An engineered Yarrowia lipolytica bacteria that secretes a large amount of L-astaxanthin and a method for producing astaxanthin by two-phase fermentation

Abstract

The present invention relates to an engineered Yarrowia lipolytica bacteria that secretes a large amount of L-astaxanthin, characterized in that the wild-type Yarrowia lipolytica is used as a starting strain, and four gene clusters GGS1-CarB-CarRP, PsCrtW-HpCrtZ, GGS1-CrtYB-CrtI and ACC1-DGA1 are randomly integrated into the wild-type Yarrowia lipolytica genome, and the endogenous gene SNQ2 of Yarrowia lipolytica is overexpressed and the MHY1 gene is knocked out. The engineered bacteria is named Yarrowia lipolytica YYYE112, the deposit number is CGMCC NO.30371, and the deposit date is April 17, 2024. The engineered bacteria provided by the present invention can be fermented with vegetable oil as a carbon source, and have the advantages of fast cell growth rate, high astaxanthin yield and high secretion capacity, which is conducive to large-scale production of astaxanthin and reduces the production and extraction costs of astaxanthin.

Description

Technical Field

[0001] The present invention relates to the technical field of genetically engineered bacteria, in particular to an engineered Yarrowia lipolytica bacterium capable of secreting a large amount of L-astaxanthin and a method for producing astaxanthin by two-phase fermentation. Background Art

[0002] Astaxanthin is a terpene unsaturated compound and a keto-type secondary carotenoid with a molecular formula of C 40 H 52 O4, a highly valuable carotenoid, is widely used in food, animal feed, health products, cosmetics and pharmaceutical industries due to its strong antioxidant, anti-inflammatory and anti-cancer activities.

[0003] Astaxanthin is classified into levorotatory, dextrorotatory, and endorotatory configurations based on optical isomerism, and these three configurations possess different antioxidant properties. Astaxanthin derived from Haematococcus pluvialis is the most antioxidant, the L-astaxanthin, but due to difficulties in cultivation and extraction, low yields, and high costs, it is difficult to scale up. Therefore, the astaxanthin currently available on the market is primarily produced through Phaffia rhodozyma fermentation and chemical synthesis. The astaxanthin produced by Phaffia rhodozyma fermentation is D-astaxanthin, while the product of chemical synthesis is a mixture of the three configurations. Astaxanthin produced by Phaffia rhodozyma fermentation and chemical synthesis is currently only used in feed.

[0004] Synthetic biology can use chassis strains such as Escherichia coli and yeast to produce astaxanthin. For example, Yarrowia lipolytica is an unconventional yeast that is widely used and has passed the General Safety Certification (GRAS) in the United States. A series of studies have confirmed that Yarrowia lipolytica has great application prospects as a production host because it can not only utilize a variety of carbon sources such as glucose and oils, but also has enough acetyl-CoA as a precursor for the biosynthesis of various terpenoid compounds. In addition, as an oil-producing yeast, it can synthesize and accumulate a large amount of lipids, which can be used to store highly hydrophobic compounds such as β-carotene (β-carotene) and astaxanthin. It also has the characteristics of fast growth and easy cultivation, which makes it very suitable for large-scale production of β-carotene or astaxanthin.

[0005] There have been many reports on genetic manipulation tools for modifying the metabolic pathways of Yarrowia lipolytica to achieve the synthesis of L-astaxanthin in Yarrowia lipolytica and increase its yield. However, since a large amount of astaxanthin is stored intracellularly, this places a burden on cell growth, which is also a problem and technical bottleneck facing the synthetic biology industry. The products of synthetic biology can be divided into intracellular and extracellular. The extraction of intracellular products usually requires cell destruction, and intracellular products are often mixed with other cellular components, requiring complex purification steps. These factors will inevitably increase the complexity and cost of the extraction process. In addition, the limited storage space inside the cell also limits the accumulation of products. Extracellular secreted products can be collected directly from the culture medium, simplifying the subsequent extraction and purification steps. Moreover, extracellular accumulation is not restricted by the intracellular space, allowing higher levels of product accumulation. Since the collection of extracellular products does not require the destruction of the cell structure, it can also be used to design a continuous fermentation production process based on this, collecting products in real time while they are secreted, effectively improving production efficiency.

[0006] To address this challenge, studies have reported using an Escherichia coli model to test the secretion effects of 19 transporters (ATP-binding cassette (ABC) transporters) from different species on zeaxanthin, canthaxanthin, β-carotene, and lycopene (from low to high hydrophobicity). The protein MsbA from Bacillus typhimurium showed the best secretion of zeaxanthin, while MsbA from Escherichia coli showed the best secretion of canthaxanthin, increasing secretion by 2.4-fold and 4.4-fold, respectively. The report also demonstrated that the secretion time of transporters increases with increasing hydrophobicity of the product (Rupak Doshi et al., 2013). Another report claims that fatty acid secretion can be enhanced by knocking out the faa1, faa2, faa3, faa4, and fat1 genes encoding acyl-CoA synthetase in Saccharomyces cerevisiae. The secreted fatty acids accounted for 50% of the detectable fatty acids, while no secreted fatty acids were detected in the parent strain (Christopher Leber, 2014). These reports indicate that it is feasible to promote the extracellular secretion of intracellular products (especially hydrophobic products) by introducing appropriate exogenous transporters into chassis strains. However, there are currently no reports or solutions for introducing exogenous transporters into specific chassis strains to promote the extracellular secretion of astaxanthin in these chassis strains. Summary of the Invention

[0007] (1) Technical issues to be resolved

[0008] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an engineered Yarrowia lipolytica that secretes a large amount of L-astaxanthin and a method for producing astaxanthin by two-phase fermentation. The engineered Yarrowia lipolytica can be fermented using vegetable oil as a carbon source, and has the advantages of fast cell growth rate, high astaxanthin yield and high secretion capacity, which is conducive to the large-scale production of astaxanthin and reduces the extraction and purification cost of astaxanthin, as well as the development of a continuous fermentation process for producing astaxanthin.

[0009] (2) Technical solution

[0010] In the first aspect, the present invention provides an engineered Yarrowia lipolytica that secretes a large amount of L-astaxanthin, which is named Yarrowia lipolytica (Yarrowia lipolytica) YYYE112, with a preservation number of CGMCC NO.30371, a preservation date of April 17, 2024, and a preservation unit of the General Microbiology Center of the China Culture Collection Administration. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] In a second aspect, the present invention provides an engineered strain of Yarrowia lipolytica that secretes a large amount of L-astaxanthin, which is obtained by randomly integrating the four gene clusters GGS1-CarB-CarRP, PsCrtW-HpCrtZ, GGS1-CrtYB-CrtI and ACC1-DGA1 into the genome of the wild-type Yarrowia lipolytica, overexpressing the endogenous gene SNQ2 of Yarrowia lipolytica and knocking out the MHY1 gene. In a third aspect, a method for constructing an engineered strain of Yarrowia lipolytica that secretes a large amount of L-astaxanthin comprises the following steps:

[0012] S1. Using overlap extension PCR, the promoter, target gene, and terminator were spliced ​​into three expression cassettes: pTEF1-GGS1-tXPR2, pGPD1-CarB-tPEX20, and pEXP1-CarRP-tLIP2. The three expression cassettes were then seamlessly connected to the linearized plasmid backbone to construct a β-carotene-producing recombinant plasmid. After digestion with NotI, the plasmid was transformed into wild-type Yarrowia lipolytica, and after screening, the β-carotene-producing engineered strain YYYE064 was obtained.

[0013] S2. Using overlap extension PCR, the promoter, target gene, and terminator were spliced ​​into a pTEFin-PsCrtW-HpCrtZ-tXPR2 expression cassette. This expression cassette was then seamlessly connected to the linearized plasmid backbone to construct an astaxanthin-producing recombinant plasmid. After digestion with NotI, the plasmid was transformed into the β-carotene-producing engineered bacterium YYYE064. After screening, the astaxanthin-producing engineered bacterium YYYE067 was obtained.

[0014] S3. Using overlap extension PCR, the promoter, target gene, and terminator were spliced ​​into three expression cassettes: pTEF1-GGS1-tXPR2, pFBA1-CrtYB-tPEX20, and pEXP1-CrtI-tLIP2. The three expression cassettes were then seamlessly connected to the linearized plasmid backbone to construct a recombinant plasmid for high astaxanthin production. After digestion with NotI, the plasmid was transformed into the astaxanthin-producing engineered bacterium YYYE067. After screening, the high-astaxanthin-producing engineered bacterium YYYE090 was obtained.

[0015] S4. Using overlap extension PCR, the promoter, target gene, and terminator were spliced ​​into two expression cassettes, pTEFin-ACC1-tXPR2 and pEXP1-DGA1-tLIP1. The two expression cassettes were then assembled with the linearized plasmid backbone by seamless connection to construct a recombinant plasmid with higher astaxanthin production. After digestion with NotI, the recombinant plasmid was transformed into the high-astaxanthin-producing engineered bacterium YYYE090. After screening, the engineered bacterium YYYE097 with higher astaxanthin production was obtained.

[0016] S5. Using PCR technology, the two expression cassettes SCR1'tRNAGly-sgRNA-tMIG1 and pTEFin-Cas9-tCYC1 were amplified from the plasmid respectively. Then, the two expression cassettes were assembled with the linearized plasmid backbone by seamless connection to construct a recombinant plasmid of an astaxanthin-producing engineered bacterium that did not produce hyphae or pseudohyphae. The recombinant plasmid was transformed into the engineered bacterium YYYE097 with higher astaxanthin production to knock out the MHY1 gene. After screening, the astaxanthin-producing engineered bacterium YYYE104 that did not produce hyphae or pseudohyphae was obtained; wherein, the sgRNA sequence was 5'-ggagatggcgagataacgga-3';

[0017] S6. Using overlap extension PCR technology, the promoter, target gene and terminator were spliced ​​into the pTEFin-SNQ2-tXPR2 expression cassette, and then the expression cassette was assembled with the linearized plasmid backbone by seamless connection to construct a recombinant plasmid that promotes astaxanthin secretion; after treatment with NotI enzyme digestion, it was transformed into the astaxanthin-producing engineered bacterium YYYE104, and after screening, the engineered bacterium YYYE112 with enhanced astaxanthin secretion was obtained.

[0018] In a fourth aspect, the present invention provides a method for producing astaxanthin based on two-phase fermentation of an engineered Yarrowia lipolytica strain, wherein the engineered Yarrowia lipolytica strain is the engineered Yarrowia lipolytica strain in the above embodiment.

[0019] Preferably, the initial culture medium for two-phase fermentation is composed of: 30-100 g / yeast powder, 20-120 g / L peptone, and 5%-30% vegetable oil (v / v); after inoculation, glucose is used as a carbon source to supplement to 150-300 g / L; ammonium chloride is used as a nitrogen source to supplement to 5-25 g / L; the fermentation cycle is 115-125 h, and glucose is added during the fermentation to maintain the glucose concentration at 120-150 g / L. The feed is reduced before the tank is lowered until the fermentation is completed, and astaxanthin is obtained from the fermented oil phase or the bacteria.

[0020] (3) Beneficial effects

[0021] (1) In the process of genetically engineering Yarrowia lipolytica, the present invention integrates the gene clusters GGS1-CarB-CarRP, PsCrtW-HpCrtZ, GGS1-CrtYB-CrtI and ACC1-DGA1 into the Yarrowia lipolytica genome by random integration, thereby gradually improving the ability of the engineered bacteria to produce astaxanthin, thereby enabling the engineered bacteria to achieve large-scale production of astaxanthin by fermentation; by integrating the SNQ2 gene into the Yarrowia lipolytica genome, the endogenous gene SN Overexpression of Q2 promotes the ability of the engineered bacteria to secrete astaxanthin outside the cell, thereby reducing the difficulty of extracting and purifying astaxanthin and enabling the continuous production of astaxanthin by fermentation of engineered bacteria. In addition, knocking out the MHY1 gene on the Yarrowia lipolytica genome prevents the engineered bacteria from producing hyphae or pseudohyphae, avoiding excessive viscosity of the fermentation broth during fermentation, improving the utilization rate of dissolved oxygen and the uniformity of the fermentation broth, reducing stirring energy consumption, and avoiding the effects on cell respiration and metabolic efficiency. This helps maintain stable fermentation conditions and increase fermentation density, thereby increasing astaxanthin production. In addition, hyphae-free bacteria are easier to separate and purify through physical methods such as centrifugation and filtration, reducing astaxanthin loss and increasing product yield. Among them, the GGS1 gene, ACC1 gene, DGA1 gene, and SNQ2 are all genes from Yarrowia lipolytica, which can reduce the adverse effects of exogenous genes on host cells.

[0022] Yarrowia lipolytica is a dimorphic yeast. Under the influence of environmental factors, it may transform from yeast form to hyphae or pseudohyphae form. Changes in cell morphology and characteristics will affect the success rate of culture and fermentation. The presence of hyphae or pseudohyphae will also affect the lipid synthesis of yeast, thereby affecting the storage of hydrophobic products in the cell. After the MHY1 gene is knocked out, Yarrowia lipolytica will only exist in yeast form without producing hyphae or pseudohyphae. Experiments have shown that knocking out MHY1 does not affect the growth of the strain and the astaxanthin content.

[0023] (2) After searching and comparing the SNQ2 gene on NCBI, it was found that the SNQ2 gene also exists in Yarrowia lipolytica, and its annotated function is the same as that of the SNQ2 gene reported in Saccharomyces cerevisiae. Subsequently, the gene sequence was amplified from the Yarrowia lipolytica genome, and then the SNQ2 gene was overexpressed in the astaxanthin-producing engineered bacteria constructed by the present invention, promoting the expression of ABC transporters, and secreting astaxanthin accumulated in the cells to the extracellular space. Compared with the existing method of introducing exogenous transporters to increase the secretion of target products to the extracellular space, the present invention can reduce the toxicity of the expression products of exogenous genes to host cells. The results of the expanded culture verification at the shake flask level showed that after overexpressing the SNQ2 gene, the astaxanthin secretion of the engineered bacteria YYYE112 increased by 49% compared with the previous (compared with the engineered bacteria YYYE104 before the overexpression of the SNQ2 gene modification), and after adding vegetable oil, the growth rate of the engineered bacteria YYYE112 was more than doubled. Further expanded culture verification was carried out at the fermentation tank level. After 120 hours of fermentation with the addition of vegetable oil, the astaxanthin production of the engineered bacteria YYYE112 reached 3426.1 mg / L, and the production of secreted astaxanthin reached 221.3 mg / L, an increase of 53.9% compared with the previous level (compared with the engineered bacteria YYYE104).

[0024] (3) The astaxanthin produced by the recombinant Yarrowia lipolytica of the present invention is L-astaxanthin, and the engineered bacteria can, on the one hand, use vegetable oil as a carbon source to accelerate cell growth and reduce fermentation time and cost; on the other hand, it can enhance the secretion capacity of astaxanthin, and the oil serves as a culture medium for the oil phase and the water phase to form a two-phase fermentation system, so that the lipophilic astaxanthin product secreted outside the cell enters the oil phase, simplifying or even eliminating the need for extraction, which can greatly reduce the cost of astaxanthin extraction and purification. At the same time, since astaxanthin is secreted from the cell, the intracellular space is released, which can promote the further production and accumulation of astaxanthin in the cell, reducing the burden on the cell, and can also utilize the affinity between oil and astaxanthin to increase the storage space of astaxanthin in the cell, greatly improving the yield of astaxanthin, providing feasibility for subsequent large-scale production of astaxanthin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are the morphological changes of the engineered bacteria under an optical microscope and the changes in the astaxanthin content in the bacteria before and after knocking out MHY1 in the genome of the engineered bacteria YYYE097 in Example 5.

[0026] Figure 2 The figures show the growth curves of the engineered bacteria before and after overexpression of the SNQ2 gene in shake flask culture in Example 7 under the conditions of medium with and without vegetable oil, and the secreted astaxanthin production under the medium with vegetable oil.

[0027] Figure 3These are the growth curves and astaxanthin production curves of the engineered bacteria in a medium containing vegetable oil before and after overexpression of the SNQ2 gene in the fermenter culture of Example 8.

[0028] Figure 4 This is the astaxanthin production curve of the engineered bacteria before and after overexpression of the SNQ2 gene in the fermentation tank culture in Example 8 in a medium containing vegetable oil. DETAILED DESCRIPTION

[0029] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0030] The experimental methods in the following examples, unless otherwise specified, are conventional methods, according to the technology or conditions described in the literature in this area or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. The gene synthesis of the present invention was completed by General Bio (Anhui) Co., Ltd., primer synthesis was completed by Guangzhou Ruibo Biotechnology Co., Ltd., and sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd.

[0031] The present invention will be further described below in conjunction with specific examples and analysis. The primers used in the following examples are shown in Table 1:

[0032]

[0033]

[0034] The sources, NCBI accession numbers and sequence numbers of the genes used in the following examples are shown in Table 2:

[0035]

[0036] The six gene sequences were synthesized by General Biotech (Anhui) Co., Ltd. to obtain the pUC57-CarB, pUC57-CarRP, pUC57-PsCrtW, pUC57-HpCrtZ, pUC57-CrtYB, and pUC57-CrtI plasmids. The following examples also use the GGS1, ACC1, DGA1, and SNQ2 genes, all from Yarrowia lipolytica.

[0037] The CarRP gene expresses phytoene synthase / lycopene cyclase. Integration of the CarB and CarRP genes into the Yarrowia lipolytica genome enhances the engineered strain's ability to produce β-carotene. The PsCrtW gene expresses β-carotene ketolase, and the HpCrtZ gene expresses β-carotene hydroxylase. Integration of the PsCrtW-HpCrtZ gene cluster into the Yarrowia lipolytica genome enables the engineered strain to produce astaxanthin. The GGS1 gene expresses geranylgeranyl diphosphate synthase, the CrtYB gene expresses phytoene synthase / lycopene cyclase, and the CrtI gene expresses phytoene dehydrogenase. Integration of the GGS1-CrtYB-CrtI gene cluster into the Yarrowia lipolytica genome enhances the engineered strain's ability to produce astaxanthin. The ACC1 gene expresses acetyl-CoA carboxylase, and the DGA1 gene expresses diacylglycerol acyltransferase. By integrating genes such as ACC1 and DGA1 into the Yarrowia lipolytica genome and overexpressing them, the engineered bacteria's ability to produce astaxanthin can be further enhanced. Targeted knockout of the MHY1 gene in the Yarrowia lipolytica genome prevents the engineered bacteria from producing hyphae or pseudohyphae. Overexpression of the SNQ2 gene, which expresses an ABC transporter, can enhance the engineered bacteria's ability to secrete astaxanthin outside the cell.

[0038] Example 1

[0039] This example uses wild-type Yarrowia lipolytica as the starting strain to construct the β-carotene-producing engineered strain YYYE064. The construction method is as follows:

[0040] 1. Using the pUC57-CarB plasmid as a template, primers F1 and R1 were used to amplify the CarB gene sequence. Using the pUC57-CarRP plasmid as a template, primers F2 and R2 were used to amplify the CarRP gene sequence. Genomic DNA was extracted from Yarrowia lipolytica using the CTAB method and used as a template to amplify the GGS1 gene sequence using primers F3 and R3. The pTEF1 gene sequence was amplified using primers F4 and R4. The pGDP1 gene sequence was amplified using primers F5 and R5. The pEXP1 gene sequence was amplified using primers F6 and R6. The tXPR2 gene sequence was amplified using primers F7 and R7. The tPEX20 gene sequence was amplified using primers F8 and R8. The tLIP2 gene sequence was amplified using primers F9 and R9.

[0041] The PCR amplification system (Table 3) and procedure are as follows (Table 4):

[0042] Table 3:

[0043] Reagents Usage template 10ng Primer F 0.4μL Primer R 0.4μL polymerase mixture 10 μL <![CDATA[Double distilled water (ddH2O)]]> up to 20μL

[0044] Table 4:

[0045]

[0046]

[0047] 2. After the enzyme digestion reaction and PCR are completed, determine whether the target band is amplified by agarose gel electrophoresis; recover the enzyme digestion product and PCR product using a gel recovery kit (Novozyme FastPure Gel DNA Extraction Mini Kit).

[0048] 3. Use overlap extension PCR to assemble the promoter, target gene, and terminator into three expression cassettes: pTEF1-GGS1-tXPR2, pGPD1-CarB-tPEX20, and pEXP1-CarRP-tLIP2. The PCR system and procedure are shown in Tables 3 and 4. Recover the three expression cassettes by agarose gel electrophoresis and a gel recovery kit.

[0049] 4. The three expression cassettes pTEF1-GGS1-tXPR2, pGPD1-CarB-tPEX20, and pEXP1-CarRP-tLIP2 were assembled with the linearized plasmid backbone using a seamless ligation kit (Novozymes ClonExpress Ultra One Step Cloning Kit) at 50°C for 30 min. The assembly system is shown in Table 5.

[0050] Table 5:

[0051] Reagents Usage Total template usage 100ng 2x ClonExpress Mix 5.0μL <![CDATA[Double distilled water (ddH2O)]]> up to 10μL

[0052] 5. Transform the assembled product into E. coli DH5α and detect by colony PCR and agarose gel electrophoresis. Select three bacterial cultures with target bands of the expected size and inoculate them into 5 mL of liquid LB medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C for 16-18 hours for plasmid extraction, enzyme digestion, and sequencing verification. The colony PCR system (Table 6) and procedure are as follows (Table 7):

[0053] Table 6:

[0054] Reagents Usage template 0 Primer F 0.2μL Primer R 0.2μL polymerase mixture 5.0μL <![CDATA[Double distilled water (ddH2O)]]> up to 10μL

[0055] Table 7:

[0056]

[0057]

[0058] 6. The plasmid was extracted using a plasmid extraction kit (Novozyme FastPure Plasmid Mini Kit). The recombinant plasmid of the β-carotene-producing engineered bacteria was successfully constructed through colony PCR, enzyme digestion identification, and sequencing verification. The enzyme digestion identification system is shown in Table 8. The reaction was carried out at 37°C for 60 minutes.

[0059] Table 8:

[0060] Reagents Usage template 200ng Restriction enzymes 0.2μL 10xbuffer 1.0 μL <![CDATA[Double distilled water (ddH2O)]]> up to 10μL

[0061] 7. The β-carotene-producing recombinant plasmid was digested with restriction endonuclease NotI at 37°C for 3 h. The amount of plasmid used was 2 μg. The enzyme digestion system is shown in Table 9:

[0062] Table 9:

[0063] Reagents Usage template 2 μg NotI 1.0μL 10x buffer 1.0μL <![CDATA[Double distilled water (ddH2O)]]> up to 10μL

[0064] 8. Next, the linearized plasmid was transformed into wild-type Yarrowia lipolytica using the lithium acetate transformation method (a commonly used standard operating method). Finally, the plasmid was spread on CM-URA auxotrophic plates and cultured at 30°C until transformants grew out.

[0065] 9. Screening of engineered bacteria producing β-carotene. The screening steps are as follows:

[0066] (1) Fifteen transformants with darker color and larger clones were selected and inoculated into 1 mL of liquid YPD medium. The culture was maintained at 30°C and 220 rpm for 24 h to obtain seed solution. The seed solution was then transferred to 5 mL of liquid YPD medium at a ratio of 1:100 (V / V) and incubated at 30°C and 220 rpm for 120 h. The YPD medium contained 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone.

[0067] (2) Prepare a standard solution of β-carotene by the following steps:

[0068] ① Accurately weigh 20 mg of β-carotene standard into a 100 mL volumetric flask, fully dissolve it with acetone and make up to volume to prepare the standard stock solution with a concentration of 200 μg / mL.

[0069] ② Accurately pipette an appropriate amount of standard stock solution and dilute it with acetone to form standard working solutions with concentrations of 0.1μg / mL, 0.5μg / mL, 1.0μg / mL, 2.0μg / mL, 5.0μg / mL, and 10.0μg / mL, respectively.

[0070] (3) Prepare 15 samples to be tested, the steps are as follows:

[0071] ① After mixing the cultured bacterial solution by pipetting, take 1 mL of the bacterial solution and place it in a 2 mL centrifuge tube. Centrifuge at 12000 rpm for 1 min, discard the supernatant, and then place the centrifuge tube in a 100℃ oven to dry the bacteria and weigh the bacteria.

[0072] ② From the cultured bacterial solution, pipette and mix thoroughly, then take 1 mL of the bacterial solution and place it in a 2 mL centrifuge tube. Centrifuge at 12000 rpm for 1 min and discard the supernatant.

[0073] ③ Add 1 mL of dichloromethane-methanol solution and thoroughly grind to completely break the cell walls. Transfer to a 50 mL centrifuge tube. Rinse the glass homogenizer with 10 mL of dichloromethane-methanol solution three times and combine the extracts. Ultrasonic extraction for 5 minutes, then centrifuge at 8000 rpm at 5°C for 5 minutes. Transfer the supernatant to a 50 mL volumetric flask and add another 10 mL of dichloromethane-methanol solution to the centrifuge tube.

[0074] ④ Repeat the above steps 3 or more times until the extracted cells are white. Combine the supernatants and dilute to 50 mL with dichloromethane-methanol solution. Then let it stand for 15 minutes.

[0075] ⑤ Accurately transfer 5 mL of the solution from the above volumetric flask into a 10 mL colorimetric tube, add 0.7 mL of sodium hydroxide-methanol solution, vortex mix, seal with nitrogen, and react in a 5°C refrigerator overnight for 12 h.

[0076] ⑥ Then add 0.4 mL of 2% phosphoric acid-methanol solution to the reaction solution to neutralize the remaining alkali. After mixing, dilute to 5 mL under nitrogen purge, filter through a 0.45 μm filter membrane, and use the filtrate as the sample solution.

[0077] (4) 15 β-carotene extracts were subjected to high performance liquid chromatography to obtain β-carotene-producing engineered bacteria YYYE064. The detection method for the β-carotene extract is as follows:

[0078] ① The supernatant containing β-carotene was analyzed using a Shimadzu 2050C HPLC equipped with a 450 nm variable wavelength detector and a C30250*4.6 nm column;

[0079] ② Mobile phase A is methanol: acetonitrile: water = 73.5:24.5:2; mobile phase B: methyl tert-butyl ether

[0080] ③ The flow rate was 1.0 mL / min, the column temperature was 30°C, and the injection volume was 20 μL;

[0081] ④ Gradient elution was performed with the mobile phase, as shown in the following table (Table 10):

[0082] Table 10:

[0083]

[0084] Example 2

[0085] This example uses the β-carotene-producing engineered bacterium YYYE064 as the starting strain to construct the astaxanthin-producing engineered bacterium YYYE067. The construction method is as follows:

[0086] 1. Using the pUC57-PsCrtW plasmid as a template, primers F10 and R10 were used to amplify the PsCrtW PCR fragment. Using the pUC57-HpCrtZ plasmid as a template, primers F11 and R11 were used to amplify the HpCrtZ PCR fragment. Using Yarrowia lipolytica genomic DNA as a template, primers F12 and R12 were used to amplify the pTEFin gene sequence, and primers F7 and R7 were used to amplify the tXPR2 gene sequence. The PCR amplification system is shown in Table 3, and the PCR amplification procedure is shown in Table 4.

[0087] 2. After the reaction, the product was recovered using a gel recovery kit. The promoter, target gene, and terminator were then spliced ​​into an expression cassette, pTEFin-PsCrtW-HpCrtZ-tXPR2, using overlap extension PCR. This expression cassette was then assembled with the linearized plasmid backbone using a seamless ligation kit. Colony PCR, enzyme digestion, and sequencing verification confirmed the successful construction of a recombinant plasmid for astaxanthin-producing engineered bacteria. For detailed steps and conditions for recombinant plasmid construction, refer to Sections 3-6 and Tables 5-8 of Example 1.

[0088] 3. The astaxanthin-producing recombinant plasmid was digested with the restriction endonuclease NotI at 37°C for 3 h, using a plasmid dosage of 2 μg (enzyme digestion system as shown in Table 9); next, the linearized plasmid was transformed into Yarrowia lipolytica YYYE064 using the lithium acetate transformation method, and finally, the plasmid was spread on CM-LEU auxotrophic plates and cultured at 30°C until transformants grew.

[0089] 4. Screening of astaxanthin-producing engineered bacteria YYYE067. The screening steps are as follows:

[0090] (1) Fifteen transformants with darker color and larger clones were selected and inoculated into 1 mL of liquid YPD medium respectively. The culture was carried out at 30°C and 220 rpm for 24 h to obtain seed solution. The seed solution was then transferred to 5 mL of liquid YPD medium at a ratio of 1:100 (V / V) and cultured at 30°C and 220 rpm for 120 h.

[0091] (2) Prepare a standard solution of astaxanthin by the following steps:

[0092] ① Accurately weigh 20 mg of astaxanthin standard into a 100 mL volumetric flask, fully dissolve it with acetone and dilute to volume to prepare the standard stock solution with a concentration of 200 μg / mL;

[0093] ②Accurately pipette an appropriate amount of standard stock solution and dilute it with acetone to the standard working solutions with concentrations of 0.1μg / mL, 0.5μg / mL, 1.0μg / mL, 2.0μg / mL, 5.0μg / mL, and 10.0μg / mL, respectively;

[0094] ③ Then, 15 test samples were prepared and the 15 astaxanthin extracts were analyzed by HPLC to obtain astaxanthin-producing engineered strain YYYE067. For the detection method, refer to Section 9 and Table 10 of Example 1. When referring to this section, replace the "β-carotene" standard and extract with the "astaxanthin" standard and extract.

[0095] Example 3

[0096] In this example, the engineered strain YYYE067 was used as the starting strain to construct the high-astaxanthin-producing engineered strain YYYE090. The construction method is as follows:

[0097] 1. Using the pUC57-CrtYB plasmid as a template, primers F13 and R13 were used to amplify the CrtYB gene sequence. Using the pUC57-CrtI plasmid as a template, primers F14 and R14 were used to amplify the CrtI gene sequence. Using Yarrowia lipolytica genomic DNA as a template, primers F15 and R15 were used to amplify the pFBA1 gene sequence. Primers F3 and R3 were used to amplify the GGS1 gene sequence. Primers F4 and R4 were used to amplify the pTEF1 gene sequence. Primers F8 and R8 were used to amplify the tPEX20 gene sequence. Primers F9 and R9 were used to amplify the tLIP2 gene sequence. Primers F6 and R6 were used to amplify the pEXP1 gene sequence. Primers F7 and R7 were used to amplify the tXPR2 gene sequence. The PCR amplification system is shown in Table 3, and the PCR amplification procedure is shown in Table 4.

[0098] 2. After the reaction, the product was recovered using a gel recovery kit. The promoter, target gene, and terminator were then spliced ​​into three expression cassettes, pTEF1-GGS1-tXPR2, pFBA1-CrtYB-tPEX20, and pEXP1-CrtI-tLIP2, using overlap extension PCR. The three expression cassettes were then assembled with the linearized plasmid backbone using a seamless ligation kit. Colony PCR, enzyme digestion, and sequencing verification confirmed the successful construction of a high-yield astaxanthin recombinant plasmid. For detailed steps and conditions for recombinant plasmid construction, refer to Sections 3-6 and Tables 5-8 of Example 1.

[0099] 3. The high-yield astaxanthin recombinant plasmid was digested with the restriction endonuclease NotI at 37°C for 3 h, with a plasmid dosage of 2 μg (enzyme digestion system as shown in Table 9); then the linearized plasmid was transformed into Yarrowia lipolytica YYYE067 using the lithium acetate transformation method, and finally spread on CM-LEU auxotrophic plates and cultured at 30°C until transformants grew.

[0100] 4. HPLC analysis was performed on the 15 astaxanthin extracts to obtain the high-yield astaxanthin engineered strain YYYE090. For the sample preparation and detection process, refer to Section 4 of Example 2, Section 9 of Example 1, and Table 10.

[0101] Example 4

[0102] In this example, the high-astaxanthin-producing engineered bacterium YYYE090 was used as the starting strain to construct an engineered bacterium YYYE097 with a higher astaxanthin production. The construction method is as follows:

[0103] 1. Using Yarrowia lipolytica genomic DNA as a template, primers F16 and R16 were used to amplify the ACC1 gene sequence, primers F17 and R17 were used to amplify the DGA1 gene sequence, primers F18 and R18 were used to amplify the tLIP1 gene sequence, primers F12 and R12 were used to amplify the pTEFin gene sequence, primers F7 and R7 were used to amplify the tXPR2 gene sequence, and primers F6 and R6 were used to amplify the pEXP1 gene sequence. The PCR amplification system is shown in Table 3, and the PCR amplification procedure is shown in Table 4.

[0104] 2. After the reaction, the product was recovered using a gel recovery kit, and the promoter, target gene, and terminator were spliced ​​into two expression cassettes, pTEFin-ACC1-tXPR2 and pEXP1-DGA1-tLIP1, using overlap extension PCR. The two expression cassettes were assembled with the linearized plasmid backbone using a seamless ligation kit. Colony PCR, enzyme digestion identification, and sequencing verification successfully constructed a recombinant plasmid with a higher yield of astaxanthin. For detailed steps and conditions for plasmid construction, refer to Sections 3-6 and Tables 5-8 of Example 1.

[0105] 3. The recombinant plasmid producing astaxanthin with higher yield was digested with the restriction endonuclease NotI at 37°C for 3 h, with a plasmid dosage of 2 μg (enzyme digestion system as shown in Table 9); next, the linearized plasmid was transformed into Yarrowia lipolytica YYYE090 using the lithium acetate transformation method, and finally spread on CM-LEU auxotrophic plates and cultured at 30°C until transformants grew.

[0106] 4. 15 astaxanthin extracts were subjected to high performance liquid chromatography to obtain engineered strain YYYE097 with higher astaxanthin production. For sample preparation and testing procedures, refer to Section 4 of Example 2, Section 9 of Example 1, and Table 10.

[0107] Example 5

[0108] This example uses the engineered strain YYYE097, which has a higher astaxanthin production, as the starting strain. The MHY1 gene in the genome of the engineered strain YYYE097 was knocked out to construct the astaxanthin-producing engineered strain YYYE104, which does not produce hyphae or pseudohyphae. The construction method is as follows:

[0109] 1. Using plasmids containing the gRNA gene fragment and the Cas9 gene fragment as templates, primers F19 and R19 were used to amplify the target fragment SCR1'tRNAGly-sgRNA-tMIG1, and primers F20 and R20 were used to amplify the target fragment pTEFin-Cas9-tCYC1. The sgRNA sequence was 5'-ggagatggcgagataacgga-3'. The PCR amplification system is shown in Table 3, and the PCR amplification procedure is shown in Table 4.

[0110] 2. After the reaction, the product was recovered using a gel recovery kit and the two expression cassettes were assembled with the linearized plasmid backbone. Colony PCR, enzyme digestion, and sequencing were performed to verify the successful construction of a recombinant plasmid that did not produce hyphae or pseudohyphae. For detailed steps and conditions for recombinant plasmid construction, refer to Sections 3-6 and Tables 5-8 of Example 1.

[0111] 3. Next, the linearized plasmid was transformed into Yarrowia lipolytica YYYE097 using the lithium acetate transformation method. The transformed bacteria were added to 5 mL of liquid CM-LEU medium, mixed by pipetting, and cultured at 30°C, 200 rpm for 96 h. After the culture was completed, a certain volume of culture solution was diluted and spread on a solid YPD plate, and cultured at 30°C until colonies grew.

[0112] 4. Screening for astaxanthin-producing engineered bacteria that do not produce hyphae or pseudohyphae. The specific method is as follows:

[0113] (1) By observing the smooth surface of transformants on the plate with the naked eye and then observing under an optical microscope, it can be seen that all transformants exist in the form of yeast after the MHY1 gene is knocked out; Figure 1 As shown in (a); without knocking out the gene, there are a lot of hyphae, such as Figure 1 As shown in (b), it can be seen that after knocking out the MHY1 gene, the astaxanthin-producing engineered bacteria YYYE104 that does not produce hyphae was finally obtained.

[0114] (2) After culturing YYYE097 and YYYE104, the astaxanthin extract was tested by HPLC. The results were as follows: Figure 1 As shown in (c), knocking out the MHY1 gene not only does not affect the astaxanthin content in the bacterial cells, but can actually increase the astaxanthin content in the bacterial cells. The astaxanthin content detection method is described in Section 9 of Example 1 or Section 4 of Example 2.

[0115] Example 6

[0116] In this example, the astaxanthin-producing engineered bacterium YYYE104, which does not produce hyphae or pseudohyphae, was used as the starting strain, and the SNQ2 gene was overexpressed to construct the engineered bacterium YYYE112 capable of increasing astaxanthin secretion.

[0117] The construction method is as follows:

[0118] 1. Using Yarrowia lipolytica genomic DNA as a template, primers F21 and R21 were used to amplify the SNQ2 gene sequence, primers F12 and R12 were used to amplify the pTEFin gene sequence, and primers F7 and R7 were used to amplify the tXPR2 gene sequence. The PCR amplification system is shown in Table 3, and the PCR amplification procedure is shown in Table 4.

[0119] 2. After the reaction, the product was recovered using a gel recovery kit. Overlap extension PCR was then used to assemble the promoter, gene, and terminator into the pTEFin-SNQ2-tXPR2 expression cassette. The expression cassette was then assembled with the linearized plasmid backbone using a seamless ligation kit. Colony PCR, enzyme digestion, and sequencing confirmed the successful construction of a recombinant plasmid capable of enhancing astaxanthin secretion. For detailed steps and conditions for recombinant plasmid construction, refer to Sections 3-6 and Tables 5-8 of Example 1.

[0120] 3. The recombinant plasmid that enhances astaxanthin secretion was digested with the restriction endonuclease NotI at 37°C for 3 h, using a plasmid dosage of 2 μg (see Table 9 for the enzyme digestion system). Next, the linearized plasmid was transformed into Yarrowia lipolytica YYYE104 using the lithium acetate method. Finally, the plasmid was plated onto CM-URA auxotrophic plates and cultured at 30°C until transformants emerged.

[0121] 4. Screening of astaxanthin-engineered bacteria with high astaxanthin secretion. The screening method is as follows:

[0122] (1) Fifteen transformants with darker color and larger colonies were selected and inoculated into 1 mL of liquid YPD medium and cultured for 48 h to obtain seed liquid. After measuring OD600, a certain volume of seed liquid was transferred to a 15 mL shake tube containing 5 mL of liquid YPD medium so that the final OD of the bacteria in the liquid was 0.05-0.1. Then, 20% vegetable oil (V / V) was added and cultured for 120 h.

[0123] (2) After the culture is completed, centrifuge at 12000rpm for 5 minutes to separate the oil phase and the bacteria, and then prepare the test sample. The preparation and detection process of the test sample refer to Part 4 of Example 2 and Part 9 and Table 10 of Example 1. After HPLC detection and screening, the engineered bacteria YYYE112 that can improve astaxanthin secretion was obtained. The applicant has deposited the engineered bacteria YYYE112 in the General Microbiology Center of the China Culture Collection Administration on April 17, 2024, with the deposit number CGMCCNO.30371.

[0124] Example 7

[0125] This example verifies the astaxanthin secretion of the engineered bacterium YYYE112, which can enhance astaxanthin secretion, through small-scale shake flask culture. The culture method is as follows:

[0126] (1) Single colonies of the astaxanthin-producing engineered bacteria YYYE104 and YYYE112 were selected and inoculated into 5 mL of liquid YPD medium, respectively, and cultured for 72 h to obtain seed solution. After measuring OD600, a certain volume of seed solution was transferred to a 250 mL Erlenmeyer flask containing 50 mL of liquid YPD medium, so that the final OD of the bacteria in the liquid was 0.05-1. 20% vegetable oil (v / v) was then added, and the culture was continued for 120 h. The astaxanthin content was detected by HPLC. The astaxanthin content was determined by reference to Section 4 of Example 2.

[0127] (2) The results are as follows Figure 2 As shown in (a), after adding vegetable oil, the growth rate of the engineered bacteria increased by more than double; after HPLC detection, the SNQ2 gene was overexpressed and after culturing with vegetable oil, the astaxanthin production of YYYE104 was 7.46 mg / L, and the astaxanthin production of YYYE112 was 14.70 mg / L. The astaxanthin production of YYYE112 was 49% higher than that of YYYE104. Figure 2 As shown in (b), it shows that overexpression of SNQ2 gene can enhance the secretion of astaxanthin.

[0128] Example 8

[0129] This example verifies the astaxanthin secretion of the engineered bacterium YYYE112, which can enhance astaxanthin secretion, through large-scale fermentation tank cultivation. The cultivation method is as follows:

[0130] (1) The engineered bacteria YYYE104 and YYYE112 stored in -80°C glycerol tubes were streaked onto solid YPD plates and cultured for 48 h. Single colonies were then picked and inoculated into 50 mL Erlenmeyer flasks containing 10 mL YPD liquid medium and cultured for 48 h to obtain the primary seed solution.

[0131] (2) The first-level seed solution was then transferred to a 300 mL Erlenmeyer flask containing 100 mL YPD liquid medium at a 10% (wt) inoculation rate and cultured for 24 h until the cell OD600 reached 10-50 to obtain the second-level seed solution.

[0132] (3) Finally, the secondary seed liquid was transferred to a 5L fermentation tank containing 3L fermentation medium at a 10% (wt) inoculation rate for fermentation. The initial medium composition was 60g / yeast powder, 60g / L peptone, and 20% vegetable oil (v / v). After inoculation, glucose was used as the carbon source and supplemented to 200g / L; ammonium chloride was used as the nitrogen source and supplemented to 20g / L. The fermentation cycle was 120h, and the glucose concentration was maintained at 130g / L. The feed was reduced before the tank was filled until the end of fermentation. Samples were retained every 24h to measure the astaxanthin production in the oil phase and the bacteria.

[0133] (4) The results are as follows Figure 3 As shown in (a) and (b), the astaxanthin production of the engineered bacteria YYYE112 was as high as 3426.1 mg / L. Figure 4 As shown, after overexpressing the SNQ2 gene, the astaxanthin production secreted by the YYYE112 engineered bacteria reached 211.3 mg / L, which was 53.9% higher than that secreted by YYYE104.

[0134] In summary, the present invention introduces multiple gene clusters into Yarrowia lipolytica to gradually enhance the ability of the engineered bacteria to accumulate astaxanthin, and produces astaxanthin by overexpressing the SNQ2 gene and two-phase fermentation. On the one hand, it can utilize vegetable oil as a carbon source, accelerate cell growth, and reduce fermentation time and cost; on the other hand, it enhances the ability of the engineered bacteria to secrete astaxanthin extracellularly, thereby reducing extraction costs; at the same time, the secretion of astaxanthin reduces the burden on the cells, promotes further accumulation of astaxanthin in the cells, and increases the storage space of astaxanthin in the cells by virtue of the affinity of oil for astaxanthin, greatly improving the astaxanthin yield, and providing technical support for the large-scale production of astaxanthin.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An engineered strain of Yarrowia lipolytica that secretes a large amount of L-astaxanthin, named Yarrowia lipolytica ( Yarrowia lipolytica )YYYE112, the deposit number is CGMCC NO.30371, the deposit date is April 17, 2024, and the depository is the General Microbiology Center of China Microbiological Culture Collection Administration.

2. A method for producing L-astaxanthin based on two-phase fermentation of Yarrowia lipolytica engineered bacteria, characterized in that: The engineered Yarrowia lipolytica is the engineered Yarrowia lipolytica in claim 1.

3. The method according to claim 2, characterized in that The initial culture medium composition of the two-phase fermentation is: 30-100g / L yeast powder, 20-120g / L peptone, and 5-30v / v% vegetable oil; after inoculation, glucose is used as a carbon source to supplement to 150-300g / L; ammonium chloride is used as a nitrogen source and supplemented to 5-25g / L; the fermentation cycle is 1 15-125h, and glucose is added during the fermentation to maintain the glucose concentration at 120-150g / L. The feed is reduced before the tank is lowered until the fermentation is completed, and L-astaxanthin is obtained from the fermentation oil phase or the bacteria.

Images