Yarrowia lipolytica engineering strain for high yield astaxanthin with oleic acid and construction method and application thereof

Abstract

The application discloses a Yarrowia lipolytica engineering bacterium for high-yield astaxanthin by using oleic acid and a construction method and application thereof. The Yarrowia lipolytica engineering bacterium is constructed by integrating acyl-CoA oxidase gene pox2, acyl-CoA oxidase gene pox3, multifunctional enzyme gene mfe1 and beta-isopropyl malate dehydrogenase gene leu2 in a high-yield astaxanthin mutant strain KY7 and knocking out a mycelium formation key gene mhy1, and the Yarrowia lipolytica engineering bacterium can efficiently produce astaxanthin by using oleic acid through strengthening a beta-oxidation pathway and morphological engineering. The Yarrowia lipolytica engineering bacterium can produce 3145.29 mg / L astaxanthin in a 5L fermenter for 168 hours, and the production rate is 18.72 mg / L·h, and has an important application prospect in the field of astaxanthin biosynthesis.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and synthetic biology, specifically relating to a lipophilic yeast engineered strain that utilizes oleic acid to produce high levels of astaxanthin, its construction method, and its application. Background Technology

[0002] Astaxanthin is a ketocarotenoid with extremely strong antioxidant activity and is widely used in medicine, health products, and aquaculture. The heterologous synthesis of astaxanthin using metabolic engineering of Yarrowia lipolytica has become a research hotspot due to its advantages such as rapid growth, ample supply of acetyl-CoA and NADPH, and abundant intracellular lipid droplets that can effectively store hydrophobic products.

[0003] The applicant's research group has established a complete heterologous astaxanthin synthesis pathway in Yarrowia lipolytica. By integrating the key β-carotene hydroxylase and ketolase genes CrtZ and CrtW through multiple copies, they constructed an engineered strain of Yarrowia lipolytica that produces astaxanthin (Wang DN, Feng J., Yu CX, et al. Integrated pathway engineering and transcriptome analysis for improved astaxanthin biosynthesis in Yarrowia lipolytica. Synth. Syst. Biotechnol. 2022, 7:1133-1141). They further verified that a mutant strain with high astaxanthin production can be obtained by using ambient pressure room temperature plasma ARTP mutagenesis combined with diphenylamine (DPA) screening strategy (Wang DN, Yu CX, Feng J., et al. Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica. Yeast 2024, 41(6):369-378). However, the aforementioned engineered and mutant strains still have limitations in further industrial applications: To maintain intracellular lipids as a reservoir for terpenoids, traditional strategies often involve downregulating the β-oxidation pathway. This prevents the strains from effectively utilizing lipids or fatty acids, blocking the metabolic flux of fatty acid degradation to acetyl-CoA (a key precursor in the mevalonate pathway), and thus limiting the synthetic flux of terpenoids. Furthermore, *Yarrowia lipolytica* readily forms mycelial states during fermentation, leading to increased fermentation broth viscosity and significantly impacting fermentation performance. Summary of the Invention

[0004] The purpose of this invention is to provide an engineered *Yersinia lipolytica* strain that produces high levels of astaxanthin from oleic acid, along with its construction method and applications. Based on an *Yersinia lipolytica* strain obtained through ARTP mutagenesis, this invention achieves efficient astaxanthin biosynthesis by enhancing the β-oxidation pathway, modifying the morphology, and optimizing the fermentation process.

[0005] The technical solution for achieving the objective of this invention is as follows:

[0006] A genetically stable and high-yield astaxanthin-producing mutant strain, Yarrowia lipolytica KY7, was deposited on April 7, 2026, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026616, located at Wuhan University, Wuhan, China.

[0007] The engineered *Yersinia lipolytica* strain that produces high levels of astaxanthin from oleic acid is an engineered strain obtained by any of the following genetic modification methods, starting with the *Yersinia lipolytica* mutant strain KY7:

[0008] (1) Integrating acyl-CoA oxidase 2 gene pox2, acyl-CoA oxidase 3 gene pox3, multifunctional enzyme 1 gene mfe1 and β-isopropylmalate dehydrogenase gene leu2;

[0009] (2) Integrate the acyl-CoA oxidase 2 gene pox2, the acyl-CoA oxidase 3 gene pox3, the multifunctional enzyme 1 gene mfe1, and the β-isopropylmalate dehydrogenase gene leu2, and knock out the morphological regulatory factor gene mhy1.

[0010] The genes pox2, pox3, mfe1, leu2, and myy1 described in this invention are all endogenous genes of *Yarrowia lipolytica*. Specifically, the nucleotide sequence of gene pox2 is shown in SEQ ID No. 1, the nucleotide sequence of gene pox3 is shown in SEQ ID No. 2, the nucleotide sequence of gene mfe1 is shown in SEQ ID No. 3, the nucleotide sequence of gene leu2 is shown in SEQ ID No. 4, and the nucleotide sequence of gene myy1 is shown in SEQ ID No. 5.

[0011] The above method for constructing the engineered Yersinia lipophila strain that utilizes oleic acid to produce high levels of astaxanthin includes the following steps:

[0012] The expression cassettes of pox2, pox3, mfe1 and leu2 of Yersinia lipolytica were assembled into a vector to construct a recombinant plasmid. After linearization, the recombinant plasmid was integrated into the genome of the starting strain KY7 to obtain the engineered Yersinia lipolytica strain KY9.

[0013] Alternatively, based on the engineered Yersinia lipolytica strain KY9, the morphological regulator myy1 can be knocked out using the CRISPR-Cas9 system to obtain the engineered Yersinia lipolytica strain KY11 that maintains the single-cell morphology of yeast.

[0014] The above-mentioned application of the engineered strain of Yersinia lipophila that utilizes oleic acid to produce high levels of astaxanthin in the production of astaxanthin.

[0015] Furthermore, the specific application method is as follows: the above-mentioned engineered yeast strain that utilizes oleic acid to produce high astaxanthin is inoculated into a fermentation medium for fermentation to produce astaxanthin.

[0016] The fermentation medium described in this invention is the YPD medium or twice the concentration of YPD medium commonly used in the culture of Yersinia lipophila. The YPD medium consists of 2% glucose, 2% peptone and 2% yeast extract.

[0017] Furthermore, the fermentation temperature was controlled at 26℃, and 20 g / L oleic acid (OA) was added as a supplementary carbon source during fermentation for 24~48 h.

[0018] Furthermore, ferrous ions (Fe3+) were added to the fermentation medium at a final concentration of 0.1–1.0 mmol / L. 2+ The preferred concentration is 0.1 mmol / L.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) The present invention uses the high-yielding mutant strain KY7, which was screened by ARTP mutagenesis and confirmed to be genetically stable through continuous passage, as the starting strain. First, the acyl-CoA oxidase genes pox2 and pox3, the multifunctional enzyme gene mfe1, and the β-isopropylmalate dehydrogenase gene leu2 are integrated into the starting strain to open up the β-oxidation pathway, enabling it to utilize fatty acids to generate acetyl-CoA. Second, the morphological regulation target mhy1 is knocked out to maintain the oval yeast morphology to improve the dissolved oxygen level, thus constructing a high-yielding astaxanthin-producing Yersinia lipolytica engineered strain.

[0021] (2) This invention optimizes the fermentation process by controlling the temperature, feeding with oleic acid, and adding ferrous ions, thereby enabling astaxanthin to be efficiently biosynthesized from oleic acid. The engineered Yeast KY11 strain of this invention was fermented in a 5L fermenter for 168 h, and the astaxanthin yield reached 3145.29 mg / L. Attached Figure Description

[0022] Figure 1 The astaxanthin yields of M2 after 3 passages and KY7 after 5 passages were compared.

[0023] Figure 2 The morphology of strains KY9 and KY11 under a microscope.

[0024] Figure 3 The study investigated the astaxanthin yield of KY7 and KY9 under YPD culture conditions, and the effect of fermentation temperature (26℃, 28℃, 30℃) on the astaxanthin yield of KY9 strain.

[0025] Figure 4The effect of oleic acid addition time (0h, 24h, 48h, 72h) on astaxanthin production of strain KY9.

[0026] Figure 5 For exogenous additives (Fe) 2+ , Mn 2+ Effects of H2O2 and TritonX-100 on astaxanthin production in strain KY9.

[0027] Figure 6 OD of strain KY11 in a 5L fermenter 600 Astaxanthin production curve. Detailed Implementation

[0028] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the following embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or in accordance with the product manual.

[0029] The plasmid pINA1269 used in the following examples is described in the literature [Madzak C, Treton B, Blanchin-Roland S. Strong hybrid promoters and integrative expression / secretion vectors for quasi-constitutive expression of heterologous proteins in theireast Yarrowia lipolytica [J]. J Mol Microbiol Biotechnol, 2000, 2(2): 207-216.].

[0030] Example 1: Obtaining high-yield mutant strain KY7 by ARTP mutagenesis + DPA screening

[0031] Astaxanthin-producing *Yarrowia lipolytica* strains were prepared into bacterial suspensions, spread on metal slides, and mutagenized using an ARTP mutagenizer. The mutagenized cells were then spread on plates containing 20 mg / L diphenylamine for screening. Single clones with darker color were selected and passaged continuously to obtain genetically stable *Yarrowia lipolytica* mutant strain KY7. This mutant strain was deposited on April 7, 2026, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026616, located at Wuhan University, Wuhan, China. For details of the ARTP mutagenization and diphenylamine screening methods, please refer to the literature [Wang DN, Yu CX, Feng J., et al. Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in *Yarrowia lipolytica*. *Yeast* 2024, 41(6):369-378].

[0032] The aforementioned literature also yielded a high-yielding mutant strain M2, with an astaxanthin yield of 181 mg / L. However, after three or more generations, the astaxanthin yield of M2 remained at 130-140 mg / L. Figure 1 As shown. To verify the genetic stability of strain KY7, five passage yield tests were conducted, and the astaxanthin yield remained at around 150 mg / L. Figure 1 As shown, the M2 strain, which has been passaged multiple times, has a higher astaxanthin yield.

[0033] Example 2: Construction of Yersinia lipolyticis engineered strain KY9 with a complemented β-oxidation pathway

[0034] 1. Using the genome of the engineered Yeast lipolyticis strain Po1f as a template, three primer pairs, P1 / P2 (SEQ ID No. 6 and SEQ ID No. 7), P3 / P4 (SEQ ID No. 8 and SEQ ID No. 9) and P5 / P6 (SEQ ID No. 10 and SEQ ID No. 11), were designed to amplify expression cassettes of the genes pox2 (SEQ ID No. 12), acyl-CoA oxidase 3 gene pox3 (SEQ ID No. 13), and multifunctional enzyme 1 gene mfe1 (SEQ ID No. 14) by overlap PCR.

[0035] 2. Primer pairs P7 / P8 (SEQ ID No. 15 and SEQ ID No. 16) were designed to amplify the expression cassette fragment of the β-isopropylmalate dehydrogenase gene leu2 (SEQ ID No. 17) from the pINA1269 plasmid.

[0036] 3. The recombinant plasmid p1269_POX2_POX3_MFE1 was constructed by seamless cloning of the pox2 expression cassette (SEQ ID No. 12), pox3 expression cassette (SEQ ID No. 13), mfe1 expression cassette (SEQ ID No. 14), and leu2 expression cassette (SEQ ID No. 17).

[0037] 4. The recombinant plasmid p1269_POX2_POX3_MFE1 was linearized into the engineered strain KY7 of Yersinia lipolytica through the restriction enzyme sites PvuI and NruI, thus obtaining the engineered strain KY9 of Yersinia lipolytica.

[0038] Example 3: Construction of morphologically engineered strain KY11

[0039] 1. Primer pairs P9 and P10 (SEQ ID No. 18 and SEQ ID No. 19) were designed to amplify the sgRNA (SEQ ID No. 20) that targets the myy1 gene, and the CRISPR plasmid pCRISPRyl_MHY1 was constructed by seamless cloning.

[0040] 2. Primer pairs P11 and P12 (SEQ ID No. 21 and SEQ ID No. 22) were designed to amplify the sequence in plasmid pCRISPRyl_MHY1 other than the sequence encoding β-isopropylmalate dehydrogenase leu2 (SEQ ID No. 23). Primer pairs P13 / P14 (SEQ ID No. 24 and SEQ ID No. 25) were designed to amplify the hygromycin B phosphotransferase gene HygR (SEQ ID No. 26). The CRISPR plasmid pCRISPRyl_MHY1_HygR, which is a selection marker for hygromycin, was constructed by seamless cloning.

[0041] 3. The CRISPR plasmid pCRISPRyl_MHY1_HygR was transformed into the engineered strain KY9 of Yersinia lipophilia to obtain the engineered strain KY11 of Yersinia lipophilia.

[0042] like Figure 2 As shown, microscopic observation confirmed that KY11 maintained its oval single-cell yeast morphology even after long-term culture, without the appearance of a large number of hyphae.

[0043] Example 4: Fermentation process optimization and yield determination

[0044] To improve the astaxanthin yield of strain KY9, fermentation optimization experiments were conducted on fermentation temperature, feeding strategy, and exogenous additives. YNB medium consisted of 2% glucose, 0.67% YNB, and 2% amino acid supplement, while YPD medium consisted of 2% glucose, 2% peptone, and 2% yeast extract.

[0045] 1. Fermentation temperature optimization and yield improvement verification of KY9 compared to KY7: Strains KY7 and KY9 were inoculated into test tubes containing 2 mL of YNB medium and cultured for 2 days. Then, 100 μL of the bacterial culture was transferred to 50 mL of YNB medium and cultured for another 2 days. The initial OD was then used as the starting point for the fermentation. 600 =0.1 g was inoculated into 50 mL of YPD medium. KY7 was cultured at 30°C for 4 days, and KY9 was cultured at 26°C, 28°C, and 30°C for 4 days, respectively. After fermentation, the bacterial culture was aspirated and astaxanthin extract was obtained using DMSO and acetone. The astaxanthin extract was filtered and analyzed by HPLC using an Agilent Zorbax SB-Aq C18 column.

[0046] The results are as follows Figure 3 As shown, under YPD culture conditions at 30℃, the astaxanthin production of KY9 was higher than that of the original strain KY7, indicating that the astaxanthin synthesis capacity of the engineered strain was improved after adding pox2, pox3, mfe1, and leu2 to KY7. Furthermore, the astaxanthin production of KY9 decreased with increasing temperature, reaching its highest accumulation at 26℃, with an astaxanthin production of 252.6 mg / L.

[0047] 2. Optimization of oleic acid addition time: The engineered strain KY9 of *Yarrowia lipolytica* was inoculated into a test tube containing 2 mL of YNB medium and cultured for 2 days. Then, 100 μL of the bacterial culture was transferred to 50 mL of YNB medium and cultured for another 2 days. Afterwards, the initial OD was used as the starting point. 600 =0.1 was inoculated into 50 mL YPD medium and cultured for 7 days. Oleic acid (20 g / L) was added to the medium at 0, 24, 48, and 72 h of fermentation. After fermentation, the bacterial culture was aspirated and astaxanthin extract was obtained using DMSO and acetone. The astaxanthin extract was filtered and analyzed by HPLC using an Agilent Zorbax SB-Aq C18 column.

[0048] The results are as follows Figure 4 As shown, the feeding strategy of adding oleic acid at 24 h of fermentation can achieve the highest astaxanthin yield and is conducive to the accumulation of cell biomass. When 20 g / L of oleic acid is added at 24 h, the astaxanthin yield of KY9 is 537.9 mg / L.

[0049] 3. Screening of exogenous additives: The engineered strain KY9 of *Yarrowia lipolyticis* was inoculated into a test tube containing 2 mL of YNB medium and cultured for 2 days. 100 μL of the bacterial culture was then transferred to 50 mL of YNB medium and cultured for 2 days. The initial OD was then used as the starting material. 600 =0.1 was inoculated into 50 mL YPD medium and cultured for 7 days. Oleic acid (20 g / L) was added to the medium after 24 hours of fermentation. After fermentation, the bacterial culture was aspirated and astaxanthin was extracted using DMSO and acetone. The astaxanthin extract was filtered and analyzed by HPLC using an Agilent Zorbax SB-Aq C18 column. Different concentrations of exogenous additives were added at 0 hours of fermentation.

[0050] The results are as follows Figure 5 As shown, the addition of ferrous ions (Fe) 2+ It has a significant promoting effect on the proportion of astaxanthin. Further gradient experiments determined that the addition of Fe at a final concentration of 0.1 mmol / L had a significant promoting effect. 2+ The effect was optimal at that time, with the astaxanthin content increasing from 44.0% to 55.8%.

[0051] Example 5: Astaxanthin yield of engineered strain KY11 in a 5L fermenter

[0052] The engineered strain of *Yarrowia lipolytica* KY11 was inoculated into test tubes containing 2 mL of YNB medium and cultured for 2 days. Then, 100 μL of the bacterial culture was transferred to 50 mL of YNB medium and cultured for another 2 days. The seed culture was then inoculated into a fermenter to achieve an initial bacterial concentration of OD0.05. 600 =0.8~1, add 0.1 mmol / L Fe to the culture medium at 0 h of fermentation. 2+ Fermentation was initiated, with samples taken every 12 hours for the first 96 hours and every 24 hours thereafter. The fermentation tank contained 2 L of liquid, the fermentation medium was twice the concentration of YPD, the pH was controlled at 5.5, the agitator speed was 900 rpm, and the aeration rate was 2 vvm. Oleic acid was continuously replenished starting at 0 h by controlling the oleic acid flow rate (average flow rate approximately 3.125 mL / h) according to the DO curve. After fermentation, the bacterial culture was aspirated, and astaxanthin extract was obtained using DMSO and acetone. The astaxanthin extract was filtered and analyzed by HPLC using an Agilent Zorbax SB-Aq C18 column.

[0053] The results are as follows Figure 6 As shown, the highest astaxanthin yield of 3145.29 mg / L can be obtained by fermentation for 168 h, with a production rate of 18.72 mg / L·h.

Claims

1. A high-yield astaxanthin and genetically stable *Yersinia lipolyticis* mutant strain KY7, characterized by: The accession number is CCTCCNO: M 2026616.

2. An engineered strain of *Yersinia lipophila* that produces high levels of astaxanthin from oleic acid, characterized in that... The *Yersinia lipolyticis* engineered strain is obtained by any of the following genetic modification methods, using the *Yersinia lipolyticis* mutant strain KY7 as the starting strain according to claim 1: (1) Integrating acyl-CoA oxidase 2 gene pox2, acyl-CoA oxidase 3 gene pox3, multifunctional enzyme 1 gene mfe1 and β-isopropylmalate dehydrogenase gene leu2; (2) Integrate the acyl-CoA oxidase 2 gene pox2, the acyl-CoA oxidase 3 gene pox3, the multifunctional enzyme 1 gene mfe1, and the β-isopropylmalate dehydrogenase gene leu2, and knock out the morphological regulatory factor gene mhy1.

3. The method for constructing the engineered *Yersinia lipophila* strain that utilizes oleic acid to produce high levels of astaxanthin, as described in claim 2, is characterized in that... Includes the following steps: The expression cassettes of pox2, pox3, mfe1 and leu2 of Yersinia lipolytica were assembled into a vector to construct a recombinant plasmid. After linearization, the recombinant plasmid was integrated into the genome of the starting strain KY7 to obtain the engineered Yersinia lipolytica strain KY9. Alternatively, based on the engineered Yersinia lipolytica strain KY9, the morphological regulator myy1 can be knocked out using the CRISPR-Cas9 system to obtain the engineered Yersinia lipolytica strain KY11 that maintains the single-cell morphology of yeast.

4. The application of the engineered yeast strain of Yersinia lipophila that utilizes oleic acid to produce high levels of astaxanthin as described in claim 2 in the production of astaxanthin.

5. The application according to claim 4, characterized in that, The specific application method is as follows: the engineered yeast strain of Yersinia lipophila that utilizes oleic acid to produce astaxanthin as described in claim 2 is inoculated into a fermentation medium for fermentation to produce astaxanthin.

6. The application according to claim 5, characterized in that, The fermentation medium is YPD medium or YPD medium at twice the concentration.

7. The application according to claim 6, characterized in that, YPD medium consists of 2% glucose, 2% peptone and 2% yeast extract.

8. The application according to claim 5, characterized in that, Fermentation temperature is controlled at 26℃.

9. The application according to claim 5, characterized in that, Add 20 g / L oleic acid as a supplementary carbon source during fermentation for 24-48 h.

10. The application according to claim 5, characterized in that, Ferrous ions (Fe2+) were added to the fermentation medium at a final concentration of 0.1–1.0 mmol / L. 2+ .

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