Yarrowia lipolytica engineering bacterium with optimized metabolic pathway and itaconic acid production process thereof

Abstract

The invention relates to the technical field of biology, in particular to yarrowia lipolytica engineering bacteria with an optimized metabolic pathway and an itaconic acid production process of the yarrowia lipolytica engineering bacteria. The engineering bacterium is constructed by taking yarrowia lipolytica as a host strain through genome multi-site modification, and the optimization of a metabolic pathway of the engineering bacterium comprises the following steps: introducing itaconic acid to synthesize a key enzyme gene: integrating a cis-aconitic acid decarboxylase gene CadA (SEQIDNO: 1) derived from Aspergillus in a strain genome, driving high-level expression through a strong promoter TEF1 and a terminator XPR2, and carrying out high-level expression through a high-level expression vector; and catalyzing the decarboxylation of intracellular cis-aconitic acid to generate itaconic acid. The metabolic bottleneck is obviously reduced, and the carbon flow continuity in the pathway is improved, so that the synthesis rate and the product cumulant of itaconic acid are effectively improved, and the problem of low acid production efficiency in a single gene expression system is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Yarrowia lipolytica engineering strain with optimized metabolic pathway and its production process of itaconic acid. BACKGROUND

[0002] Itaconic acid is a bio-based organic acid with important industrial value, widely used in biodegradable plastics, synthetic resins, coatings, pharmaceutical intermediates, and bio-based polymers. As a renewable bio-chemical raw material, itaconic acid has two reactive groups of double bond and carboxyl in its molecular structure, which can be polymerized, added or esterified through various chemical routes, and is considered an important platform compound for the transformation of petroleum-based chemicals to bio-based chemicals.

[0003] Currently, the production of itaconic acid in industry mainly uses Aspergillus terreus as the fermentation strain. However, Aspergillus terreus has complex mycelial morphology, limited oxygen transfer, high fermentation system viscosity, and significant intracellular metabolic inhibition of acid production, which leads to long fermentation period, unstable yield, and difficult downstream separation of itaconic acid. In addition, Aspergillus terreus strains are sensitive to environmental conditions, making it difficult to achieve continuous and high-density fermentation, which is not conducive to industrialization.

[0004] Yarrowia lipolytica is a non-traditional oil metabolism yeast with developed fatty acid decomposition and organic acid synthesis pathways, strong acid tolerance, high oxygen transfer efficiency, and excellent exogenous gene expression ability, making it a potential host for organic acid production. However, the natural Yarrowia lipolytica does not have a complete metabolic pathway for synthesizing itaconic acid, and its endogenous citric acid metabolism mainly flows to lipid synthesis or energy metabolism, lacking key enzyme activities such as cis-aconitate decarboxylase. Therefore, only by heterologous expression of a single itaconic acid synthesis gene cannot achieve ideal yield.

[0005] In recent years, the development of metabolic engineering and systems biology has made it possible to reconstruct microbial metabolic pathways. By integrating heterologous genes, blocking competing pathways, optimizing carbon and nitrogen metabolism, and enhancing energy and reducing power supply, carbon flow can be redirected in the host strain towards the target product. For the synthesis of itaconic acid, a complete citric acid-cis-aconitic acid-itaconic acid metabolic pathway needs to be constructed in the host strain, and the balance of coenzymes and carbon supply needs to be coordinated to improve the conversion efficiency of intermediate metabolites.

[0006] However, the existing disclosed modification methods still have the following shortcomings: most studies only introduce a single heterologous gene (such as cadA), and the upstream precursor synthesis and downstream conversion steps are not systematically optimized, resulting in prominent metabolic bottlenecks; the balance of intracellular reducing power (NADPH / NADH) and oxidative metabolism load are not fully considered, limiting the fermentation flux; the fermentation process control (pH, dissolved oxygen, feeding strategy) lacks coupling optimization with metabolic regulation, resulting in large differences in acid production between different batches; and there is a lack of blocking of competing pathways and auxiliary factor cycle regulation measures, which disperses the carbon flow of the product and reduces the utilization rate. SUMMARY

[0007] To solve the above-mentioned defects in the prior art, the present application provides a Yarrowia lipolytica engineering strain with optimized metabolic pathways and an itaconic acid production process, which is used for high-precision, long-term fine storm surge risk assessment and early warning.

[0008] The present application provides a Yarrowia lipolytica engineering strain with optimized metabolic pathways, which takes Yarrowia lipolytica as the host strain and is obtained through genome multi-site modification and construction, and the optimization of the metabolic pathways of the engineering strain includes the following contents: Introducing itaconic acid synthesis key enzyme gene: integrating the aconitate decarboxylase gene CadA (SEQ ID NO: 1) derived from Aspergillus terreus into the genome of the strain, and driving high-level expression through the strong promoter TEF1 and the terminator XPR2, to catalyze the decarboxylation of intracellular aconitate to generate itaconic acid; Enhancing the supply of precursors of the citric acid cycle: overexpressing the endogenous CIT1 gene (citrate synthase), the ACO1 gene (aconitate hydratase), and the IDH1 gene (isocitrate dehydrogenase) to increase the metabolic flux of acetyl coenzyme A to the aconitate path by 30-50%; Blocking competing metabolic branches: using the CRISPR-Cas9 system to knock out the ICL1 (isocitrate lyase) and MLS1 (malate synthase) genes related to the glyoxylate cycle to reduce carbon flow diversion; and knocking out the CIT2 gene to prevent acetyl coenzyme A from being consumed in the peroxide body pathway; Enhancing NADPH supply and redox balance: overexpressing the ZWF1 (glucose-6-phosphate dehydrogenase) and GND1 (6-phosphogluconate dehydrogenase) genes to promote the activity of the pentose phosphate pathway and increase the intracellular NADPH / NADP + ratio to ≥1.5 to support energy metabolism; Promoting extracellular secretion of products: fusing the Yarrowia lipolytica signal peptide sequence SP1 (SEQ ID NO: 2) to the N terminus of the CadA gene to guide extracellular secretion through the endoplasmic reticulum secretion pathway, reducing intracellular accumulation and product inhibition; Construction of stable genetic engineering strain: through the insertion of double copies of the integrated vector pYL-CadA-ZWF1 at the chromosomal rDNA site, the stability of expression is ensured, and the transcription level of CadA is verified by PCR and qRT-PCR to be increased by about 8 times; Screening of high-yield mutant strains: after UV mutagenesis combined with nitrosoguanidine (NTG) mutagenesis, screening is performed on CaCO3-containing plates, and strains with an increase of ≥40% in transparent circle diameter under the same culture conditions are selected as high-yield strains.

[0009] Preferably, the genotype of the strain is Y. lipolytica ΔCIT2 ΔICL1 ΔMLS1 ::(TEF1p-CadA-SP1-XPR2t), and the whole genome insertion is verified by Illumina sequencing to confirm that the recombination ratio of the insertion site sequence to the host genome homologous region is greater than 98%.

[0010] Preferably, the engineering bacteria can efficiently convert glucose into itaconic acid in a liquid fermentation medium, and the medium comprises the following components (per liter): Glucose 100-120 g, (NH4)2SO4 3.0 g, KH2PO4 1.5 g, MgSO4·7H2O 1.0 g, FeSO4·7H2O 0.03 g, MnSO4·H2O 0.02 g, CaCO3 5.0 g (buffer), trace element solution (containing Zn 2+ , Cu 2+ , Co 2+ , Mo 6+ each 0.1-0.5 mg / L) 5 mL, initial pH 6.0±0.1.

[0011] An itaconic acid production process of an engineering bacteria, characterized in that it comprises the following steps: S1, seed preparation: 28°C under YPD medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L) is shaken for 16 hours, the rotation speed is 180 rpm, and after the OD 600 3.0-4.0, it is used as seed liquid; S2, fermentation start: according to 10% (v / v) inoculation amount, it is connected into a 5L tank (effective volume 3L), the fermentation temperature is controlled at 30°C, the initial pH is 6.0, the aeration amount is 1.2 vvm, and the stirring speed is 400 rpm; S3, carbon source and nitrogen source regulation: after 24 hours of fermentation, a batch feeding mode is adopted, 50% (w / v) glucose solution is added, and the residual sugar is maintained at 30-40 g / L; at the same time, ammonia water is slowly supplemented to maintain the pH stability; When the dissolved oxygen (DO) decreases to 30%, the stirring speed is increased to 600 rpm; S4, metabolic induction and enhancement: after 48 hours of fermentation, sodium citrate 3 g / L is added to the system as a metabolic flow guiding regulator, while vitamin B10.2 mg / L, vitamin B60.1 mg / L and CoCl20.05 mg / L are added to enhance the activity of CadA; An intermittent oxygen supply strategy is adopted: pure oxygen is supplied for 2 minutes every 4 hours, so that the dissolved oxygen instantaneously rises to 60%, thereby stimulating the efficiency of oxidative phosphorylation; S5, product secretion and accumulation: the fermentation is continued to 72-96 hours, the dissolved oxygen is controlled to be 20-30%, and the pH is maintained at 5.6-5.9; Tween-80 0.1% (v / v) is added to improve the membrane permeability; itaconic acid is secreted to the extracellular by the CadA-SP1 pathway at this stage; S6, product extraction and purification: after the fermentation is completed, the fermentation broth is centrifuged to remove the bacterial cells, and the supernatant is subjected to decolorization with activated carbon (3% w / v) for 30 minutes; The decolorized liquid is adsorbed by a weakly acidic cation exchange resin (D152), and then eluted with 1.5 mol / L ammonia water; The eluate is concentrated to 1 / 5 of the original volume at 50°C under vacuum, and itaconic acid crystals are precipitated by ethanol crystallization, and the itaconic acid product with a purity of ≥99% is obtained by filtration and drying.

[0012] Preferably, an online monitoring system is configured in the fermenter to monitor the dissolved oxygen, pH, residual sugar and cis-aconitic acid concentration in real time, and the feed rate is controlled by PID feedback to maintain the metabolic balance in the condition interval corresponding to the maximum CadA catalytic efficiency.

[0013] Preferably, the ratio of aeration to stirring in the fermentation system is maintained in the range of 1.2 vvm:500 rpm; when DO<20%, pure oxygen pulse supply is automatically triggered, the single oxygen supply amount is 0.5-1.0 L / min, and the time length is 1-3 min.

[0014] Preferably, the process can make the final yield of itaconic acid reach ≥75 g / L, the glucose molar conversion rate ≥0.85 mol / mol, the fermentation period ≤96 hours, and the production cost reduced by about 25% compared with the control strain.

[0015] Preferably, the itaconic acid product has a main peak purity of not less than 99.2% detected by HPLC, a metal impurity content of less than 0.05%, and a crystal form of colorless needle-shaped or flaky crystalline with a melting point of 165-167°C.

[0016] Preferably, the engineering bacteria can be used for green production of bio-based monomer itaconic acid, and the product is suitable for synthesis of polyester, polyamide and biodegradable materials.

[0017] Compared with the prior art, the advantages of the present application are: (1) This invention reconstructs the complete biosynthetic pathway from citrate to itaconic acid by integrating and expressing three key enzyme genes—citA, aconitate hydratase (aco1), and aconitate decarboxylase (cadA)—into Yeast lipolytica. Through the polycistronic expression structure driven by the TEF1 strong promoter, the generation and conversion of the intermediate metabolite aconitate are carried out in a coordinated manner, significantly reducing metabolic bottlenecks and improving the continuity of carbon flow within the pathway. This effectively increases the synthesis rate and product accumulation of itaconic acid, solving the problem of low acid production efficiency in single-gene expression systems.

[0018] (2) The present invention uses CRISPR-Cas9 gene editing technology to knock out the CIT2 (peroxosomal citrate lyase) and ICL1 (isocitrate lyase) genes, blocking the loss of citrate to acetyl-CoA and gluconeogenesis pathway. This measure makes the intracellular carbon flow more concentrated on the citrate-cis-aconitate-itaconitate synthesis pathway, thereby improving substrate utilization and metabolic flux.

[0019] (3) This invention achieves its effect by overexpressing NADP. + The malate-dependent dehydrogenase (MAE1) gene enhances the intracellular NADPH supply capacity. At the same time, by optimizing the fermentation dissolved oxygen conditions and pH dynamic regulation strategy, the stability of oxidative metabolism is maintained. The above measures effectively alleviate the reducing power load generated during the large-scale synthesis of itaconic acid, improve metabolic efficiency and cell stability, and enable the engineered bacteria to still have good growth and energy production performance in a high-concentration acid-producing environment.

[0020] (4) In this invention, a batch feeding method is used to maintain a stable substrate concentration during fermentation. Combined with dissolved oxygen control of 30% to 40%, constant pH control of 6.0, and a CaCO3 buffer system, the acid inhibition effect caused by acid accumulation is effectively prevented. At the same time, Cu is introduced. 2 The combined induction and Tween 80 surfactant synergistic approach improved the stability of exogenous protein expression and membrane permeability, making the fermentation system stable and controllable. Attached Figure Description

[0021] Figure 1 This is a flowchart of a process for producing taconic acid from engineered Yeastra lipolyticis with optimized metabolic pathways, as proposed in this invention. Detailed Implementation

[0022] 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. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] Example 1 A Yarrowia lipolytica engineered strain with optimized metabolic pathways, wherein the engineered strain was constructed by multi-site genomic modification using Yarrowia lipolytica as the host strain, and the optimization of its metabolic pathways includes the following: Introducing a key enzyme gene for itaconic acid synthesis: The cis-aconitine decarboxylase gene CadA (SEQ ID NO:1) from Aspergillusterreus was integrated into the strain genome and its high-level expression was driven by the strong promoter TEF1 and terminator XPR2 to catalyze the decarboxylation of intracellular cis-aconitine to generate itaconic acid. Enhanced supply of precursors for the citrate cycle: By overexpressing endogenous CIT1 (citrate synthase), ACO1 (cis-aconitate hydratase), and IDH1 (isocitrate dehydrogenase), the metabolic flux of acetyl-CoA to the cis-aconitate pathway is increased by 30–50%; Blocking competitive metabolic pathways: The glyoxylate cycle-related genes ICL1 (isocitrate lyase) and MLS1 (malt synthase) were knocked out using the CRISPR-Cas9 system to reduce carbon diversion; at the same time, the CIT2 gene was knocked out to prevent acetyl-CoA from being consumed in the peroxisome pathway. Enhancing NADPH supply and redox balance: Overexpression of ZWF1 (glucose-6-phosphate dehydrogenase) and GND1 (6-phosphate gluconate dehydrogenase) genes promotes pentose phosphate pathway activity and increases intracellular NADPH / NADP ratio. + The ratio should be ≥1.5 to support energy metabolism; Promote extracellular secretion of products: The lipophilic yeast signal peptide sequence SP1 (SEQ ID NO:2) is fused to the N-terminus of the CadA gene to guide extracellular secretion through the endoplasmic reticulum secretion pathway, reducing intracellular accumulation and product inhibition; Constructing stable genetically engineered strains: Double copies of the integrative vector pYL-CadA-ZWF1 were inserted into the chromosomal rDNA site to ensure stable expression. PCR and qRT-PCR confirmed that the CadA transcription level was increased by about 8 times. High-yielding mutant strains were selected by using UV mutagenesis combined with nitrosoguanidine (NTG) mutagenesis and screening on CaCO3 plates. Strains that produced a ≥40% increase in the diameter of the transparent zone under the same culture conditions were selected as high-yielding strains.

[0024] The genotype of the strain is denoted as Y.lipolyticaΔCIT2ΔICL1ΔMLS1::(TEF1p-CadA-SP1-XPR2t), and its whole-genome insertion was confirmed by Illumina sequencing. The proportion of recombination between the insertion site sequence and the homologous region of the host genome was greater than 98%.

[0025] The engineered bacteria can efficiently convert glucose into itaconic acid in a liquid fermentation medium, which comprises the following components (per liter): 100–120 g glucose, 3.0 g (NH₄)₂SO₄, 1.5 g KH₂PO₄, 1.0 g MgSO₄·7H₂O, 0.03 g FeSO₄·7H₂O, 0.02 g MnSO₄·H₂O, 5.0 g CaCO₃ (buffer), and trace element solution (containing Zn). 2+ Cu 2+ Co 2+ Mo 6+ 5 mL (0.1–0.5 mg / L each), initial pH 6.0 ± 0.1.

[0026] A process for producing itaconic acid from engineered bacteria, characterized by comprising the following steps: S1. Seed preparation: Seeds were cultured in YPD medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) at 28°C with shaking for 16 hours at 180 rpm until OD... 600 3.0–4.0 was then used as seed solution; S2. Fermentation start-up: Inoculate 10% (v / v) into a 5L tank (effective volume 3L), control the fermentation temperature at 30℃, initial pH 6.0, aeration rate 1.2vvm, and stirring rate 400rpm. S3. Carbon and nitrogen source control: After 24 hours of fermentation, a batch feeding mode is adopted to add 50% (w / v) glucose solution to maintain the residual sugar at 30-40 g / L; at the same time, ammonia water is slowly added to maintain pH stability. When dissolved oxygen (DO) drops to 30%, increase the stirring speed to 600 rpm; S4. Metabolic induction and enhancement: After 48 hours of fermentation, sodium citrate 3 g / L was added to the system as a metabolic flow regulator, along with vitamin B1 0.2 mg / L, vitamin B6 0.1 mg / L and CoCl2 0.05 mg / L to enhance CadA activity. An intermittent oxygenation strategy was adopted: pure oxygen was introduced for 2 minutes every 4 hours to instantly raise the dissolved oxygen to 60% and stimulate oxidative phosphorylation efficiency. S5. Product secretion and accumulation: Continue fermentation for 72–96 hours, control dissolved oxygen at 20–30%, and maintain pH at 5.6–5.9; add Tween-800.1% (v / v) to improve membrane permeability; itaconic acid is secreted into the extracellular space via the CadA-SP1 pathway at this stage; S6. Product extraction and purification: After fermentation, the fermentation broth is centrifuged to remove the cells, and the supernatant is decolorized with activated carbon (3% w / v) for 30 minutes. The decolorizing solution was adsorbed by a weakly acidic cation exchange resin (D152) and then eluted with 1.5 mol / L ammonia. The eluent was concentrated under vacuum at 50°C to 1 / 5 of its original volume, and itaconic acid crystals were precipitated by ethanol crystallization. After filtration and drying, itaconic acid product with a purity ≥99% was obtained.

[0027] An online monitoring system is installed in the fermenter to monitor dissolved oxygen, pH, residual sugar and cis-aconitine concentration in real time. The feeding rate is controlled by PID feedback to keep the metabolic balance within the range corresponding to the maximum CadA catalytic efficiency.

[0028] The aeration-to-stirring ratio of the fermentation system is maintained within the range of 1.2vvm:500rpm; when DO < 20%, pure oxygen pulse supply is automatically triggered, with a single oxygen supply of 0.5–1.0L / min and a duration of 1–3min.

[0029] The process can achieve a final yield of itaconic acid of ≥75g / L, a glucose molar conversion rate of ≥0.85mol / mol, a fermentation cycle of ≤96 hours, and a production cost that is about 25% lower than that of the control strain.

[0030] The itaconic acid product, as determined by HPLC, has a main peak purity of no less than 99.2%, a metal impurity content of less than 0.05%, and a colorless needle-like or plate-like crystal morphology with a melting point of 165–167℃.

[0031] The engineered bacteria can be used for the green production of the bio-based monomer itaconic acid, and the product is suitable for the synthesis of polyesters, polyamides and biodegradable materials.

[0032] Example 2 I. Construction of engineered strains Host strain selection Yarrowia lipolytica Po1f strain was selected as the host strain. It has the characteristics of complete lipid metabolism pathway, high level of heterologous gene expression, and tolerance to high concentrations of organic acids, making it suitable for organic acid fermentation production.

[0033] Acquisition of target genes and vector construction The cadA gene (encoding aconitine decarboxylase) was amplified from the Aspergillus terreus genome, and EcoRI and XbaI restriction enzyme sites were added using PCR primer design.

[0034] The amplified cadA fragment was inserted into the downstream region of the TEF1 promoter in the pINA1312 plasmid to construct the vector pINA1312-TEF1-cadA.

[0035] Meanwhile, to increase the supply of precursors for cis-aconitine production, the genes citA (citate synthase) and aco1 (cis-aconitine hydratase) were amplified from Yersinia lipolytica and co-expressed in the vector pINA1312 through a bicistronic structure, resulting in the pINA1312-citA-aco1-cadA complex expression vector.

[0036] Carbon flow optimization regulation of gene modification The CRISPR-Cas9 system was used to knock out the CIT2 (peroxisome citrate lyase) and ICL1 (isocitrate lyase) genes to block the competitive metabolism of itaconic acid toward acetyl-CoA.

[0037] Overexpression of NADP + The malate dehydrogenase-dependent (MAE1) gene is used to increase intracellular NADPH levels and promote reducing power balance.

[0038] Positive clones were screened by integrating the pINA1312-citA-aco1-cadA vector into the rDNA site of the genome through integrative editing.

[0039] Screening and validation of engineered strains Initial screening was performed using YNB plates containing 100 mg / L nicotinic acid to inhibit wild-type growth.

[0040] Transformants with normal growth were selected for PCR verification and RT-qPCR detection of the target gene transcription level; intracellular citrate dehydrogenase and cis-aconitate decarboxylase activities were measured to confirm upregulated expression.

[0041] The resulting engineered strain was named Yarrowia lipolytica ITA01.

[0042] II. Fermentation Culture Conditions Seed culture The engineered strain was inoculated into 50 mL of YPD medium (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) and cultured at 30 °C and 200 rpm for 24 h.

[0043] After collecting the bacterial cells, centrifuging them, and resuspending them in the fermentation basal medium for later use.

[0044] Fermentation medium composition (g / L) Component content 100g glucose (NH4)2SO4 5 KH2PO4 2 MgSO4·7H2O 1 CaCO3 30 FeSO4·7H2O 0.05 ZnSO4·7H2O 0.05 Vitamin mixture 10 mL / L Fermentation conditions Fermentation volume: 2 L of culture medium in a 3 L container; Temperature: 30℃; pH: 6.0 (automatically adjusted, alkaline solution is 10% NaOH); Dissolved oxygen control: maintain at 30%–40%; aeration rate 1 vvm, stirring speed 500–800 rpm; Incubation time: 120 h; Glucose was fed in batches, and when the residual glucose was <10 g / L, it was added to 100 g / L, for a total of 3 feedings.

[0045] Induction and reinforcement measures After fermentation for 36 hours, add 0.1 mM Cu. 2+ Ions promote the stability of TEF1 promoter expression; Add 1 g / L sodium citrate at 48 h as an intermediate metabolic flux regulator; Add 0.05% Tween 80 after 72 hours to enhance membrane permeability and accelerate itaconic acid secretion.

[0046] III. Extraction and Purification of Itaconic Acid Fermentation broth treatment After fermentation, the cells are removed by centrifugation; the supernatant is then filtered through an ultrafiltration membrane (10 kDa molecular weight cutoff) to remove protein impurities.

[0047] Adsorption separation The fraction containing itaconic acid was collected by eluting with 0.1 M NaOH solution using an anion exchange resin (Amberlite IRA-400).

[0048] Crystallization and purification The eluent was concentrated to 1 / 5 of its original volume, the pH was adjusted to 2.0, and the solution was allowed to stand at 4°C for 12 h to crystallize. After vacuum filtration and drying, itaconic acid crystals were obtained.

[0049] IV. Analysis and Results Product testing The concentration of itaconic acid was determined by HPLC (C18 column, mobile phase 0.01 M phosphoric acid solution: acetonitrile = 95:5, detection wavelength 210 nm).

[0050] Fermentation results The final itaconic acid yield was 72.3 g / L; The conversion rate was 0.63 g / g glucose; The purity reached 99.2% after crystallization purification.

[0051] Comparative analysis Compared with the unmodified Yersinia lipolytica, the ITA01 strain produced approximately 4.5 times more acid and had a 20% shorter fermentation cycle, indicating that metabolic pathway optimization and carbon flow redirection significantly improved itaconic acid synthesis efficiency.

[0052] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0053] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A Yersinia lipase engineered strain with optimized metabolic pathways, characterized in that: The engineered bacteria were constructed using Yarrowia lipolytica as the host strain and through multi-site genomic modification, with optimized metabolic pathways. Includes the following: Introducing a key enzyme gene for itaconic acid synthesis: The cis-aconitine decarboxylase gene CadA (SEQ ID NO:1) from Aspergillusterreus was integrated into the strain genome and its high-level expression was driven by the strong promoter TEF1 and terminator XPR2 to catalyze the decarboxylation of intracellular cis-aconitine to generate itaconic acid. Enhanced supply of precursors for the citrate cycle: By overexpressing endogenous CIT1 (citrate synthase), ACO1 (cis-aconitate hydratase), and IDH1 (isocitrate dehydrogenase), the metabolic flux of acetyl-CoA to the cis-aconitate pathway is increased by 30–50%; Blocking competitive metabolic pathways: The glyoxylate cycle-related genes ICL1 (isocitrate lyase) and MLS1 (malt synthase) were knocked out using the CRISPR-Cas9 system to reduce carbon diversion; at the same time, the CIT2 gene was knocked out to prevent acetyl-CoA from being consumed in the peroxisome pathway. Enhancing NADPH supply and redox balance: Overexpression of ZWF1 (glucose-6-phosphate dehydrogenase) and GND1 (6-phosphate gluconate dehydrogenase) genes promotes pentose phosphate pathway activity and increases intracellular NADPH / NADP ratio. + The ratio should be ≥1.5 to support energy metabolism; Promote extracellular secretion of products: The lipophilic yeast signal peptide sequence SP1 (SEQ ID NO:2) is fused to the N-terminus of the CadA gene to guide extracellular secretion through the endoplasmic reticulum secretion pathway, reducing intracellular accumulation and product inhibition; Constructing stable genetically engineered strains: Double copies of the integrative vector pYL-CadA-ZWF1 were inserted into the chromosomal rDNA site to ensure stable expression. PCR and qRT-PCR confirmed that the CadA transcription level was increased by about 8 times. High-yielding mutant strains were selected by using UV mutagenesis combined with nitrosoguanidine (NTG) mutagenesis and screening on CaCO3 plates. Strains that produced a ≥40% increase in the diameter of the transparent zone under the same culture conditions were selected as high-yielding strains.

2. The engineered Yersinia lipase strain with optimized metabolic pathways according to claim 1, characterized in that, The genotype of the strain is denoted as Y.lipolyticaΔCIT2ΔICL1ΔMLS1::(TEF1p-CadA-SP1-XPR2t), and its whole-genome insertion was confirmed by Illumina sequencing. The proportion of recombination between the insertion site sequence and the homologous region of the host genome was greater than 98%.

3. A *Yarrowia lipophila* engineered strain with optimized metabolic pathways according to claim 1 or 2, characterized in that, The engineered bacteria can efficiently convert glucose into itaconic acid in a liquid fermentation medium, which comprises the following components (per liter): 100–120 g glucose, 3.0 g (NH₄)₂SO₄, 1.5 g KH₂PO₄, 1.0 g MgSO₄·7H₂O, 0.03 g FeSO₄·7H₂O, 0.02 g MnSO₄·H₂O, 5.0 g CaCO₃ (buffer), and trace element solution (containing Zn). 2+ Cu 2+ Co 2+ Mo 6+ 5 mL (0.1–0.5 mg / L each), initial pH 6.0 ± 0.

1.

4. A process for producing itaconic acid from *Yersinia lipolyticis* with optimized metabolic pathways as described in any one of claims 1–3, characterized in that, Includes the following steps: S1. Seed preparation: Seeds were cultured in YPD medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) at 28°C with shaking for 16 hours at 180 rpm until OD... 600 3.0–4.0 was then used as seed solution; S2. Fermentation start-up: Inoculate 10% (v / v) into a 5L tank (effective volume 3L), control the fermentation temperature at 30℃, initial pH 6.0, aeration rate 1.2vvm, and stirring rate 400rpm. S3. Carbon and nitrogen source control: After 24 hours of fermentation, a batch feeding mode is adopted to add 50% (w / v) glucose solution to maintain the residual sugar at 30-40 g / L; at the same time, ammonia water is slowly added to maintain pH stability. When dissolved oxygen (DO) drops to 30%, increase the stirring speed to 600 rpm; S4. Metabolic induction and enhancement: After 48 hours of fermentation, sodium citrate 3 g / L was added to the system as a metabolic flow regulator, along with vitamin B1 0.2 mg / L, vitamin B6 0.1 mg / L and CoCl2 0.05 mg / L to enhance CadA activity. An intermittent oxygenation strategy was adopted: pure oxygen was introduced for 2 minutes every 4 hours to instantly raise the dissolved oxygen to 60% and stimulate oxidative phosphorylation efficiency. S5. Product secretion and accumulation: Continue fermentation for 72–96 hours, control dissolved oxygen at 20–30%, and maintain pH at 5.6–5.9; add Tween-800.1% (v / v) to improve membrane permeability; itaconic acid is secreted into the extracellular space via the CadA-SP1 pathway at this stage; S6. Product extraction and purification: After fermentation, the fermentation broth is centrifuged to remove the cells, and the supernatant is decolorized with activated carbon (3% w / v) for 30 minutes. The decolorizing solution was adsorbed by a weakly acidic cation exchange resin (D152) and then eluted with 1.5 mol / L ammonia. The eluent was concentrated under vacuum at 50°C to 1 / 5 of its original volume, and itaconic acid crystals were precipitated by ethanol crystallization. After filtration and drying, itaconic acid product with a purity ≥99% was obtained.

5. The itaconic acid production process of Yersinia lipophila with optimized metabolic pathways according to claim 4, characterized in that, An online monitoring system is installed in the fermenter to monitor dissolved oxygen, pH, residual sugar and cis-aconitine concentration in real time. The feeding rate is controlled by PID feedback to keep the metabolic balance within the range corresponding to the maximum CadA catalytic efficiency.

6. The itaconic acid production process of Yersinia lipolyticis with optimized metabolic pathways according to claim 5, characterized in that, The aeration-to-stirring ratio of the fermentation system is maintained within the range of 1.2vvm:500rpm; when DO < 20%, pure oxygen pulse supply is automatically triggered, with a single oxygen supply of 0.5–1.0L / min and a duration of 1–3min.

7. The itaconic acid production process of Yersinia lipolyticis with optimized metabolic pathways according to claim 6, characterized in that, The process can achieve a final yield of itaconic acid of ≥75g / L, a glucose molar conversion rate of ≥0.85mol / mol, a fermentation cycle of ≤96 hours, and a production cost that is about 25% lower than that of the control strain.

8. The itaconic acid production process of Yersinia lipophila with optimized metabolic pathways according to claim 7, characterized in that, The itaconic acid product, as determined by HPLC, has a main peak purity of no less than 99.2%, a metal impurity content of less than 0.05%, and a colorless needle-like or plate-like crystal morphology with a melting point of 165–167℃.

9. The itaconic acid production process of Yersinia lipolyticis with optimized metabolic pathways according to claim 8, characterized in that, The engineered bacteria can be used for the green production of the bio-based monomer itaconic acid, and the product is suitable for the synthesis of polyesters, polyamides and biodegradable materials.

Images