CN116396877A - Zeaxanthin-producing yarrowia lipolytica and application thereof - Google Patents

Zeaxanthin-producing yarrowia lipolytica and application thereof Download PDF

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CN116396877A
CN116396877A CN202310219327.7A CN202310219327A CN116396877A CN 116396877 A CN116396877 A CN 116396877A CN 202310219327 A CN202310219327 A CN 202310219327A CN 116396877 A CN116396877 A CN 116396877A
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zeaxanthin
yarrowia lipolytica
producing
yield
genetically engineered
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CN116396877B (en
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刘振
毛相朝
张桂林
陈静
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Ocean University of China
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Abstract

The invention discloses a zeaxanthin-producing yarrowia lipolytica and application thereof, and belongs to the technical field of genetic engineering. The zeaxanthin-producing yarrowia lipolytica is classified and named yarrowia lipolytica OUC-Zea8-A7ZGL, and is preserved in China Center for Type Culture Collection (CCTCCNO), wherein the preservation number is: m20222062, date of preservation: 2022, 12, 23. The strain is applied to the preparation of zeaxanthin. The invention successfully obtains the genetic engineering bacteria of high-yield zeaxanthin by transferring heterologous genes, over-expressing key enzymes, regulating key metabolic nodes, assembling and expressing modularized enzymes and other strategies, and the yield can reach 816.9mg/L. The strain has the advantages of rapid growth and propagation, high product concentration, stable and reliable reaction and the like, has potential of industrial production, and has good application prospect.

Description

Zeaxanthin-producing yarrowia lipolytica and application thereof
Technical Field
The invention relates to a zeaxanthin-producing yarrowia lipolytica and application thereof, belonging to the technical field of genetic engineering.
Background
Zeaxanthin (zeaxanthin) is a carotenoid, a dihydroxy derivative of beta-carotene, widely found in nature in fruits, vegetables, bacteria and microalgae, but humans and animals cannot synthesize zeaxanthin and can only be taken from food. The ingested zeaxanthin is commonly found in the skin and eyes of humans and animals, and has a certain protective effect on the skin and eyes; in addition, zeaxanthin has antioxidant, antiinflammatory, anticancer, and neuroprotective effects. The safety of zeaxanthin has been demonstrated and is widely used in pharmaceuticals, sunscreens, food additives, feed additives, and the like.
The current production method of zeaxanthin includes chemical synthesis and natural resource extraction. The zeaxanthin products produced by chemical synthesis have low activity and risk of residual hazardous agents. The natural resource extraction method mainly extracts from plants and algae, but the content of zeaxanthin contained in the plants and algae in the nature is generally low, and the production raw materials are easily influenced by climatic environment.
With the development of scientific technologies such as synthetic biology and genetic engineering, microbial fermentation methods are becoming an effective way to produce natural products. Yarrowia lipolytica is a well-established safety (GRAS) microorganism, rich in lipids and acetyl-coa, an excellent chassis organism for carotenoid production. However, the construction of recombinant engineering bacteria for producing zeaxanthin has the following difficulties: the synthesis path of zeaxanthin is complex, and the expressed genes of enzymes involved in the synthesis path are recombined into engineering bacteria to be converted for a plurality of times, so that the difficulty is high and the success rate is low; after the heterologous gene is transferred to enable the genetically engineered bacteria to realize the synthesis of the product, the intermediate strain is also required to be transformed and screened for multiple times so as to obtain the genetically engineered bacteria with high yield. There is no report on construction of recombinant engineering bacteria producing zeaxanthin.
Disclosure of Invention
Aiming at the prior art, the invention constructs recombinant engineering bacteria through genetic engineering and screens to obtain a strain of yarrowia lipolytica with high zeaxanthin yield. The invention also provides application of the zeaxanthin-producing yarrowia lipolytica in preparing zeaxanthin.
The invention is realized by the following technical scheme:
a strain of zeaxanthin-producing yarrowia lipolytica, classified and named yarrowia lipolytica OUC-Zea8-A7ZGL, is preserved in China center for type culture Collection with the preservation number CCTCCNO: m20222062, date of preservation: 2022, 12, 23.
The biological characteristics of the zeaxanthin-producing yarrowia lipolytica are: aerobic yeast was cultured on YPD agar medium, and the colony was red, wrinkled and had a hairlike edge. The cells have three forms of yeast type, hypha type and pseudo hypha type.
The use of said zeaxanthin-producing yarrowia lipolytica for the preparation of zeaxanthin.
Further, in specific applications, zeaxanthin is obtained by culturing zeaxanthin-producing yarrowia lipolytica and extracting. The specific mode of the culture can be as follows: seed solution of zeaxanthin-producing yarrowia lipolytica is inoculated in YPD medium in an amount of 1%, and subjected to shaking-bed induced fermentation at 30℃and 220rpm for 48-120 hours. The specific way of extraction can be as follows: taking a fermentation broth of the cultured yarrowia lipolytica, centrifuging, discarding the supernatant, placing the fermentation broth in a grinding tube, and adding zirconia grinding beads for grinding; centrifuging, filtering the supernatant to remove impurities to obtain an extract, wherein the extract contains zeaxanthin.
The invention relates to a zeaxanthin-producing yarrowia lipolytica, which comprises the following construction methods: yarrowia lipolytica PO1h (host strain suitable for expression of foreign protein in the prior art) is used as an initial strain, pMT015 is used as an expression vector, and phytoene dehydrogenase gene carB and phytoene synthase ∈are sequentially transferredLycopene cyclase bifunctional enzyme gene carRP, mevalonate kinase gene ERG12S, isopentenyl pyrophosphate isomerase gene IDI and geranylgeranyl pyrophosphate synthase gene GGS1 assembled by modularized enzyme RIAD-RIDD, geranyl pyrophosphate synthase/farnesyl pyrophosphate synthase gene ERG20Y assembled by modularized enzyme RIAD-RIDD MT And a geranylgeranyl pyrophosphate synthase gene GGS1, an acetyl-CoA acetyltransferase/HMG-CoA reductase gene mvaE and an HMG-CoA synthase gene mvaS MT The beta-carotene hydroxylase gene crtZ, the oxidoreductase gene RFNR1 and finally the carrP and crtZ genes were transferred. Transformants with the highest carotenoid yield were selected as hosts for the next transformation after each transformation.
The invention constructs recombinant engineering bacteria, and the intermediate bacterial strain is transformed for a plurality of times, for example: over-expressing key enzymes to enhance the rate limiting step in the synthetic pathway, engineering the enzymes at key nodes to increase enzyme activity, assembling and expressing part of the enzymes to partition metabolic reactions to regulate metabolic flows, and adding cofactors to enhance related redox reactions. Finally, the invention successfully screens and obtains the genetic engineering bacteria of high-yield zeaxanthin.
The invention successfully obtains the yarrowia lipolytica genetic engineering strain capable of producing zeaxanthin at high yield through strategies such as transferring heterologous genes, over-expressing key enzymes, regulating key metabolic nodes, assembling and expressing modularized enzymes and the like. The yield of zeaxanthin of the genetically engineered strain of yarrowia lipolytica for producing zeaxanthin with high yield can reach 816.9mg/L, and the high-efficiency synthesis of zeaxanthin in the genetically engineered strain is realized. The genetically engineered bacterium has the advantages of rapid growth and propagation, high product concentration, stable and reliable reaction and the like, has potential of industrial production, and has good application prospect.
The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
The zeaxanthin-producing yarrowia lipolytica is classified and named Yarrowialipolytica OUC-Zea8-A7ZGL and is preserved in China center for type culture collection, and the preservation number is CCTCCNO: m20222062, date of preservation: 2022, 12, 23, deposit address: eight-way university of Wuhan China center for type culture collection, mail code 430072, wuhan, hubei province.
Fig. 1: a synthetic pathway for zeaxanthin.
Fig. 2: the production of carotenoids (lycopene, beta-carotene, zeaxanthin) of the key engineering bacteria (Car 1-8) is schematically shown.
Fig. 3: beta-carotene production by transformant (Car 1) is schematically shown.
Fig. 4: schematic of carotenoid production by transformant (Car 2).
Fig. 5: schematic of carotenoid production by transformant (Car 3).
Fig. 6: schematic of carotenoid production by transformant (Car 4).
Fig. 7: schematic of carotenoid production by transformant (Car 5).
Fig. 8: schematic of carotenoid production by transformant (Car 6).
Fig. 9: schematic of carotenoid production by transformant (Car 7).
Fig. 10: schematic of carotenoid production by transformant (Car 8).
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents and materials used in the examples below are conventional instruments, reagents and materials known in the art and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods and detection methods known in the prior art unless otherwise specified.
The shake flask fermentation method adopted by the invention comprises the following steps: 50mL of YPD medium was prepared in a 250mL shake flask, and the seed solution of the genetically engineered bacterium was inoculated at 1% and cultured at 30℃for 5 days at 220 rpm.
The extraction method of zeaxanthin adopted by the invention comprises the following steps: 1mL of fermentation broth is placed in a 2mL grinding tube, 10000 Xg is centrifuged for 2min, the supernatant is discarded, a small amount of 0.52mm zirconia grinding beads and 1mL methyl tertiary butyl ether are added into the grinding tube, and a full-automatic sample rapid grinding instrument is used for crushing and extracting engineering bacteria under the following grinding conditions: the frequency is set to be 65Hz, the running time is 2min each time, the interruption time after each running is 10s, and the running times are 8 times. After finishing grinding, the grinding tube is centrifuged for 2min at 10000 Xg, the supernatant is sucked by a syringe, and the impurities are removed by using a nylon filter membrane with the size of 0.22 mu m to obtain an extract.
The method for carrying out HPLC analysis on the extract liquid comprises the following steps: c18 chromatographic column (250×4.6mm,5 μm), column temperature 35 ℃, sample injection amount 10 μL, total flow rate 1mL/min, gradient elution method, mobile phase A of tetrahydrofuran, mobile phase B of acetonitrile/ethyl acetate/water (74/16/10), gradient elution time program of 0min, mobile phase B of 100%; at 10min, mobile phase B was 100%; at 12min, mobile phase B was 30%; at 18min, mobile phase B was 30%; at 20min, mobile phase B was 100%; at 25min, mobile phase B was 100%. Lycopene was detected at 472nm, beta-carotene at 450nm and zeaxanthin at 455 nm.
In the present invention, the synthetic pathway of zeaxanthin is shown in FIG. 1.
The meaning of the english abbreviations referred to in the present invention is as follows:
MvaE: acetyl-CoA acetyltransferase/HMG-CoA reductase.
MvaS MT : HMG-CoA synthase.
HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme A.
MVA: mevalonic acid.
ERG12S: mevalonate kinase.
MVA-5-P: mevalonate-5-phosphate.
IDI: isopentenyl pyrophosphate isomerase.
IPP: isoprene pyrophosphoric acid.
DMAPP: dimethyl allyl pyrophosphoric acid.
ERG20Y MT : geranyl pyrophosphate synthase/farnesyl pyrophosphate synthase.
FPP: farnesyl pyrophosphate.
GGS1: geranylgeranyl pyrophosphate synthase.
GGPP: geranylgeranyl pyrophosphate.
CarRP: phytoene synthase/lycopene cyclase.
CarB: phytoene dehydrogenase.
CrtZ: beta-carotene hydroxylase.
EXAMPLE 1 construction of beta-carotene-producing yarrowia lipolytica genetically engineered bacterium
(1) The carB gene and the carRP gene are derived from Mucor circinelloides, the nucleotide sequence of the carB gene is shown as SEQ ID NO.1, and the nucleotide sequence of the carRP gene is shown as SEQ ID NO.2 after codon optimization.
(2) The pMT015 plasmid is used as expression vector, and two expression cassettes with promoter and terminator are respectively P GPD -T LIP1 And P TEF -T XPR2 The promoter is followed by a kozak sequence "GCCACC". The screening mark of the pMT015 plasmid is URA3, and the promoter and terminator are P respectively LEU2 And T LEU2 And have LoxP sites at both ends. Construction of the carB and carRP genes to the P of plasmid pMT015 by seamless cloning, respectively GPD -T LIP1 And P TEF -T XPR2 On the expression cassette, the pMT015-carB-carRP plasmid was obtained.
(3) Linearizing a carB-carRP-ura3 expression cassette in a pMT015-carB-carRP plasmid by using PCR, transferring into yarrowia lipolytica PO1h by using a PEG/LiAc chemical conversion method to obtain genetically engineered yarrowia lipolytica strain producing beta-carotene, selecting 10 transformants with the highest yield (the same applies below), comparing the beta-carotene yields of the transformants, and screening out the genetically engineered strain OUC-Car1-9ZGL with the highest yield as shown in figure 3.
(4) And carrying out shake flask fermentation, extraction and HPLC detection on the obtained genetically engineered bacterium OUC-Car1-9ZGL. The result of the production is shown in FIG. 2, and the yield of beta-carotene of the genetically engineered bacterium OUC-Car1-9ZGL is 19.9mg/L.
Example 2 Metabolic engineering strategies to increase beta-carotene production in yarrowia lipolytica genetically engineered bacteria
(1) The ERG12S gene is derived from Saccharomyces cerevisiae. The IDI and GGS1 genes are derived from yarrowia lipolytica itself. ERG20Y MT Is also derived from yarrowia lipolytica, but the 87 th amino acid is mutated from phenylalanine to serine, and the nucleotide sequence is shown as SEQ ID NO. 3. Modular enzymes RIAD and RIDD are from document KangW, maT, liuM, et al, modular enzyme for enhancement of cascades biological enzyme and enzyme for use in a method of screening a plant]Nature Communications,2019,10 (1): 4248, codon optimized, nucleotide sequences shown as SEQ ID NO.8 and SEQ ID NO. 9. The Linker1 and Linker2 nucleotide sequences are shown as SEQ ID NO.10 and SEQ ID NO. 11. mvaE and mvaS MT The gene is derived from enterococcus faecalis, and the nucleotide sequence is shown as SEQ ID NO.4 and SEQ ID NO.5 after codon optimization.
(2) The pMT015 plasmid is used as an expression vector, and the ERG12S gene is constructed to the P of the plasmid pMT015 by a seamless cloning method TEF -T XPR2 On the expression cassette, the pMT015-ERG12S plasmid was obtained. The IDI, linker1 and RIAD sequences were constructed by seamless cloning to the P of plasmid pMT015 GPD -T LIP1 On the expression cassette, GGS1, linker2 and RIDD sequences are constructed to P of plasmid pMT015 by a seamless cloning method TEF -T XPR2 On the expression cassette, pMT015-IDI-RIAD-GGS1-RIDD plasmid was obtained. Based on pMT015-IDI-RIAD-GGS1-RIDD plasmid, the IDI gene is replaced by ERG20Y by seamless cloning MT Gene, and obtaining pMT015-ERG20Y MT -the RIAD-GGS1-RIDD plasmid. mvaE and mvaS MT The genes are respectively constructed to P of plasmid pMT015 by a seamless cloning method GPD -T LIP1 And P TEF -T XPR2 On the expression cassette, pMT015-mvaE-mvaS was obtained MT A plasmid.
(3) The pUB4-CRE plasmid is transferred into genetically engineered bacterium OUC-Car1-9ZGL by using a PEG/LiAc chemical conversion method, and the URA3 screening mark is removed so as to recycle the mark.
(4) Linearizing an ERG12S-URA3 expression cassette in a pMT015-ERG12S plasmid by using PCR, and transferring the expression cassette into the genetically engineered bacterium obtained in the step (3) by using a PEG/LiAc chemical conversion method to obtain the genetically engineered bacterium with higher beta-carotene yield.
(5) Repeating the step (3) and the step (4), and carrying out IDI-RIAD-GGS1-RIDD-URA3 and ERG20Y MT RIAD-GGS1-RIDD-URA3 and mvaE-mvaS MT Sequentially transferring the URA3 gene fragments into genetically engineered bacteria to obtain genetically engineered bacteria with higher beta-carotene yield. Comparing the carotenoid yields of the transformants obtained in the 4-time screening process, and screening out the genetically engineered bacteria OUC-Car5-3ZGL with the highest yield as shown in FIGS. 4, 5, 6 and 7.
(6) And carrying out shake flask fermentation, extraction and HPLC detection on the obtained genetically engineered bacterium OUC-Car5-3ZGL. The result of the production is shown in FIG. 2, and the yield of beta-carotene of the genetically engineered bacterium OUC-Car5-3ZGL is 536.8mg/L.
Example 3 construction of zeaxanthin-producing yarrowia lipolytica genetically engineered bacteria
(1) The crtZ gene is derived from Pantoea ananatis, and the nucleotide sequence is shown as SEQ ID NO.6 after codon optimization.
(2) The crtZ gene is constructed to the P of the plasmid pMT015 by using the pMT015 plasmid as an expression vector through a seamless cloning method TEF -T XPR2 On the expression cassette, the pMT015-crtZ plasmid was obtained.
(3) Repeating the step (3) and the step (4) in the example 2, transferring the crtZ-URA3 gene fragment into genetically engineered bacteria OUC-Car5-3ZGL to obtain zeaxanthin-producing yarrowia lipolytica genetically engineered bacteria, comparing the yield of each transformant, and screening out the genetically engineered bacteria OUC-Zea6-12ZGL with the highest yield as shown in figure 8.
(4) And carrying out shake flask fermentation, extraction and HPLC detection on the obtained genetically engineered bacterium OUC-Zea6-12ZGL. The results of the production are shown in FIG. 2, and the zeaxanthin yield of the genetically engineered bacterium OUC-Zea6-12ZGL is 326.5mg/L.
Example 4 metabolic engineering strategies to increase zeaxanthin production in yarrowia lipolytica genetically engineered bacteria
(1) The RFNR1 gene is derived from Arabidopsis thaliana, and the nucleotide sequence is shown as SEQ ID NO.7 after codon optimization.
(2) To be used forThe pMT015 plasmid is used as an expression vector, and the RFNR1 gene is constructed to the P of the plasmid pMT015 by a seamless cloning method TEF -T XPR2 On the expression cassette, pMT015-RFNR1 plasmid was obtained. Construction of the CarRP, linker1 and RIAD fragments by means of seamless cloning to the P of the plasmid pMT015 TEF -T XPR2 On the expression cassette, crtZ, linker2 and RIDD fragments were constructed to P of plasmid pMT015 by seamless cloning GPD -T LIP1 On the expression cassette, the pMT015-carRP-RIAD-crtZ-RIDD plasmid was obtained.
(3) The steps (3) and (4) in example 2 were repeated, and the RFNR1-URA3 and carRP-RIAD-crtZ-RIDD-URA3 gene fragments were sequentially transferred into the genetically engineered bacterium OUC-Car6-12ZGL to obtain a zeaxanthin-producing yarrowia lipolytica genetically engineered bacterium, and the yields of the transformants were compared, and as shown in FIGS. 9 and 10, the genetically engineered bacterium OUC-Zea8-7ZGL (CCTCCM 20222062) having the highest yield was selected.
(4) And carrying out shake flask fermentation, extraction and HPLC detection on the obtained genetically engineered bacterium OUC-Zea8-7 ZGL. The results of the production are shown in FIG. 2, and the zeaxanthin yield of the genetically engineered bacterium OUC-Zea8-7ZGL is 816.9mg/L.
The invention constructs recombinant engineering bacteria, and carries out transformation and screening on intermediate strains for 8 times, finally obtains the recombinant engineering bacteria-OUC-Zea 8-7ZGL for producing zeaxanthin with high yield, and the zeaxanthin yield of the transformant is up to 816.9mg/L and is far higher than that of other transformants. The invention preserves the transformant OUC-Zea8-7ZGL, and the transformant is preserved in China Center for Type Culture Collection (CCTCCNO) with the preservation number of: m20222062, date of preservation: 2022, 12, 23, deposit address: eight-way university of Wuhan China center for type culture collection, mail code 430072, wuhan, hubei province.
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.

Claims (5)

1. A strain of zeaxanthin-producing yarrowia lipolytica, classified and named yarrowia lipolytica OUC-Zea8-A7ZGL, is preserved in China center for type culture Collection with the preservation number CCTCCNO: m20222062, date of preservation: 2022, 12, 23.
2. Use of the zeaxanthin-producing yarrowia lipolytica of claim 1 for the preparation of zeaxanthin.
3. The use according to claim 2, characterized in that: culturing the zeaxanthin-producing yarrowia lipolytica, and extracting to obtain zeaxanthin.
4. The use according to claim 3, wherein the culturing is performed in the following manner: seed solution of zeaxanthin-producing yarrowia lipolytica is inoculated in YPD medium in an amount of 1%, and subjected to shaking-bed induced fermentation at 30℃and 220rpm for 48-120 hours.
5. The use according to claim 3, characterized in that the extraction is performed in a specific way: taking a fermentation broth of the cultured yarrowia lipolytica, centrifuging, discarding the supernatant, placing the fermentation broth in a grinding tube, and adding zirconia grinding beads for grinding; centrifuging, filtering the supernatant to remove impurities to obtain an extract, wherein the extract contains zeaxanthin.
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