raised in the background art: first, the heterologous metabolic pathway and the host metabolic network have poor coordination, causing accumulation of intermediate metabolites; second, the transmembrane transport of cis-aconitic acid is limited, and the product is retained in the mitochondria; third, the fixed expression system lacks dynamic regulation ability, making it difficult to balance the needs of the growth and acid production stages; fourth, the reducing power and coenzyme regeneration are insufficient, limiting the high-throughput synthesis efficiency; fifth, the traditional single carbon source process has obvious inhibition effect and short fermentation stage.
[0005] Technical solution: a recombinant Yarrowia lipolytica strain with high yield of itaconic acid comprises the following components: The recombinant Yarrowia lipolytica strain with high itaconic acid production is obtained by genome metabolic reconstruction from a wild type Yarrowia lipolytica strain, and the recombinant Yarrowia lipolytica strain with high itaconic acid production comprises a cis-aconitate decarboxylase gene cadA derived from Aspergillus terreus ATCC20542 inserted into the genome of Yarrowia lipolytica, and the cis-aconitate decarboxylase gene cadA is placed between a TEF1 promoter and a CYC1 terminator to form a stable expression cassette; A mitochondrial targeting signal peptide sequence derived from the first 30 amino acid residues of the CIT1 gene is fused to the N terminus of the cis-aconitate decarboxylase gene cadA, so as to direct the cis-aconitate decarboxylase to be located in the mitochondrial matrix; The isocitrate dehydrogenase gene IDH1 and the isocitrate lyase gene ACL1 in the genome of Yarrowia lipolytica are knocked out, so as to block the carbon flow branch of isocitrate to alpha-ketoglutarate and acetyl-CoA, thereby enhancing the accumulation of cis-aconitate; The citrate synthase gene CIT1 and the aconitate hydratase gene ACO1 in the genome of Yarrowia lipolytica are overexpressed, so as to enhance the metabolic flux of citrate to cis-aconitate; The alcohol dehydrogenase gene ADH1 in the genome of Yarrowia lipolytica is knocked out, so as to inhibit the carbon flow loss of the ethanol synthesis branch; The NADH oxidase gene noxE derived from Lactococcus lactis subsp, cremoris MG1363 is introduced and placed between the PGK1 promoter and the ADH1 terminator, so as to rebalance the intracellular NADH oxidation; The recombinant Yarrowia lipolytica strain with high itaconic acid production reaches an itaconic acid yield of more than 12 g / L after being fermented for 72 hours under the conditions of glucose as the only carbon source, 30°C, a dissolved oxygen mass fraction of 15% to 25%, and a pH of 5.5 to 6.0.
[0006] Preferably, the cadA expression cassette is integrated into the rDNA multicopy repeat sequence region of the Yarrowia lipolytica chromosome by homologous recombination, and the length of the fragment for homologous recombination is 800 to 1000 bp, so as to realize stable multicopy integration and high-level expression.
[0007] Preferably, the mitochondrial targeting signal peptide sequence and the cis-aconitate decarboxylase gene cadA are assembled by seamless ligation PCR, and the length of the ligation region is controlled to be within 15 to 25 nucleotides, so as to ensure that the translation frame is continuous and the activity of the cis-aconitate decarboxylase is not less than 95% of the original enzyme activity.
[0008] Preferably, the knockout of the alcohol dehydrogenase gene ADH1 and the isocitrate lyase gene ACL1 is achieved by a CRISPR-Cas9 system, which comprises a plasmid pCAS-KLE901 carrying a Cas9 protein coding sequence and an sgRNA expression cassette, wherein the sgRNA target sequences are located in the 2nd exon and the 3rd exon of the alcohol dehydrogenase gene ADH1, respectively, and the knockout efficiency is not less than 80%.
[0009] Preferably, the NADH oxidase gene noxE is integrated at the URA3 site of the Yarrowia lipolytica chromosome, and the expression level after integration is detected by real-time quantitative PCR, and the relative transcription level is not less than 2.5 times that of the reference isocitrate lyase gene ACL1.
[0010] Preferably, the method for constructing a recombinant Yarrowia lipolytica strain with high itaconic acid yield comprises the following steps: S1. Selecting a wild-type Yarrowia lipolytica strain as the starting strain; S2. Knocking out the IDH1 gene and the ACL1 gene respectively by using a CRISPR-Cas9 system; S3. Constructing a cadA expression cassette and integrating it into the rDNA site by homologous recombination; S4. Linking the mitochondrial targeting signal peptide sequence to the N terminus of the cadA gene by fusion PCR technology; S5. Constructing a noxE overexpression vector and integrating it into the URA3 site; S6. Obtaining a recombinant Yarrowia lipolytica strain with high itaconic acid yield by G418 resistance screening, PCR verification and sequencing confirmation.
[0011] Preferably, the homologous recombination integration uses a double-arm recombination fragment, the left arm and the right arm are 900 bp and 850 bp in length respectively, the integration fragment is amplified and purified by PCR and transformed into Yarrowia lipolytica cells in a linear form, and the integration success rate is not less than 85%.
[0012] Preferably, in the fermentation culture, a semi-continuous fermentation process with a carbon-nitrogen ratio of 20:1 is used, and when the residual sugar concentration decreases to below 5 g / L, the feed is supplemented with sugar, and the glucose concentration in the culture medium is maintained in the range of 20-30 g / L, so that the final itaconic acid yield is increased to more than 35 g / L, and the conversion rate is not less than 0.32 g / g.
[0013] Preferably, the citrate transmembrane carrier protein gene CIT2 is further overexpressed in the Yarrowia lipolytica genome, so that the transmembrane transport rate of cis-aconitic acid is increased by 1.8 times compared with the unmodified strain, thereby improving the extracellular accumulation rate of itaconic acid.
[0014] Preferably, a high-yield itaconic acid recombinant Yersinia lipolytica strain is used in aerobic fermentation in a synthetic medium containing glucose, nitrogen source, phosphate and trace elements. When the culture temperature is 28-32℃, the dissolved oxygen mass fraction is 15%-25%, and the stirring speed is 400-600 rpm, the final itaconic acid yield in the fermentation system reaches 35-40 g / L. After filtration, acidification, crystallization and drying of the fermentation broth, the obtained itaconic acid has a purity of ≥99.5%, which is suitable for the preparation of biodegradable polymers, plasticizers and pharmaceutical intermediates.
[0015] Compared with the prior art, the advantages of this invention are: (1) In terms of host selection, Yarrowia lipolytica strains with targeted weakening of lipid metabolism pathways were used as chassis cells, which improved the allocation efficiency of carbon flow to the tricarboxylic acid cycle compared with conventional Yarrowia lipolytica Po1f or W29 strains.
[0016] (2) By stably integrating heterologous genes cadA, mttA and cis-aconitatedecarboxylase at multiple sites on chromosomes, the instability and metabolic burden of plasmid vectors are avoided, and long-term stable genetic expression is achieved.
[0017] (3) Introduce a mitochondrial-cytoplasmic dual-positioning transport system to improve the transmembrane transport efficiency of aconitine and overcome the product retention problem caused by the transmembrane transport bottleneck in the existing technology.
[0018] (4) By utilizing dynamic metabolic regulatory elements (pH-responsive promoters and substrate-sensing regulatory factors), the expression ratio of key enzyme genes is automatically adjusted at different fermentation stages to achieve dynamic optimal allocation of carbon source utilization and energy balance.
[0019] (5) By reconstructing the acetyl-CoA redistribution pathway, a high-throughput connection from fatty acid β-oxidation to itaconic acid synthesis is achieved, significantly improving the product synthesis rate.
[0020] (6) An enhanced NADPH regeneration module (through overexpression of zwf and malicenzyme) is introduced to enhance the supply of reducing power and solve the problem of insufficient electron transfer during itaconic acid synthesis.
[0021] (7) A quantitative introduction strategy driven by metabolic flux analysis is adopted, and gene copy number and promoter strength are precisely controlled through a computational model to achieve the coupling optimization of itaconic acid production and cell growth.
[0022] (8) By using a carbon source co-utilization mechanism (glucose + glycerol synergistic metabolism), substrate conversion efficiency and fermentation stage sustainability are improved, thus improving the acid buildup inhibition problem of traditional single carbon source systems.
[0023] (9) A two-stage dissolved oxygen control strategy is adopted in the fermentation process, namely, high dissolved oxygen in the growth stage promotes biomass accumulation, and oxygen restriction in the acid production stage induces product accumulation, thus solving the problem of by-product generation caused by oxygen limitation in conventional processes.
[0024] (10) At the application level, the recombinant lipophilic yeast strain that produces high itaconic acid achieves efficient bioconversion of renewable carbon sources (such as waste glycerol and biodiesel byproducts), expanding the raw material adaptability and industrial application scenarios of green itaconic acid manufacturing. Attached Figure Description
[0025] Fig. 1 This is a schematic diagram of the component composition of a recombinant lipophilic yeast strain that produces high itaconic acid according to the present invention. Fig. 2 This is a schematic diagram of the construction process of a recombinant lipophilic yeast strain that produces high itaconic acid according to the present invention. Detailed Implementation
[0026] For examples, please refer to Figs. 1-2 A recombinant lipophilic yeast strain that produces high itaconic acid comprises the following components: The high-itaconic acid-producing recombinant Yersinia lipolytica strain was obtained by genome metabolic reconstruction of wild-type Yersinia lipolytica strain. A high-itaconic acid-producing recombinant Yersinia lipolytica strain includes the insertion of the cis-aconitine decarboxylase gene cadA from AspergillusterreusATCC20542 into the Yersinia lipolytica genome, and the use of codon-optimized nucleotide sequence. The cis-aconitine decarboxylase gene cadA is placed between the TEF1 promoter and the CYC1 terminator to form a stable expression cassette. A mitochondrial targeting signal peptide sequence derived from the first 30 amino acid residues of the CIT1 gene is fused to the N-terminus of the cis-aconitine decarboxylase gene cadA to direct cis-aconitine decarboxylase to the mitochondrial matrix. Knocking out the isocitrate dehydrogenase gene IDH1 and the isocitrate lyase gene ACL1 in the genome of Yersinia lipolytica blocks the carbon flow branch from isocitrate to α-ketoglutarate and acetyl-CoA, thereby enhancing the accumulation of cis-aconitate. Overexpression of the citrate synthase gene CIT1 and the cis-aconitate hydratase gene ACO1 in the genome of Yersinia lipophila enhances the metabolic flux from citrate to cis-aconitate. Knocking out the alcohol dehydrogenase gene ADH1 in the genome of Yersinia lipophilia inhibits carbon flow loss in the ethanol synthesis pathway. The NADH oxidase gene noxE, derived from Lactococcus lactis subsp. cremoris MG1363, was introduced and placed between the PGK1 promoter and the ADH1 terminator to rebalance intracellular NADH oxidation. A recombinant Yersinia lipolytica strain with high itaconic acid production achieved an itaconic acid yield of over 12 g / L after fermentation for 72 hours under the conditions of glucose as the sole carbon source, 30°C, dissolved oxygen content of 15%–25%, and pH of 5.5–6.0.
[0027] Specifically, using *Yarrowia lipolyticis* Po1f as the starting strain, IDH1 and ACL1 gene deletion mutants were constructed using the CRISPR-Cas9 system and verified by PCR and sequencing. Subsequently, codon optimization software (such as GeneOptimizer) was used to optimize the cadA gene of cis-aconitine decarboxylase from *Aspergillusterreus* ATCC20542, matching its GC content to the host gene preferences of *Yarrowia lipolyticis*. A pTEF1-cadA-pCYC1 expression cassette was constructed, and a mitochondrial targeting signal peptide sequence (MSTSKKRTFFRLTSLVRSPKTSLAGLTS) derived from the first 30 amino acid residues of the CIT1 gene was seamlessly integrated at its N-terminus, localizing the enzyme to the mitochondrial matrix. This expression cassette was integrated into the rDNA repetitive sequence region via homologous recombination, and positive clones were screened, with expression levels confirmed by RT-qPCR. Further, the ADH1 gene was knocked out, and the noxE gene from *Lactococcus lactis subsp. cremoris* MG1363 (driven by the PGK1 promoter) was introduced to achieve intracellular redox balance. The strain was inoculated into YND medium (30 g / L glucose, 1 g / L nitrogen source, pH 5.8) and cultured at 30°C with shaking for 72 hours. The itaconic acid yield was 12.6 g / L, which was significantly higher than that of the unmodified strain.
[0028] The cadA expression cassette integrates into the rDNA multiple copy repeat region of the Yersinia lipolyticis chromosome via homologous recombination. The length of the homologous recombination fragment is 800-1000 bp, so as to achieve stable multiple copy integration and high-level expression.
[0029] Specifically, a two-arm homologous recombination strategy was used to construct the integrated fragment. The left and right arms were derived from the regions flanking the rDNA repetitive sequence, with lengths of 920 bp and 870 bp, respectively. After PCR amplification and purification, the fragment was assembled with a cadA expression cassette using Gibson chromatography to form a linear integrated fragment. This fragment was introduced into *Yarrowia lipolytica* cells via electroporation, and positive colonies were selected using YPD plates (G418 200 μg / mL). PCR detection of the integration-specific fragments flanking the rDNA site confirmed multi-copy integration via sequencing. RT-qPCR showed that the relative expression level of cadA was approximately 3.5 times higher than that of the single-copy integrated strain, ensuring high-level and stable expression.
[0030] The mitochondrial targeting signal peptide sequence and the cis-aconitine decarboxylase gene cadA were assembled by seamless PCR, with the length of the linker region controlled within 15-25 nucleotides to ensure the continuity of the translation framework and maintain the cis-aconitine decarboxylase activity at no less than 95% of the original enzyme activity.
[0031] Specifically, seamless assembly PCR was used to fuse the mitochondrial targeting signal peptide sequence with the cis-aconitine decarboxylase gene cadA. Primers were designed to ensure 15–25 bp overlap both upstream and downstream. A high-fidelity enzyme (Phusion DNA Polymerase) was used in the PCR system. After amplification, the enzyme was purified and cloned into the pTEF1-CYC1 backbone vector. The translated fusion enzyme activity assay showed that it retained more than 95% of the catalytic activity compared to wild-type cadA (measured in U / mg). Enzyme localization was verified using a mitochondrial fluorescent labeling co-localization assay.
[0032] The knockout of the alcohol dehydrogenase gene ADH1 and the isocitrate lyase gene ACL1 was achieved using the CRISPR-Cas9 system, which includes the plasmid pCAS-KLE901 carrying the Cas9 protein coding sequence and the sgRNA expression cassette. The sgRNA target sequences are located in the second exon and the third exon of the alcohol dehydrogenase gene ADH1, respectively, with a knockout efficiency of not less than 80%.
[0033] Specifically, the CRISPR-Cas9 system uses plasmid pCAS-KLE901, with the Cas9 sequence driven by the TEF1 promoter. sgRNA sequences were designed to target exon 2 of ADH1 (5'-GCTGATCGGATTCCAGTGGA-3') and exon 3 of ACL1 (5'-TGGTTGCTTCCGACTTACCA-3'), respectively. After transformation into bacterial cells using the lithium-ion method, positive clones were selected on G418 resistant plates. Genotyping verification confirmed the deletion mutation through PCR product sequencing, achieving a knockout efficiency of 82%–88%. Simultaneously, a significant decrease in the NADH / NAD+ ratio was detected, indicating an improvement in redox balance.
[0034] The NADH oxidase gene noxE was integrated into the URA3 site of the Yersinia lipolytica chromosome. The expression level after integration was detected by real-time quantitative PCR, and the relative transcription level was not less than 2.5 times that of the reference isocitrate lyase gene ACL1.
[0035] Specifically, a PGK1p-noxE-ADH1t expression cassette was constructed, using the URA3 site as the integration target region and employing 800bp upstream and downstream homologous arms for integration. After transformation, positive strains were screened in URA-deficient medium, and RT-qPCR analysis showed that the noxE expression level was 2.6 times that of the ACL1 gene. Biochemical analysis revealed that the residual NADH concentration in the fermentation broth decreased by approximately 40% compared to the control strain, indicating restored oxidative balance and promoting efficient itaconic acid synthesis.
[0036] The method for constructing a recombinant lipophilic yeast strain that produces high itaconic acid includes the following steps: S1. Wild-type strain of Yersinia lipolytica was selected as the starting strain; S2. Knock out the IDH1 and ACL1 genes respectively using the CRISPR-Cas9 system; S3. Construct the cadA expression cassette and integrate it into the rDNA site via homologous recombination; S4. A mitochondrial targeting signal peptide sequence was ligated to the N-terminus of the cadA gene using fusion PCR technology; S5. Construct a noxE overexpression vector and integrate it into the URA3 site; S6. A recombinant Yersinia lipolytica strain producing high itaconic acid was obtained through G418 resistance screening, PCR verification, and sequencing confirmation.
[0037] Specifically, the steps S1-S6 were performed sequentially: First, wild-type *Yarrowia lipolytica* strains were cultured in YPD medium; then, the IDH1 and ACL1 genes were knocked out using the pCAS-KLE901 vector; next, the pTEF1-cadA-pCYC1 expression cassette was constructed using the Gibson assembly method and integrated via rDNA homologous arms; the mitochondrial targeting signal peptide was ligated to the cadA gene using fusion PCR; the PGK1p-noxE-ADH1t vector was constructed and integrated into the URA3 site; after G418 resistance screening, PCR verification, sequencing, and RT-qPCR confirmation, a stable, genetically high-yielding itaconic acid-producing recombinant strain was obtained. Each step was repeated three times to ensure reliable results.
[0038] Homologous recombination integration uses a two-arm recombination fragment with left and right arms of 900bp and 850bp in length, respectively. The integrated fragment is amplified and purified by PCR and transformed into Yeast lipolyticis cells in a linear form, with an integration success rate of no less than 85%.
[0039] Specifically, homologous arm DNA was amplified from the Yersinia lipophila genome template using high-fidelity PCR. The amplified DNA was then purified by agarose gel electroporation, and the linearized DNA fragment was introduced into cells via electroporation. Integrative strains were screened using YPD plates containing 200 μg / mLG418, with a PCR positive rate of approximately 86%. Southern blot validation showed that multiple copies of the integrated fragment were inserted into the expected location, and genetic stability remained unchanged after 10 generations of passage.
[0040] A semi-continuous fermentation process with a carbon-to-nitrogen ratio of 20:1 was adopted in the fermentation culture. When the residual sugar concentration dropped to below 5 g / L, sugar was added to maintain the glucose concentration in the culture medium in the range of 20-30 g / L, so that the final yield of itaconic acid could be increased to more than 35 g / L and the conversion rate could not be less than 0.32 g / g.
[0041] Specifically, semi-continuous fed-batch fermentation was conducted in a 5L bioreactor at a carbon-to-nitrogen ratio of 20:1. The initial glucose concentration was 25 g / L, and the nitrogen source was ammonium sulfate at 1.25 g / L. Feeding was controlled via online residual sugar monitoring; when the concentration fell below 5 g / L, 25 g / L glucose solution was automatically added. The pH was maintained at 5.8 ± 0.2, and the dissolved oxygen concentration was maintained at 20%. After 72 hours, the itaconic acid concentration reached 35.8 g / L, with a yield of 0.33 g / g, and the byproduct ethanol was almost undetectable.
[0042] Furthermore, overexpression of the citrate transmembrane carrier protein gene CIT2 in the genome of Yersinia lipolytica increased the transmembrane transport rate of cis-aconitine by 1.8 times compared with the unmodified strain, thereby enhancing the extracellular accumulation rate of itaconic acid.
[0043] Specifically, the citrate transmembrane carrier protein gene CIT2 was overexpressed under the control of the strong promoter GPD1p, with the integration site selected from the LEU2 region. Fluorescence tracing experiments demonstrated a 1.8-fold increase in the transmembrane transport rate of aconitic acid. The extracellular chalcanic acid accumulation rate in the fermentation broth reached 1.2 mmol / (L·h), approximately 85% higher than that of the unmodified strain, significantly shortening the accumulation phase.
[0044] A high-yield itaconic acid recombinant Yersinia lipolytica strain was developed. This strain underwent aerobic fermentation in a synthetic medium containing glucose, nitrogen source, phosphate, and trace elements. At a culture temperature of 28–32℃, dissolved oxygen content of 15%–25%, and stirring speed of 400–600 rpm, the final itaconic acid yield reached 35–40 g / L. After filtration, acidification, crystallization, and drying, the itaconic acid obtained had a purity ≥99.5%, making it suitable for the preparation of biodegradable polymers, plasticizers, and pharmaceutical intermediates.
[0045] Specifically, the recombinant strain constructed above was inoculated into YND medium containing glucose (40 g / L), yeast extract (1 g / L), (NH4)2SO4 (2 g / L), KH2PO4 (1 g / L), and trace elements, and fermented under aeration at 28–32 °C and 400–600 rpm. Dissolved oxygen was maintained at 15%–25%, and the pH of the fermentation broth was maintained at 5.8. After 72–96 hours, the itaconic acid concentration reached 38 g / L. The fermentation broth was adjusted to pH 2.0 with sulfuric acid, cooled, and crystallized. After centrifugation, washing, and vacuum drying, the obtained itaconic acid had a purity of 99.6%. The chemical purity of the obtained product was confirmed by Fourier transform infrared (FTIR) analysis, and it can be directly used to prepare biodegradable polylactic acid-itaconic acid copolymers and pharmaceutical-grade plasticizer intermediates.
[0046] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.
Claims
1. A recombinant lipophilic yeast strain that produces high itaconic acid, characterized in that, The recombinant lipophilic yeast strain that produces high itaconic acid comprises the following components: The high-itaconic acid-producing recombinant Yersinia lipolytica strain was obtained by genome metabolic reconstruction of wild-type Yersinia lipolytica strain. The high-itaconic acid-producing recombinant Yersinia lipolytica strain includes the insertion of the cis-aconitine decarboxylase gene cadA from AspergillusterreusATCC20542 into the Yersinia lipolytica genome, and the use of a codon-optimized nucleotide sequence. The cis-aconitine decarboxylase gene cadA is placed between the TEF1 promoter and the CYC1 terminator to form a stable expression cassette. A mitochondrial targeting signal peptide sequence derived from the first 30 amino acid residues of the CIT1 gene is fused to the N-terminus of the cis-aconitine decarboxylase gene cadA to enable cis-aconitine decarboxylase to be directed to the mitochondrial matrix. Knocking out the isocitrate dehydrogenase gene IDH1 and the isocitrate lyase gene ACL1 in the genome of the aforementioned Yersinia lipolytica blocks the carbon flow branch from isocitrate to α-ketoglutarate and acetyl-CoA, thereby enhancing the accumulation of cis-aconitate. Overexpression of the citrate synthase gene CIT1 and the cis-aconitate hydratase gene ACO1 in the genome of the lipophilic yeast enhances the metabolic flux from citrate to cis-aconitate. Knocking out the alcohol dehydrogenase gene ADH1 in the genome of the aforementioned Yersinia lipophila inhibits carbon flow loss in the ethanol synthesis pathway. The NADH oxidase gene noxE, derived from Lactococcus lactis subsp. cremoris MG1363, was introduced and placed between the PGK1 promoter and the ADH1 terminator to rebalance intracellular NADH oxidation. The recombinant Yersinia lipolytica strain with high itaconic acid production achieved an itaconic acid yield of over 12 g / L after fermentation for 72 hours under the conditions of glucose as the sole carbon source, 30°C, dissolved oxygen content of 15%–25%, and pH of 5.5–6.
0.
2. The recombinant lipophilic yeast strain with high itaconic acid production according to claim 1, characterized in that, The cadA expression cassette is integrated into the rDNA multiple copy repeat region of the Yersinia lipolyticis chromosome via homologous recombination. The length of the homologous recombination fragment is 800-1000 bp, so as to achieve stable multiple copy integration and high-level expression.
3. The recombinant lipophilic yeast strain with high itaconic acid production according to claim 1, characterized in that, The mitochondrial targeting signal peptide sequence and the cis-aconitine decarboxylase gene cadA are assembled by seamless PCR, with the length of the linker region controlled within 15 to 25 nucleotides to ensure the continuity of the translation framework and maintain the cis-aconitine decarboxylase activity at no less than 95% of the original enzyme activity.
4. The recombinant lipophilic yeast strain with high itaconic acid production according to claim 1, characterized in that, The knockout of the alcohol dehydrogenase gene ADH1 and the isocitrate lyase gene ACL1 is achieved using a CRISPR-Cas9 system. The CRISPR-Cas9 system includes a plasmid pCAS-KLE901 carrying a Cas9 protein-coding sequence and an sgRNA expression cassette. The sgRNA target sequences are located in exon 2 and exon 3 of the alcohol dehydrogenase gene ADH1, respectively, with a knockout efficiency of not less than 80%.
5. The recombinant lipophilic yeast strain with high itaconic acid production according to claim 1, characterized in that, The NADH oxidase gene noxE was integrated into the URA3 site of the Yersinia lipolytica chromosome. The expression level after integration was detected by real-time quantitative PCR, and the relative transcription level was not less than 2.5 times that of the reference isocitrate lyase gene ACL1.
6. A method for constructing a recombinant lipophilic yeast strain that produces high itaconic acid, characterized in that, The method for constructing the recombinant lipophilic yeast strain that produces high itaconic acid includes the following steps: S1. Wild-type strain of Yersinia lipolytica was selected as the starting strain; S2. Knock out the IDH1 and ACL1 genes respectively using the CRISPR-Cas9 system; S3. Construct the cadA expression cassette and integrate it into the rDNA site via homologous recombination; S4. The mitochondrial targeting signal peptide sequence was ligated to the N-terminus of the cadA gene using fusion PCR technology; S5. Construct a noxE overexpression vector and integrate it into the URA3 site; S6. A recombinant Yersinia lipolytica strain producing high itaconic acid was obtained through G418 resistance screening, PCR verification, and sequencing confirmation.
7. The method for constructing a recombinant lipophilic yeast strain with high itaconic acid production according to claim 6, characterized in that, The homologous recombination integration uses a two-arm recombination fragment, with the left arm and right arm being 900bp and 850bp in length, respectively. The integrated fragment is amplified and purified by PCR and transformed into Yeast lipolyticis cells in a linear form, with an integration success rate of no less than 85%.
8. The method for constructing a recombinant lipophilic yeast strain with high itaconic acid production according to claim 1, characterized in that, A semi-continuous fermentation process with a carbon-to-nitrogen ratio of 20:1 was adopted in the fermentation culture. When the residual sugar concentration dropped to below 5 g / L, sugar was added to maintain the glucose concentration in the culture medium in the range of 20-30 g / L, so that the final yield of itaconic acid could be increased to more than 35 g / L and the conversion rate could not be less than 0.32 g / g.
9. The method for constructing a recombinant lipophilic yeast strain with high itaconic acid production according to claim 1, characterized in that, Furthermore, overexpression of the citrate transmembrane carrier protein gene CIT2 in the genome of Yersinia lipolytica increased the transmembrane transport rate of cis-aconitine by 1.8 times compared with the unmodified strain, thereby enhancing the extracellular accumulation rate of itaconic acid.
10. Application of a recombinant lipophilic yeast strain that produces high itaconic acid, characterized in that, The aforementioned high-yield itaconic acid recombinant Yersinia lipolytica strain was used for aerobic fermentation in a synthetic medium containing glucose, nitrogen source, phosphate, and trace elements. When the culture temperature was 28–32℃, the dissolved oxygen mass fraction was 15%–25%, and the stirring speed was 400–600 rpm, the final itaconic acid yield in the fermentation system reached 35–40 g / L. After filtration, acidification, crystallization, and drying, the itaconic acid obtained had a purity ≥99.5%, making it suitable for the preparation of biodegradable polymers, plasticizers, and pharmaceutical intermediates.