CN116731886A - Engineering bacteria that produce glycosylated astaxanthin and their construction methods and applications - Google Patents

Abstract

The invention relates to engineering bacteria for producing glycosylated astaxanthin, a construction method and application thereof, and belongs to the technical field of fermentation engineering. The invention discloses an engineering bacterium for producing glycosylated astaxanthin, which is named OUC-AdGHB1-7CJ and is preserved in China general microbiological culture Collection center (CGMCC) No.25446 in the year 08 and 01 of 2022, and is classified as yarrowia lipolytica Yarrowia lipolytica. The engineering bacteria are applied to the preparation of glycosylated astaxanthin. The invention also discloses a construction method of the engineering bacteria. The engineering bacteria for producing glycosylated astaxanthin can achieve the yield of glycosylated astaxanthin of 27.274mg/L. The invention provides feasibility for the high-efficiency synthesis of other glycosylated carotenoids in microorganisms, and lays a foundation for the industrial production of glycosylated astaxanthin.

Description

Engineering bacteria that produce glycosylated astaxanthin and their construction methods and applications

Technical field

The invention relates to an engineering bacterium that produces glycosylated astaxanthin and its construction method and application, and belongs to the technical field of fermentation engineering.

Background technique

Glycosylated astaxanthin, also known as astaxanthin glucoside, is a rare natural carotenoid. It is formed by the dehydration condensation of astaxanthin with two molecules of glucose under the catalysis of glycosyltransferase CrtX. Astaxanthin has strong antioxidant properties and red coloring properties. It is the only carotenoid that can penetrate the blood-brain and blood-retina barriers and have a positive effect on the central nervous system and brain function. It has an important role in the food, feed, pharmaceutical and cosmetics industries. Wide range of applications. However, due to the hydrophobicity of astaxanthin, its application will be limited. Glycosylated astaxanthin can increase the polarity of the molecule and reduce its hydrophobicity through glycosylation, making it easier to be absorbed by the human body as a food additive and drug. . In addition, glycosylation of carotenoids has also led to structural diversity and several other benefits, such as increased bioavailability, enhanced efficacy as food supplements and pharmaceuticals, and improved photostability and bioactivity of carotenoids (e.g., anti- oxidative activity).

Glycosylated astaxanthin is sparsely distributed in nature (mainly distributed in some bacteria). There is still a lack of research on the properties of glycosylated astaxanthin, and its health effects on humans or animals are also poorly understood. The content of naturally synthesized glycosylated astaxanthin that exists in nature is very low, and it is difficult to extract and separate it. Biosynthesis through metabolic engineering is an effective way to obtain glycosylated astaxanthin, but to date, only a few studies have achieved the biosynthesis of carotenoid glycosides in E. coli and several natural microorganisms, and these studies have only produced Detectable carotenoid glycosides, far from the minimum requirements for industrial applications.

Yarrowia lipolytica is an unconventional yeast that has been certified as a GRAS (generally recognized as safe) strain by the US FDA. It has a wide substrate spectrum, tolerance to various environmental stresses, and Lactoacetyl-CoA has attracted a lot of attention in the field of synthetic biology manufacturing in recent years due to its sufficient supply, high-density fermentation, and safety to the human body.

Contents of the invention

In view of the above-mentioned existing technology, in order to realize the industrial production of glycosylated astaxanthin, the present invention constructs an engineering bacterium that produces glycosylated astaxanthin, and provides its construction method, as well as in the preparation of glycosylated astaxanthin. Applications. The present invention also obtains an engineering bacterium that produces glycosylated astaxanthin through screening of engineering bacteria.

The present invention is achieved through the following technical solutions:

An engineered bacterium that produces glycosylated astaxanthin, named OUC-AdGHB1-7CJ, has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on August 1, 2022, with the deposit number CGMCC NO.25446 , classified as Yarrowia lipolytica.

The biological characteristics of the above-mentioned engineered bacteria that produce glycosylated astaxanthin are: cultured on YPD solid medium at 30°C for 24 hours, the colonies are round, brick red, wrinkled, with hairy edges and dry texture. In the natural state, they are generally yeast morphology and hyphal morphology coexist.

Application of the above engineered bacteria producing glycosylated astaxanthin in the preparation of glycosylated astaxanthin. The engineering bacteria producing glycosylated astaxanthin of the present invention can prepare glycosylated astaxanthin with high efficiency, which is far superior to other transformants, and has huge advantages and application prospects when used to prepare glycosylated astaxanthin. .

Further, for specific application, the above engineered bacteria producing glycosylated astaxanthin are cultured and glycosylated astaxanthin is extracted. The specific method of culturing can be as follows: picking colonies of engineering bacteria that produce glycosylated astaxanthin, inserting them into YPD culture medium, and culturing them to obtain seed culture fluid; inserting the seed culture fluid into YPD culture medium, and culturing them to obtain Fermentation broth; take the fermentation broth, centrifuge to obtain the bacterial cell precipitate and fermentation supernatant, and extract the bacterial cell precipitate to obtain glycosylated astaxanthin.

Further, the culture conditions of the seed culture liquid are: 30°C, 200 rpm for 24 hours.

Further, the inoculum amount of the seed culture solution is 2% (volume ratio).

Further, the culture conditions of the fermentation broth are: 30°C, 200 rpm for 84 hours.

The YPD culture medium is a commercial culture medium existing in the prior art, and its ingredients include: 1% yeast extract, 2% peptone, 2% glucose, and the balance is water.

An engineered bacterium that produces glycosylated astaxanthin. The host is β-carotene-producing Yarrowia lipolytica. Its genome contains the following genes ①②⑤ or ③④⑤:

①The encoding gene HBFD of carotenoid 4-hydroxy-β-ring 4-dehydrogenase (HBFD), the nucleotide sequence is shown in SEQID NO.2;

②The coding gene tr53-CBFD for carotenoid β-ring 4-dehydrogenase (tr53-CBFD) with chloroplast transit peptide removed, the nucleotide sequence is shown in SEQ ID NO.3; its promoter is the hp4d promoter , the nucleotide sequence is shown in SEQ IDNO.7;

③The encoding gene crtZ of β-carotene hydroxylase (crtZ), the nucleotide sequence is shown in SEQ ID NO.10;

④The encoding gene crtW of β-carotene ketolase (crtW), the nucleotide sequence is shown in SEQ ID NO.9;

⑤The coding gene crtX for glycosyltransferase (crtX), the nucleotide sequence is shown in SEQ ID NO.8.

Among them, CBFD and HBFD are derived from Adonis aestivali, crtW is derived from Brevundimonas sp. SD212, and crtZ and crtX are derived from Pantoea ananatis. The process of producing glycosylated astaxanthin is: β-carotene is converted into astaxanthin through the catalysis of CBFD and HBFD or CrtW and CrtZ, and then converted into glycosylated astaxanthin through the catalysis of CrtX.

The construction method of the above-mentioned engineering bacteria producing glycosylated astaxanthin includes the following steps:

(1) Construct a recombinant plasmid containing the following nucleotide fragments: hp4d promoter; tr53-CBFD-RIDD fragment; HBFD-RIAD fragment;

Among them, RIDD is the coding gene for the short peptide RIDD, and the nucleotide sequence is as shown in SEQ ID NO.5; RIAD is the coding gene for the short peptide RIAD, and the nucleotide sequence is as shown in SEQ ID NO.6;

Or: construct a recombinant plasmid containing the following nucleotide fragments: crtZ, crtW;

(2) Construct a recombinant plasmid containing crtX;

(3) Transform β-carotene-producing Yarrowia lipolytica with the recombinant plasmid constructed in step (1) to obtain a transformant; then transform the transformant with the recombinant plasmid constructed in step (2) to obtain glycosylated shrimp. Cyanin-containing engineering bacteria.

SEQ ID NO.3：

5’-ATGTCTGTGGCTGGAAGAACTCGAAATCTGGACATCCCCAAATCGAGGAGGAGGAGGAACGTGGAGGAG CTGATTGAGCAGACCGACTCTGACATCGTGCACATCAAGAAGACCCTGGGCGGCAAGCAGTCTAAGCGACCCACTGGATCTATCGTGGCCCCTGTGTCTTGCCTGGGCATTCTGTCTATGATCGGCCCCGCCGTTTATTTCAAGTTCTCTCGACTGATGGAGGGCGGCGACATCCCCGTT GCAGAGATGGGCATTACTTTTGCTACCTTTGGTGGCCGCCGCCGTGGGCACTGAATTTCTGTCTGCTTGGGTGCACAAGGAGCTGTGGCACGAGTCTCTGTGGTACATCCACAAGTCTCACCACCGATCTCGAAAGGGCCGATTCGAGTTCAACGACGTGTTCGCCATCATCAACGCCCTGCCCGCTATTGCCCTTATCAACTACGGCTTCTCTAACGAGGGCCTGCTGCCCGGAGCTTGTTTTGGAGTTGGACTGGGAACCACTGT GTGTGGCATGGCTTATCTTTCTGCACAACGGCCTGTCTCACCGACGATTCCCCGTGTGGCTGATTGCCAATGTGCCCTATTTCCACAAGCTGGCCGCCGCCCATCAAATCCATTCTGGAAAGTTTCAGGGCGTGCCCTTTGGCCTGTTTCTGGGCCCTAAAGAGCTGGAGGAGGTGCGAGGAGGAACTGAGGAGCTGGAAAGAGTGATTTCTAGAACCACCAAGCGAACCCAGCCCTCTACCTAA-3’.

SEQ ID NO.4:

5’-ATGGGCGGAACTGGCAAAGTGGGAGGATCTACTGCCCTGGCTCTGTCTAAATTCTCTCCCGACCTGCGACTG GTGATCGGCGGAAGAAATCGAGAGAAAGGCGACGCCGTGGTGTCTAAGCTGGGCGAAAATTCTGAGTTCGTGGAGGTGAACGTGGACTCTGTGCGATCTCTGGAGTCTGCCCTGGAGGACGTGGATCTGGTGGTGCATGCTGCTGGACCTTTTCAAGCTGAGAAGTGTACC GTGCTGGAGGCTGCCATTTCTACCCGAACTGCCTACTGGACGTGTGCGACAATACCTCTTACTCTATGCAGGCCAAGTCTTTCCACGACAAGGCCGTGGCCGCCAATGTGCCTGCCATTACTACTGCCGGAATCTTCCCTGGCGTGTCTAACGTGATCGCCGCCGAACTGGTGAGATCTGCTCGAGATGAAAACACCGAGCCCCAGAGACTGAGATTTTCTTACTTCACCGCCGGCTCTGGCGGCGCTGGACCTACTTCTC TGGTGACTTCTTTTCTGCTGCTGGGCGAGGAGGTGGTGGCCTATTCTGAAGGAGAGAAAGTGGAGCTGAAACCCTACACCGGCAAGCTGAACATCGACTTCGGCAAGGGCGTGGGCAAACGAGATGTGTATCTGTGGAACCTGCCCGAGGTGCGATCTGGCCATGAAATTCTGGGCGTGCCTACCGTGTCTGCCAGATTTGGCA CTGCCCCCTTTTTTTGGAACTGGGCCATGGTGGCCATGACCACCCTGCTGCCTC CTGGAATTCTGCGAGATAGAAATAAAATCGGCATGCTGGCCAACTTCGTGTACCCCTCTGTGCAGATCTTCGACGGCATCGCCGGAGAGTGTCTGGCCATGAGAGTGGATCTGGAGTGCGCCAATGGAAGAAACACCTTCGGCATCCTGTCTCACGAGCCGACTGTCTGTGCTGGTGGGCACTTCTACTGCCGGTTTGCCATGGCCATCCTGGAGGGATCTACCCAGCCTGGCGTTTGGTTTCCTGAAGAACCCGG CGGAATTGCCATCTCTGATCGAGAGCTGCTGCTGCAGAGAGCCTCTCAGGGAGCTATTAATTTCATCATGAAGCAG-3’.

SEQ ID NO.5：

5’-GGAGGAGGCGGATCTGGAGGAGGAGGATTGGAGGAGGAGGATGCGGATCTCTGAGAGAATGTGAACTGTAT GTGCAGAAGCACAACATCCAGGCCCTGCTGAAGGACTCTATCGTGCAGCTGTGCACCGCCCGACCTGAAAGACCTATGGCTTTTCTGAGAGAGTACTTCGAGCGACTGGAGAAGGAGGAGGCCAAGTAA-3’.

SEQ ID NO.6：

5’-GGAGGAGGAGGATCTGGAGGAGGAGGATCTGGAGGAGGAGGATGCGGACTGGAACAATATGCTAATCAGCTG GCTGATCAGATTATCAAGGAGGCCACCGAGGGCTGCTAA-3’.

SEQ ID NO.7:

5’-GCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTA CATTATCGAGACCGTTGTTCCCGCCCACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGACCGTTGTTCCCGCCCACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCA GTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGACCGTTGTTCCCGCCCACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGACCGTTGTTCCCGCCCACCTCGATCCGGCATGCACTGATCACGGGCAAAAGTGCGTATATATACAAGAGCGTTTGCCAGCCACAGATTTTCACTCCACACACC ACATCACACATACAACCACACACATCCACGATG-3’.

SEQ ID NO.8：

5’-ATGAGCCATTTCGCGGCGATCGCACCGCCTTTTTACAGCCATGTTCGCGCATTACAGAATCTCGCTCAGGAA CTGGTCGCGCGCGGTCATCGGGTGACCTTTATTCAGCAATACGATATTAAACACTTGATCGATAGCGAAACCATTGGATTTCATTCCGTCGGGACAGACAGCCATCCCCCCGGCGCGTTAACGCGCGTGCTACACCTGGCGGCTCATCCTCTGGGGCCGTCAATGCTGAAGCTCATCAATGAAATG GCGCGCACCACCGATATGCTGTGCCGCGAACTCCCCAGGCATTTAACGATCTGGCCGTCGATGGCGTCATTGTTGATCAAATGGAACCGGCAGGCGCGCTCGTTGCTGAAGCACTGGGACTGCCGTTTATCTCTGTCGCCTGCGCGCTGCCTCTCAATCGTGAACCGGATATGCCCCTGGCGGTTATGCCTTTCGAATACGGGACCAGCGACGCGGCTCGCGAACGTTATGCCGCCAGTGAAAAAATTTATGGGCTAA TGCGTCGTCATGACCGTGTCATTGCCGAACACAGCCACAGAATGGGCTTAGCCCCCCGGCAAAAGCTTCACCAGTGTTTTTCGCCACTGGCGCAAATCAGCCAGCTTGTTCC TGAACTGGATTTTCCCCGCAAAGCGTTACCGGCTTGTTTTCATGCCGTCGGGCCTCTGCGCGAAACGCACGCACCGTCAACGTCTTCATCCCGTTATTTTACATCCTCAGAAAAACCCGGATTTTCGCCTCGCTGGGCACGCTTCAGGGAC ACCGTTATGGGCTGTTTAAAACGATAGTGAAAGCCTGTGAAGAAATTGACGGTCAGCTCCTGTTAGCCCACTGTGGTCGTCTTACGGACTCTCAGTGTGAAAGAGCTGGCGCGAAGCCGTCATACACAGGTGGTGGATTTTGCCGATCAGTCAGCCGCGCTGTCTCAGGCGCAGCTGGCGATCACCCACGGCGGCATGAATACGGTACTGGACGCGATTAATTACCGGACGCCCCTTTTAGCGCTTCCGCTGGCCTTT GATCAGCCCGGCGTCGCGTCACGCATCGTTTATCACGGCATCGGCAAGCGTGCTTCCCGCTTTACCACCAGCCATGCTTTGGCCTCGTCAGATGCGTTCATTGCTGACCAACGTCGACTTTCAGCAGCGCATGGCGAAAATCCAGACAGCCCTTCGTTTGGCAGGGGGCACCATGGCCGCTGCCGATATCATTGAGCAGGTTATGTGCACCGGTCAGCCTGTCTTAAGTGGGAGCGGCTATGCAACCGCATTATGA- 3’.

SEQ ID NO.9:

5’-ATGACCGCCGCCGTCGCCGAGCCCCGAATCGTCCCCCGACAGACCTGGATTGGCCTGACCCTGGCCGGCATG ATTGTGGCCGGCTGGGGCTCCCTCCACGTCTACGGTGTCTACTTCCACCGATGGGGCACCTCTTCCCTCGTGATTGTTCCCGCTATTGTCGCTGTTCAAACCTGGCTGTCTGTTGGACTTTTTATTGTCGCTCACGACGCCATGCATGGTTCTCTGGCTCCTGGTAGACCCGACT TAACGCTGCCGTTGGTAGATTGACCTTGGGTCTCTACGCTGGCTTTAGATTTGATAGGCTGAAAACCGCACACCATGCCCATCACGCCGCTCCTGGTACTGCTGATGATCCTGATTTTTATGCTCCTGCACCTCGTGCTTTTCTCCCTTGGTTCCTCAACTTCTTCCGAACCTACTTCGGCTGGCGAGAGATGGCCGTCCTGACCGCTCTCGTGCTGATCGCCCTGTTCGGCCTCGGCGCCCGACCCGCCAACCTG CTGACCTTCTGGGCCGCCCCGCCCTCCTGTCCGCCCTGCAGCTGTTCACCTTCGGCACCTGGCTCCCCCACCGACACACCGACCAGCCCTTCGCCGACGCTCACCACGCCCGATCCTCTGGTTACGGCCCCGTCCTGTCGCTGCTGACCTGCTTCCACTTCGGCCGACACCACGAGCACCACCTGACCCCCTGGCGACCCTGGTGGCGACTGTGGCGAGGTGAGTCGTAA-3’.

SEQ ID NO.10:

5’-ATGCTGTGGATTTGGAACGCCCTGATTGTCTTTGTTACTGTTATCGGTATGGAAGTGATTGCTGCTCTCGCT CACAAGTACATTATGCACGGCTGGGCTGGGGCTGGCACCTGTCCCACCACGAGCCCCGAAAGGGCGCCTTCGAGGTCAACGACCTGTACGCCGTCGTCTTCGCCGCCCTGTCCATCCTGCTGATCTACCTGGGCTCCACCGGCATGTGGCCACTGCAGTGGATCGGTGCCGGAA TGACCGCCTACGGCCTGCTGTACTTCATGGTCCACGACGGCCTGGTCCACCAGCGATGGCCCTTCCGATACATCCCCCGAAAGGGCTACCTGAAGCGACTGTACATGGCCCACCGAATGCACCACGCCGTCCGAGGCAAGGAGGGCTGCGTCTCTTTCGGCTTCCTGTACGCCCCCCTGTCCAAGCTGCAGGCCACCCTGCGAGAGCGACATGGTGCTAGAGCTGGCGCCGCCCGAGACGCTCAGGGAGGGTGAGGAT GAGCCTGCTTCCGGAAAGTAA-3’.

Application of the engineered bacteria producing glycosylated astaxanthin in the preparation of glycosylated astaxanthin. For specific applications, engineered bacteria that produce glycosylated astaxanthin are cultured and glycosylated astaxanthin is extracted.

A method for preparing glycosylated astaxanthin: culturing the above-mentioned engineering bacteria producing glycosylated astaxanthin or the engineering bacteria producing glycosylated astaxanthin constructed by the above method, and extracting the glycosylated astaxanthin white.

Further, the method for preparing glycosylated astaxanthin specifically includes: inserting the engineered bacteria producing glycosylated astaxanthin into the culture medium, cultivating it to obtain a seed culture liquid; inserting the seed culture liquid into the culture medium, Culture the fermentation broth to obtain the fermentation broth; take the fermentation broth, centrifuge to obtain the bacterial cell precipitate and fermentation supernatant, and extract the bacterial cell precipitate to obtain glycosylated astaxanthin. The conditions for culturing the seed culture liquid can be: 30°C, 200 rpm for 24 hours; the inoculum amount of the seed culture liquid can be 2%; the culture medium can be YPD culture medium; the fermentation liquid The culture conditions can be: 30°C, 200 rpm for 84 hours.

The present invention uses Yarrowia lipolytica as a host to synthesize carotenoids, which has unique advantages: first, Yarrowia lipolytica can synthesize a large amount of acetyl-CoA as the precursor of the MVA pathway, which is more conducive to the accumulation of carotenoids. Secondly, a large amount of lipids accumulate in yeast cells, forming a good hydrophobic space that can potentially store lipophilic carotenoids. Thirdly, Yarrowia lipolytica has low requirements for the growth environment, can use a variety of low-cost carbon sources as its culture substrate, has high osmotic pressure and is extremely tolerant to various pH values. In addition, the genetic background of this strain is clear, and relatively complete genetic and metabolic modification tools have been developed in recent years. It is an ideal industrial host strain for accumulating glycosylated astaxanthin and other carotenoids.

The present invention produces glycosylated astaxanthin in Yarrowia lipolytica for the first time. Under the combined action of CBFD and HBFD or CrtW and CrtZ, β-carotene can be synthesized into astaxanthin. This synthesis pathway is unique compared with the astaxanthin synthesis pathway in microorganisms and algae. Currently, this pathway is only found in the large intestine. Bacilli. HBFD only acts on carotenoids with a 4-hydroxy-β ring. At the same time, CBFD cannot hydroxylate the No. 3 carbon of the unmodified β ring or the 4-hydroxy-β ring. This feature allows it to be strictly controlled. The sequence of catalytic reactions contributes to the efficient synthesis of astaxanthin without the production of intermediate products with hydroxyl groups, which is very beneficial for the production, extraction and purification of glycosylated astaxanthin.

The present invention uses synthetic biology technology to introduce genes required for the synthesis of glycosylated astaxanthin into β-carotene-producing Yarrowia lipolytica, obtain a strain that produces glycosylated astaxanthin, and regulate it through metabolic engineering strategies The production of astaxanthin in the engineering strain ultimately improves the production of glycosylated astaxanthin by yeast fermentation. The glycosylated astaxanthin produced by the engineering strain OUC-AdGHB1-7CJ screened in the present invention The production of astaxanthin reached 27.274mg/L, making it the engineering strain with the highest production of glycosylated astaxanthin. This provides the feasibility for the efficient synthesis of other glycosylated carotenoids in microorganisms and lays the foundation for industrial production.

Various terms and phrases used herein have their ordinary meanings known to those skilled in the art.

Description of drawings

The engineered bacterium that produces glycosylated astaxanthin of the present invention has a strain name of OUC-AdGHB1-7CJ, and the date of preservation is August 1, 2022. The preservation unit is the General Microbiology Center of the Chinese Microbial Culture Collection Committee, and the preservation number is CGMCC NO.25446, classified as Yarrowia lipolytica, is deposited at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

Figure 1: Liquid phase detection chart of F-A1 and F-A2 strain fermentation samples.

Figure 2: Liquid phase detection chart of fermentation samples of strains F-A3, F-A4, F-A5, F-X1, F-X2, and F-X3.

Figure 3: Schematic diagram of the determination results of astaxanthin production of strains F-A2, F-A3 and F-A4.

Figure 4: Schematic diagram of the determination results of astaxanthin production of F-A5 strain.

Figure 5: Schematic diagram of the determination results of glycosylated astaxanthin production by F-X3 strain.

Figure 6: Schematic diagram of the determination results of glycosylated astaxanthin production by F-X1, F-X2, and F-X3 strains.

Detailed ways

The present invention will be further described below in conjunction with the examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications can be made to the present invention without departing from the spirit and scope of the invention.

Unless otherwise specified, the instruments, reagents, and materials involved in the following examples are conventional instruments, reagents, and materials that are already in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples are all conventional experimental methods and detection methods existing in the prior art unless otherwise specified.

The Yarrowia lipolytica strain Polh and the pMT015 plasmid involved in the present invention were donated by Wang Shi'an of Qingdao Institute of Bioenergy and Engineering, Chinese Academy of Sciences.

The strain F0 involved in the present invention is obtained by introducing carB (phytoene dehydrogenase) and carRP (tomato red cyclase/phytoene synthase) genes into Yarrowia lipolytica Polh. Obtained, its β-carotene yield is 21.2 mg/L. The specific construction method is as follows: carB and carRP genes were entrusted to Sangon Bioengineering (Shanghai) Co., Ltd. for full gene synthesis. The synthesized genes were used as templates and PCR amplification was performed to obtain carB. and carRP gene fragments, using pMT015 plasmid as a template, PCR amplified the plasmid skeleton, used homologous recombination seamless splicing technology for connection, transformed the recombinant plasmid into E. coli DH5α competent cells, and used a rapid plasmid miniprep kit to extract the plasmid . The plasmid was linearized by PCR, and the linear DNA fragment was randomly inserted into the Yarrowia lipolytica genome using chemical transformation. The strain with the highest β-carotene production was screened out through high-performance liquid chromatography, which is F0. References for specific methods involved in constructing strain F0: Zhang Guilin. Construction and regulation of β-carotene synthesis pathway in Yarrowia lipolytica: [Master's thesis]. Qingdao: Ocean University of China, 2022.

The strain F1 involved in the present invention is transformed into strain F0 by transforming the ERG12S gene derived from Saccharomyces cerevisiae, the modular IDI-GGS1 gene, the modular ERG20Y-GGS1 gene and the mvaE-mvaSMT gene derived from Enterococcus faecalis. Among them, the strain with the highest beta-carotene production was screened through high-performance liquid chromatography, and its beta-carotene production was 607.1 mg/L. References for specific construction methods: Zhang Guilin. Construction and regulation of β-carotene synthesis pathway in Yarrowia lipolytica: [Master's thesis]. Qingdao: Ocean University of China, 2022.

The basic descriptions of relevant strains and constructed strains used in the present invention are shown in Table 1. The specific primers used in the present invention are shown in Table 2.

Table 1

strain Genotype description or purpose source E. coli DH5α Clone plasmid TsingKe F0(Y.lipolytica PO1h-β) carB-carRP Lab build F-A1-1 CBFD-HBFD-URA The invention constructs F-A1-2 tr53-CBFD-tr45-HBFD-URA The invention constructs F-A2 tr53-CBFD-HBFD-URA The invention constructs F-A3 tr53-CBFD-RIDD-HBFD-RIAD-URA The invention constructs F-A4 P _hp4d -tr53-CBFD-RIDD-HBFD-RIAD-URA The invention constructs F-A5 crtW-crtZ-URA The invention constructs F-X1 tr53-CBFD-HBFD+crtX-URA The invention constructs F-X2 P _hp4d -tr53-CBFD-RIDD-HBFD-RIAD+crtX-URA The invention constructs F-X3 crtW-crtZ-URA+crtX-URA The invention constructs Pub4-cre Cre plasmid Lab build

Table 2

Example 1 Construction of astaxanthin-producing strain F-A1

The two enzymes CBFD and HBFD jointly catalyze the reaction of β-carotene to generate astaxanthin. Therefore, in this example, the CBFD and HBFD genes are further integrated on the basis of the β-carotene-producing engineering strain F0 to construct an astaxanthin-producing chassis. strains.

(1) Construction of pMT-1 plasmid

(1) The CBFD gene derived from Calendula officinale was codon-optimized based on the preference of Yarrowia lipolytica. The optimized nucleotide sequence is shown in SEQ ID NO.1, commissioned by Beijing Liuhe BGI Technology Ltd. artificially synthesized this gene fragment. Using the synthesized gene fragment as a template, the CBFD fragment of 921 bp was amplified by PCR using primers CBFD-1-F/CBFD-1-R and P525 enzyme.

Using plasmid pMT015 as a template, PCR amplification was performed with primers PMT-1-F/PMT-1-R to obtain 8422 bp plasmid backbone 1, which was recovered using a PCR recovery kit. The recovered CBFD fragment and plasmid backbone 1 were used Homologous recombination seamless splicing technology was used to connect the plasmid pMT-a.

(2) The HBFD gene derived from Calendula officinale was codon-optimized based on the preference of Yarrowia lipolytica. The optimized nucleotide sequence is shown in SEQ ID NO.2 and was commissioned by Beijing Liuhe BGI. Technology Co., Ltd. artificially synthesized the gene fragment. Using the synthesized gene fragment as a template, the HBFD fragment of 1221 bp was amplified by PCR using primers HBFD-1-F/HBFD-1-R and P525 enzyme.

Use plasmid pMT-a as a template and use primers PMT-2-F/PMT-2-R to perform PCR amplification to obtain 9187 bp plasmid backbone 2. Use a PCR recovery kit to recover the recovered HBFD fragment and plasmid backbone. 2 Use homologous recombination seamless splicing technology to connect and obtain plasmid pMT-1.

SEQ ID NO.1：

5’-ATGGCAATTTCAGTGTTTCAGTTCAGGTTATTCTTTCTACAAGAATCTCTTGTTGGACTCAAAACCAAATATT CTCAAACCCCCATGCCTGCTATTCTCTCCAGTTGTGATCATGTCGCCTATGAGAAAGAAAAAGAAACATGGTGATCCATGTATCTGCTCCGTTGCAGGGAGAACAAGGAACCTTGATATTCCTCAAATTGAAGAAGAGGAAGAGAATGTGGAAGAACTAA TAGAACAGACCGATTCT GACATAGTGCATATAAAGAAAACACTAGGGGGGGAAACAATCAAAACGGCCCACTGGCTCCATTGTCGCACCCGTATCTTGTCTTGGGATCCTTTCAATGATTGGACCTGCTGTTTACTTCAAGTTTTCACGGCTAATGGAGGGTGGAGATATACCTGTAGCAGAAATGGGGATTACGTTTGCCACCTTTGTTGCTGCTGCTGTTGGCACGGAGTTTTTGTCAGCATGGGTTCACAAAGAACTCTGGCACGAGTCTTTGTGGTA CATTCACAAGTCTCACCATCGGTCACGAAAAGGCCGCTTCGAGTTCAATGATGTGTTTGCTATTATTAACGCGCTTCCCGCTATTGCTCTTATCAATTATGGATTCTCCAATGAAGGCCTCCTTCCTGGAGCGTGCTTTGGTGTCGGTCTTGGAACAACAGTCTGTGGTATGGCTTACATTTTTCTTCACAATGGCCTATCACACCGAAGGTTCCCAGTATGGCTTATTGCGAACGTCCTTATTTCCACAAGCTGGCTG CAGCTCACCAAATACACCACTCAGGAAAATTTCAGGGTGTACCATTTGGCCTGTTCCTTGGACCCAAGGAATTGGAAGAAGTAAGAGGAGGCACTGAAGAGTTGGAGAGGGTAATCAGTCGTACAACTAAACGAACGCAACCATCTACC-3’.

SEQ ID NO.2：

5’-ATGGCTCCTGTGCTGCTGGGACTGAAGCCTACTCTGTCTACTGGCTCTGTGGTGAAGGAGACTAACGTGGGGC TCTACCCTGGCCTCTCCTCTGAATAAAACCCAGAACTCTCGAGTGCTGGTGCTGGGCGGAACTGGCAAAGTGGGAGGATCTACTGCCCTGGCTCTGTCTAAATTCTCTCCCGACCTGCGACTGGTGATCGGCGGAAGAAATCGAGAGAAAGGCGACGCTGTGGTCTAAGC TGGGCGAAAATTCTGAGTTCGTGGAGGTGAACGTGGACTCTGTGCGATCTCTGGAGTCTGCCCTGGAGGACGTGGATCTGGTGGTGCATGCTGCTGGACCTTTTCAACAAGCTGAGAAGTGTACCGTGCTGGAGGCTGCCATTTCTACCCGAACTGCCTACGTGGACGTGTGCGACAATACCTCTTACTCTATGCAGGCCAAGTCTTTCCACGACAAGGCCGTGGCCGCCAATGTGCCTGCCATTACTACTGCCGGAATCTTC CCTGGCGTGTCTAACGTGATCGCCGCCGAACTGGTGAGATCTGCTCGAGATGAAAACACCGAGCCCCAGAGACTGAGATTTTCTTACTTCACCGCCGGCTCTGGCGGCGCTGGACCTACTTCTCTGGTGACTTCTTTTCTGCTGCTGGGCGAGGAGGTGGTGGCCTATTCTGAAGGAGAGAAAGTGGAGCTGAAACCCTACACCGGCAAGCTGAACATCGACTTCGGCAAGGGCGTGGGCAAACGAGATGTGTATCTG TGGAACCTGCCCGAGGTGCGATCTGGCCATGAAATTCTGGGCGTGCCTACCGTGTCTGCCAGATTTGGCACTGCCCCCTTTTTTTGGAACTGGGCCATGGTGGCCATGACCACCCTGCTGCCTCCTGGAATTCTGCGAGATAGAAATAAAATCGGCATGCTGGCCAACTTCGTGTACCCCTCTGTGCAGATCTTCGACGGCATCGCCGGAGAGAGTGTCTGGCCATGAGAGTGGATCTGGAGTGCGCCAATGGAAGAAACACC TTCGGCATCCTGTCTCACGAGCGACTGTCTGTGCTGGTGGGCACTTCTACTGCCGGTTTGCCATGGCCATCCTGGAGGGATCTACCCAGCCTGGCGTTTGGTTTCCTGAAGAACCCGGCGGAATTGCCATCTCTGATCGAGAGCTGCTGCTGCAGAGAGCCTCTCAGGGAGCTATTAATTTCATCATGAAGCAG-3’.

(2) Construction of astaxanthin-producing strain F-A1

Linearize the pMT-1 plasmid through PCR, transform the linearized target fragment into strain F0, and obtain the engineering strain F-A1, ferment it, and determine the production of astaxanthin and other carotenoids. The specific steps are as follows:

(A) Plasmid linearization

Using pMT-1 plasmid as a template, use primers 1-F/1-R to linearize the target fragment by PCR, and use a PCR recovery kit to recover the linearized target fragment.

(B)Conversion

The recovered linearized fragments are chemically transformed into β-carotene-producing Yarrowia lipolytica strain F0. The specific steps are as follows:

(1) Plug F0 into YPD medium (10 mL) and culture it in a temperature-controlled shaker at 30°C and 200 rpm for 36 hours;

(2) Collect an appropriate amount of bacterial cells, resuspend in about 1 mL of 1×TE buffer, centrifuge at 3000g for 2 minutes, discard the supernatant, resuspend in about 1 mL of 0.1M LiAc solution, incubate at 30°C for 1 hour, and centrifuge at 3000g for 2 minutes. Discard the supernatant;

(3) Add about 200 μL of 0.1M LiAc solution and resuspend to the appropriate concentration (refer to the competent concentration of E.coli DH5α and other competent cells), and dispense 40 μL/tube;

(4) Add 3 μL fish sperm DNA and 3 μg linear target fragment to each competent tube, mix by pipetting, and incubate at 30°C for 15 minutes;

(5) Add 350 μL PEG-LiAc (315 μL 50% PEG and 35 μL 1M LiAc) and 16 μL 1M DTT, and incubate at 30°C for 1 hour;

(6) Add 40 μL DMSO, heat shock at 39°C for 10 minutes, add 600 μL LiAc, and let stand at room temperature for 30 minutes;

(7) Centrifuge at 3000g for 2 minutes, discard part of the supernatant, and leave about 100 μL of the remaining bacterial solution, mix well, apply SD-URA plate, and incubate in a 30°C incubator for 3 days with an inversion;

(8) Pick 20 single colonies with darker red color from the long bacterial URA plate and insert them into the new SD-URA plate, and incubate them upside down in the 30°C incubator for 24 hours.

(C) Strain fermentation and determination of cell dry weight

Pick 10 colonies from the SD-URA plate in (8) above and insert them into 10 mL of YPD medium, and culture them at 30°C and 200 rpm for 24 hours to obtain a seed culture medium. Add 1 mL of seed culture solution to YPD medium (50 mL) and culture at 30°C and 200 rpm for 84 hours.

After the fermentation is completed, take 1 mL of the bacterial liquid in a bacteria preservation tube containing 500 μL of 50% glycerol and store it at -20°C. Then take 2 mL of the bacterial liquid and add it to a 2 mL weighed EP tube. Centrifuge at 12,000 rpm for 2 min. Discard the supernatant and open the lid. Place in an 80°C oven for 24 hours, weigh, continue to place for 1 hour, weigh again until there is no change in weight, then take it out, and calculate the dry weight of the cells based on the weight change.

(D) Extraction and detection of fermentation products

Add 500 μL of the above remaining bacterial liquid into a 2 mL grinding tube, centrifuge at 12,000 rpm for 3 min, discard the supernatant, add 1 mL of methyl tert-butyl ether, mix well, and grind in the grinder according to the program of 65 Hz, 120 s/0 Hz, 10 s, 10 cycles. After grinding, centrifuge at 12,000 rpm for 3 minutes, put the supernatant solution into a 5 mL centrifuge tube, blow dry the supernatant with nitrogen blowing, and reconstitute it with 500 μL acetone solution. The reconstituted liquid is filtered with a 0.22 μm organic filter. , for detection.

Fermentation products were detected using high performance liquid chromatography. The detection conditions are chromatographic column: C18 liquid chromatography column; column temperature: 35°C; flow rate: 0.9mL/min; injection volume: 20μL; detection wavelength: 470nm; detection time: 45min; mobile phase: A water, B acetonitrile, Tetrahydrofuran (1:1), the elution gradient is as follows (min-%A): 0-95; 5-95; 15-20; 24-20; 25-0; 35-0; 40-95; 45-95.

The peak elution times of glycosylated astaxanthin, astaxanthin, β-carotene, and lycopene were 18.4, 23.2, 34.0, and 33.3 minutes respectively.

Accurately weigh a certain amount of astaxanthin standard, dissolve it in acetone, and dilute it to different times. Use the above method to measure the peak areas of different concentrations of the standard using high performance liquid chromatography. Draw a standard curve based on the concentration and peak area of the standard. Substitute the peak area of the extracted sample of the bacteria to be tested by HPLC into the standard curve to obtain the astaxanthin content of the bacteria to be tested (mg/L, specifically the astaxanthin content per liter of bacterial liquid).

The liquid phase test results showed that none of the 10 strains produced astaxanthin. The liquid phase test chart of the fermentation broth of one strain, F-A1, is shown in Figure 1. Considering that CBFD and HBFD are derived from plants, TargetP-2.0 was used to analyze the complete protein sequence and predict the sequences of the chloroplast transit peptides of CBFD and HBFD. CBFD and HBFD may have chloroplast transit peptides that affect the expression of the enzyme. Therefore, in order to obtain astaxanthin-producing strains, the following experiments will remove the chloroplast transit peptide sequences of CBFD and HBFD.

Example 2 Construction of astaxanthin-producing strain F-A2 with removal of chloroplast transit peptide

(1) Construction of CBFD and HBFD plasmid pMT-2 that removes chloroplast transit peptides

Use the TargetP-2.0 website to predict the chloroplast transit peptides of the amino acid sequences of CBFD and HBFD, and based on the prediction results, remove the chloroplast transit peptide sequences of CBFD and HBFD respectively, and use the primers CBFD-2-F/CBFD-2-R to predict the chloroplast transit peptides of CBFD and HBFD. The gene was amplified as a template to obtain a tr53-CBFD fragment of 768 bp (the nucleotide sequence excluding the chloroplast transit peptide sequence is shown in SEQ ID NO.3), and primers HBFD-2-F/HBFD-2-R were used to HBFD-based Because the tr45-HBFD fragment was obtained by template amplification (the nucleotide sequence with the chloroplast transit peptide sequence removed is shown in SEQ ID NO. 4).

Use primers PMT-3-F/PMT-3-R to amplify plasmid pMT-1 as a template to obtain 9507 bp plasmid backbone 3. The amplified tr53-CBFD fragment and plasmid backbone 3 were recovered by PCR, and homologous recombination was used. Seamless splicing technology is used to connect and construct pMT-2-1. Using primers PMT-10-F/PMT-10-R and plasmid pMT-2-1 as a template, plasmid backbone 10 was amplified. The amplified tr45-HBFD fragment and plasmid backbone 10 were recovered by PCR, and homologous recombination was used. Seamless splicing technology is used to connect and build pMT-2-2.

(2) Effect of chloroplast transit peptides removing CBFD and HBFD on astaxanthin production

Linearize pMT-2-1 and pMT-2-2 plasmids by PCR using primers 1-F/1-R, and transform the linearized target fragment into strain F0 to obtain engineering bacteria F-A2-1 and F- A2-2 (Note: After transforming strain F0 with pMT-2-1, apply SD-URA-deficient medium. After 3 days, pick the 20 transformants with the darkest red color and transfer them to the new SD-URA-deficient medium for 24 hours. Then, the 10 transformants with the deepest red color were picked and inserted into 50 mL liquid YPD medium, and cultured at 200 rpm and 30°C for 84 hours; all 10 strains produced astaxanthin, and the strain with the highest astaxanthin production was named F-A2. -1) (Similarly, F-A2-2 and the strains in the following examples, unless otherwise stated, are all screened in this way), ferment them, and measure the production of astaxanthin and other carotenoids. .

The liquid phase detection diagram of the fermentation broth of F-A2-1 and F-A2-2 strains is shown in Figure 1. The F-A2-1 strain produced astaxanthin. The measurement results of astaxanthin content are shown in Figure 3. Strain F-A2-1 produced 0.597mg/L (0.054mg/g DCW) astaxanthin, but strain F-A2-2 did not produce astaxanthin, indicating that CBFD It is active when removing the predicted chloroplast transit peptide, while HBFD is inactive when removing the predicted chloroplast transit peptide. Strain F-A2-1 also accumulates more β-carotene, and the conversion rate of β-carotene is low. This may be due to the low expression levels of CBFD and HBFD. To increase the production of astaxanthin, it is necessary to control CBFD and HBFD. Make a transformation.

Example 3 Construction of astaxanthin production-improving strains F-A3 and F-A4

Constructing multi-enzyme complexes can prevent the diffusion of intermediate products, increase the yield of final products, and control the flux of metabolites. Modular assembly of CBFD and HBFD can be achieved through a pair of short peptide tags (RIAD and RIDD). Expressing the tr53-CBFD-RIDD-HBFD-RIAD enzyme complex in the β-carotene-producing strain F0 can increase the production of astaxanthin; the increase in promoter strength can increase the transcription level of the gene, thereby increasing the expression of the enzyme. , replacing the promoter of CBFD with the stronger hp4d can also increase the yield of astaxanthin. The specific method is as follows:

(1) Construction of CBFD and HBFD modular assembly plasmid pMT-3

The gene sequence of the short peptide RIDD (the nucleotide sequence of which is shown in SEQ ID NO.5) and the gene sequence of RIAD (the nucleotide sequence of which is shown in SEQ ID NO.6) were together with tr53-CBFD and HBFD. Synthesis (entrusted to Beijing Liuhe Huada Gene Technology Co., Ltd. for artificial synthesis), using the synthesized fragment as a template, using primers CBFD-3-F/CBFD-3-R and P525 enzyme to PCR amplify the tr53-CBFD-RIDD fragment of 1032 bp ; Use primers HBFD-3-F/HBFD-3-R and P525 enzyme to PCR amplify the 1374bp HBFD-RIAD fragment.

Use primers PMT-4-F/PMT-4-R to amplify plasmid pMT015 as a template to obtain plasmid backbone 4. The amplified tr53-CBFD-RIDD fragment and plasmid backbone 4 were recovered by PCR, and homologous recombination was used to seamlessly Splicing technology was used to connect and construct plasmid pMT-b.

Use primers PMT-5-F/PMT-5-R to amplify plasmid pMT-b as a template to obtain plasmid backbone 5. The amplified HBFD-RIAD fragment and plasmid backbone 5 were recovered by PCR, and homologous recombination was used to seamlessly Splicing technology was used to connect and construct pMT-3.

(2) Construction of plasmid pMT-4 with CBFD replacement promoter

The hp4d sequence (its nucleotide sequence is shown in SEQ ID NO.7) was synthesized by Beijing Liuhe BGI Technology Co., Ltd. and used as a template to amplify the hp4d fragment using primers hp4d-1-F/hp4d-1-R.

The primers PMT-6-F/PMT-6-R were used to amplify plasmid pMT-3 as a template to obtain plasmid backbone 6. The amplified hp4d fragment and plasmid backbone 6 were recovered by PCR, and homologous recombination seamless splicing technology was used. Ligation was performed to construct pMT-4.

(3) Effects of CBFF and HBFD modular assembly and CBFD promoter replacement on astaxanthin production

The pMT-3 plasmid was linearized by PCR using primers 1-F/1-R, and the linearized target fragment was transformed into strain F0 to obtain engineering strain F-A3, which was fermented to determine astaxanthin and other substances. Carotene production.

The pMT-4 plasmid was linearized by PCR using primers 1-F/1-R, and the linearized target fragment was transformed into strain F0 to obtain engineering strain F-A4, which was fermented to determine astaxanthin and other substances. Carotene production.

All the above were carried out according to the method in Example 1.

The liquid phase detection diagram of the fermentation broth of F-A3 and F-A4 strains is shown in Figure 2A. Both F-A3 and F-A4 strains produced astaxanthin. The measurement results of astaxanthin content are shown in Figure 3. It can be seen that the astaxanthin production of F-A3 and F-A4 strains are 1.112mg/L and 1.480mg/L respectively, which are respectively higher than those of F-A2-1 strain. It increased by 86.3% and 1.48 times, among which the F-A4 strain had the highest astaxanthin production of 1.480mg/L, and the unit cell production was 0.142mg/g DCW.

Example 4 Construction of glycosylated astaxanthin-producing strains F-X1 and F-X2

The crtX gene from Pantoea ananatis ATCC 19321, which has been verified by in vitro enzymology to synthesize glycosylated astaxanthin using UDP-glucose and astaxanthin, was transformed into astaxanthin-producing Yarrowia lipolytica F-A2-1 and F In -A4, glycosylated astaxanthin is produced. The specific method is as follows:

(1) Construction of pMT-5 plasmid

The crtX gene derived from Pantoea ananatis ATCC 19321 was codoned and the optimized nucleotide sequence is shown in SEQ ID NO.8. Beijing Liuhe Huada Gene Technology Co., Ltd. was entrusted to synthesize the gene fragment. The synthesized gene fragment is Template, use primers crtX-1-F/crtX-1-R to amplify a 1296bp crtX fragment.

Using primers PMT-9-F/PMT-9-R and using plasmid pMT015 as a template, an 8275 bp plasmid backbone 9 was amplified. The amplified crtX fragment and plasmid backbone 9 were recovered by PCR, and homologous recombination seamless splicing technology was used. Ligation was performed to construct pMT-5.

(2) Construction of glycosylated astaxanthin-producing strains F-X1 and F-X2

The pMT-5 plasmid was linearized by PCR using primers 2-F/2-R, and the linearized target fragment was transformed into strain F-A2-1 to obtain engineering strain F-X1. The linearized target fragment was transformed into Strain F-A4, the engineering strain F-X2 was obtained, fermented, and the production of glycosylated astaxanthin and other carotenoids was measured.

All the above were carried out according to the method in Example 1.

The liquid phase detection diagram of the fermentation broth of F-X1 and F-X2 strains is shown in Figure 2B. At 18.4 minutes, both F-X1 and F-X2 strains produced glycosylated astaxanthin. The measurement results of glycosylated astaxanthin content are shown in Figure 6. The yield of glycosylated astaxanthin in F-X1 strain is 0.741mg/L, and the yield of glycosylated astaxanthin in F-X2 strain is 1.157mg. /L, which is 56.2% higher than that of F-X1 strain.

Example 5 Construction of astaxanthin-producing strain F-A5 derived from bacteria CrtW and CrtZ

(1) Construction of crtW and crtZ plasmid pMT-6

The crtW gene derived from Brevundimonas sp.SD212 and the crtZ gene derived from Pantoea ananatis were codon-coded and the optimized nucleotide sequences are shown in SEQ ID NO.9 and 10 respectively, and were commissioned to be synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. For this gene fragment, use the synthesized gene fragment as a template, use primers crtW-1-F/crtW-1-R and crtZ-1-F/crtZ-1-R to amplify the crtW and crtZ fragments respectively, and use primer PMT-7 -F/PMT-7-R used plasmid pMT015 as a template to amplify plasmid backbone 7, and used homologous recombination seamless splicing technology to connect the crtW fragment and backbone 7 to construct plasmid pMT-c;

Plasmid backbone 8 was amplified using primers PMT-8-F/PMT-8-R using plasmid pMT-c as a template. The amplified crtZ fragment and plasmid backbone 8 were recovered by PCR, and homologous recombination seamless splicing technology was used. Ligation was performed to construct pMT-6.

(2) Construction of astaxanthin-producing strains derived from bacteria CrtW and CrtZ

The pMT-6 plasmid was linearized by PCR using primers 1-F/1-R, and the linearized target fragment was transformed into strain F1 to obtain engineering strain F-A5 (a total of 10 strains were obtained, each named F-A5 -1～F-A5-10), ferment it, and measure the production of astaxanthin and other carotenoids.

All the above were carried out according to the method in Example 1.

The liquid phase detection chart of the strain fermentation broth is shown in Figure 2A. The F-A5 strain produced astaxanthin (the F-A5 strain in Figure 2A, specifically F-A5-8), as shown in Figure 4. Astaxanthin production The highest strain, F-A5-8, produced 79.077mg/L (7.567mg/gDCW) astaxanthin.

Example 6 Construction of glycosylated astaxanthin-producing strain F-X3 derived from bacterial pathways

The crtX gene from Pantoea ananatis ATCC 19321, which has been verified by in vitro enzymology to synthesize glycosylated astaxanthin using UDP-glucose and astaxanthin, was transferred into astaxanthin-producing Yarrowia lipolytica F-A5 to produce sugar. Kylated astaxanthin, the specific method is as follows:

The pMT-6 plasmid was linearized by PCR using primers 2-F/2-R, and the linearized target fragment was transformed into strain F-A5-8 to obtain engineering strain F-X3 (a total of 10 strains were obtained, temporarily named (F-X3-1～F-X3-10), ferment them (pick 10 colonies and insert them into 10mL YPD medium respectively, culture them at 30℃, 200rpm for 24h to obtain the seed culture liquid; take 1mL seed culture liquid and add Into 50mL YPD medium, culture at 30°C, 200rpm for 84h), and measure the production of glycosylated astaxanthin and other carotenoids.

All the above were carried out according to the method in Example 1.

The liquid phase detection chart of the strain fermentation broth is shown in Figure 2B. It can be seen that strain F-X3 produces glycosylated astaxanthin. The determination results of glycosylated astaxanthin content of F-X3 strain are shown in Figure 5 and Figure 6 (F-X3 in Figure 6 is F-X3-7 strain), in which the glycosylated astaxanthin content of F-X3-7 strain The yield of cyanin is the highest, 27.274 mg/L, which is significantly higher than that of other strains (the yields of the other 9 strains F-X3-1, 2, 3, 4, 5, 6, 8, 9, and 10 are: 8.079 mg respectively) /L, 7.975mg/L, 7.445mg/L, 7.066mg/L, 6.909mg/L, 6.666mg/L, 7.035mg/L, 9.044mg/L, 7.514mg/L), the present invention has carried out Preserved, the strain is officially named OUC-AdGHB1-7CJ, the preservation date is August 1, 2022, the preservation unit is the General Microbiology Center of the Chinese Microbial Culture Collection Committee, the preservation number is CGMCC NO.25446, and the classification name is Lipolytica Yarrowia lipolytica is deposited at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art will be within the scope of the appended claims.

Claims (10)

1. An engineering bacterium for producing glycosylated astaxanthin, which is characterized in that: the strain is named OUC-AdGHB1-7CJ, and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.25446 in the year 08 and 01, and is named yarrowia lipolytica Yarrowia lipolytica in a classification.

2. Use of the engineered bacterium for producing glycosylated astaxanthin according to claim 1 for the preparation of glycosylated astaxanthin.

3. The use according to claim 2, characterized in that: in specific application, engineering bacteria for producing glycosylated astaxanthin are cultured, and the glycosylated astaxanthin is obtained by extraction.

4. The use according to claim 3, wherein the culturing is performed in the following manner: inoculating engineering bacteria producing glycosylated astaxanthin into a culture medium, and culturing to obtain seed culture solution; inoculating the seed culture solution into a culture medium, and culturing to obtain a fermentation broth; and (3) taking fermentation liquor, centrifuging to obtain bacterial precipitate and fermentation supernatant, and extracting the bacterial precipitate to obtain the glycosylated astaxanthin.

5. The use according to claim 3, wherein the conditions of the culture of the seed culture are: culturing at 30 ℃ for 24 hours at 200 rpm; the inoculation amount of the seed culture solution is 2%; the culture conditions of the fermentation broth are as follows: culturing at 30℃for 84 hours at 200 rpm.

6. An engineering bacterium for producing glycosylated astaxanthin, which is characterized in that: the host is yarrowia lipolytica producing beta-carotene, and the genome of the host comprises the following genes (1) (2) (5) or (3) (4) (5):

(1) the nucleotide sequence of the coding gene HBFD of carotenoid 4-hydroxy-beta-ring 4-dehydrogenase is shown as SEQ ID NO. 2;

(2) the coding gene tr53-CBFD of carotenoid beta-ring 4-dehydrogenase with chloroplast transit peptide removed has a nucleotide sequence shown as SEQ ID NO. 3; the promoter is an hp4d promoter, and the nucleotide sequence is shown in SEQ ID NO. 7;

(3) the nucleotide sequence of the coding gene crtZ of the beta-carotene hydroxylase is shown as SEQ ID NO. 10;

(4) the nucleotide sequence of the coding gene crtW of the beta-carotene ketolase is shown as SEQ ID NO. 9;

(5) the coding gene crtX of glycosyltransferase has a nucleotide sequence shown in SEQ ID NO. 8.

7. The method for constructing the engineering bacterium for producing glycosylated astaxanthin according to claim 1 or 6, comprising the steps of:

(1) Constructing a recombinant plasmid containing the following nucleotide fragments: the hp4d promoter; tr53-CBFD-RIDD fragment; an HBFD-RIAD fragment;

wherein RIDD is the coding gene of short peptide RIDD, and the nucleotide sequence is shown as SEQ ID NO. 5; RIAD is the coding gene of short peptide RIAD, and the nucleotide sequence is shown as SEQ ID NO. 6;

or: constructing a recombinant plasmid containing the following nucleotide fragments: crtZ, crtW;

(2) Constructing a recombinant plasmid containing crtX;

(3) Transforming the recombinant plasmid constructed in the step (1) into yarrowia lipolytica producing beta-carotene to obtain a transformant; and (3) transforming the transformant by using the recombinant plasmid constructed in the step (2) to obtain the engineering bacteria for producing glycosylated astaxanthin.

8. The use of the engineered bacterium for producing glycosylated astaxanthin according to claim 6 for the preparation of glycosylated astaxanthin.

9. The use according to claim 8, characterized in that: in specific application, engineering bacteria for producing glycosylated astaxanthin are cultured, and the glycosylated astaxanthin is obtained by extraction.

10. A method of preparing glycosylated astaxanthin, characterized by: culturing the engineering bacterium for producing glycosylated astaxanthin according to claim 1 or the engineering bacterium for producing glycosylated astaxanthin according to claim 6, and extracting to obtain glycosylated astaxanthin.