CN115261244B

CN115261244B - Culture medium combination and fermentation process for high-yield canthaxanthin by utilizing yarrowia lipolytica

Info

Publication number: CN115261244B
Application number: CN202210764029.1A
Authority: CN
Inventors: 陈正杰; 方毅; 刘敏
Original assignee: Shandong Weiyan Biotechnology Co ltd
Current assignee: Shandong Weiyan Biotechnology Co ltd
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2024-02-23
Anticipated expiration: 2042-06-30
Also published as: CN115261244A

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a culture medium combination and a fermentation process for producing canthaxanthin by utilizing yarrowia lipolytica. The slant culture medium in the culture medium combination comprises 100-190g/L malt extract powder, 15-20g/L agar and 0.05-0.2g/L chloramphenicol; the seed culture medium comprises 5-15g/L glucose, 5-20g/L soybean peptone, 1-10g/L potassium nitrate, 2-12g/L ammonium sulfate and 2-10g/L potassium dihydrogen phosphate; the fermentation medium comprises 120-300g/L glucose, 15-45g/L peptone, 5-20g/L corn steep liquor, 2-12g/L ammonium sulfate, 1-6g/L magnesium sulfate, 1-6g/L manganese sulfate and 20-110g/L rapeseed oil. The invention can realize higher level of 5g/L fermentation titer on a 100L fermentation tank, and has wide industrial application prospect.

Description

Culture medium combination and fermentation process for high-yield canthaxanthin by utilizing yarrowia lipolytica

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a culture medium combination and a fermentation process for producing canthaxanthin by utilizing yarrowia lipolytica.

Background

Cantharidin yellow is an important carotenoid, is dark purple crystal or crystalline powder, is unstable to oxygen and light, and needs to be stored in a light-shielding container filled with inert gas. Cantharidin yellow is widely existing in living bodies in nature, and is often used as a colorant in foods, feeds, cosmetics and the like because of its good coloring effect. Meanwhile, cantharidin yellow is widely applied to the medical field due to the physiological function of cantharidin yellow, is similar to vitamin E, astaxanthin, beta-carotene and the like in the aspect of quenching free radicals, but has the effect of nearly twice that of beta-carotene and fifty times that of vitamin E, and is also called super vitamin E. It has effects in enhancing immunity, delaying aging, preventing senile diseases, and resisting cancer.

The method for obtaining the spot chelating yellow mainly comprises the steps of extracting natural raw materials, chemically synthesizing and biologically fermenting and producing. Since the cantharidin yellow content in various species is very trace, the cantharidin yellow cannot be produced in large quantity by a manual extraction method; at present, chemical synthesis production is mainly adopted in industrial production: including the holochemical synthesis and the beta-carotene oxidation. The whole chemical synthesis method for preparing cantharis yellow has the disadvantages of long route and generally higher cost; the beta-carotene oxidation method is a method for carrying out one-step oxidation by taking beta-carotene as a raw material, has the characteristics of convenient operation, simple equipment and the like compared with the method, is a main method for industrially producing cantharidin yellow, and is divided into a solid-phase grinding method and a double-liquid-phase oxidation method. The method overcomes the defects of the two methods and has the advantages of simple production process, short period, low cost, good product quality and the like. However, the biological fermentation method needs to obtain a proper high-yield strain, and then the strain is developed through a fermentation process to realize high-efficiency production. There are no other published reports of the fermentation process for producing cantharidin yellow.

Disclosure of Invention

Aiming at the technical problem that a method for efficiently producing cantharidin yellow through a fermentation process is lacking at present, the invention provides a culture medium combination and a fermentation process for producing cantharidin yellow by utilizing yarrowia lipolytica, which can realize a higher level of 5g/L of fermentation titer on a 100L fermentation tank and have wide industrialized application prospect.

In a first aspect, the invention provides a combination of media for high production of canthaxanthin by yarrowia lipolytica, comprising a slant medium, a seed medium and a fermentation medium;

wherein, the slant culture medium comprises 100-190g/L malt extract powder, 15-20g/L agar and 0.05-0.2g/L chloramphenicol;

the seed culture medium comprises 5-15g/L glucose, 5-20g/L soybean peptone, 1-10g/L potassium nitrate, 2-12g/L ammonium sulfate and 2-10g/L potassium dihydrogen phosphate;

the fermentation medium comprises 120-300g/L glucose, 15-45g/L peptone, 5-20g/L corn steep liquor, 2-12g/L ammonium sulfate, 1-6g/L magnesium sulfate, 1-6g/L manganese sulfate and 20-110g/L rapeseed oil.

Further, the slant culture medium comprises 130g/L malt extract powder, 20g/L agar and 0.1g/L chloramphenicol;

the seed culture medium comprises 15g/L of glucose, 10g/L of soybean peptone, 2g/L of potassium nitrate, 5g/L of ammonium sulfate and 3g/L of potassium dihydrogen phosphate;

the fermentation medium comprises 260g/L of glucose, 30g/L of peptone, 10g/L of corn steep liquor, 5g/L of ammonium sulfate, 2.5g/L of magnesium sulfate, 2.5g/L of manganese sulfate and 100g/L of rapeseed oil.

In a second aspect, the invention provides a fermentation process for combining high-yield spot chelation yellow by using the culture medium, which comprises the following steps:

(1) Inoculating the frozen and preserved yarrowia lipolytica genetically engineered bacteria into a slant culture medium for activation;

(2) Inoculating the activated strain into a seed culture medium to obtain mature seed liquid;

(3) Inoculating the seed liquid into a fermentation medium, and performing ventilation and feed supplement regulation to obtain fermentation liquid.

Further, the step (2) is specifically to culture at 24-28 ℃ under the conditions of a ventilation ratio of 1vvm and a rotation speed of 200-300r/min to obtain mature seed liquid.

Further, the step (3) is specifically to inoculating the seed liquid into a fermentation medium in an inoculum size of 3% -15%, adjusting the pH value to 5.0-5.3, and fermenting and culturing at the temperature of 24-28 ℃ and the aeration ratio of 1-2.5vvm and the rotating speed of 200-800r/min to obtain the fermentation liquid.

Further, in the step (3), a part of glucose and/or rapeseed oil is added into the culture medium at the beginning of fermentation, and the rest of glucose and/or rapeseed oil is added during the fermentation.

Further, the glucose supplement rate is 4-8.5 mL/(h.L), and the rapeseed oil supplement rate is 1-2 mL/(h.L).

Further, the yarrowia lipolytica genetically engineered bacterium comprises a polynucleotide BSW for encoding beta-carotene ketolase, and the sequence of the polynucleotide BSW is shown as SEQ ID NO. 83. Preferably, the yarrowia lipolytica genetically engineered bacterium is MC-CTX4, and the construction method thereof is described in the Chinese patent application CNC 202210057473. X.

The invention has the beneficial effects that:

the invention provides a special culture medium combination for producing spot yellow by utilizing yarrowia lipolytica, which comprises a carbon source: oils and fats such as rapeseed oil, saccharides such as glucose, etc.; nitrogen source: corn steep liquor, peptone, etc.; ammonium salts such as ammonium sulfate; nitrates such as potassium nitrate, ammonium nitrate, etc.; and various phosphates, various metal salts such as manganese salts, magnesium salts, and the like. Meanwhile, the invention also provides a method for producing the spot-chelating yellow with high yield, which can ferment and produce cantharidin yellow fermentation liquor with the concentration of up to 5g/L, thereby greatly reducing the threshold of producing cantharidin yellow by a fermentation method.

Detailed Description

In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

Yarrowia lipolytica MC209 used in the invention is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and has a preservation address of North Silu No. 1, no. 3, a preservation date of 2021, 11 months and 08, and a preservation number of CGMCC No.23758.

Yarrowia lipolytica MYA-2613 used in the present invention was purchased from ATCC; pMD19-T simple vector was purchased from TAKARA corporation; one Step Fusion Cloning Mix kit is available from TOROIVD company.

The BSW gene, the carB gene (genebank accession number: AJ 238028), the carRP gene (genebank accession number: AJ 250827) and the leu selection marker (genebank accession number: M37309) used in the present invention were synthesized by Hongsu Biotechnology Co., ltd, wherein the BSW gene, the carB gene and the carRP gene refer to polynucleotides of the genes.

The recoverable hisG-URA3-hisG selection marker used in the present invention can be constructed by the method described in the literature (Voth WP, richards JD, shaw JM, et al Yeast vectors for integration at the HO locus.nucleic acids Res. 2001,29 (12): E59), wherein URA3 (genebank accession number: AJ 306421) is synthesized by Suzhou Biotech Co., ltd., and added with hisG sequences (genebank accession number: AF 324729) at both ends thereof, and inserted into pUC57 vector to obtain pUC-HUH.

GGS1 gene (polynucleotide sequence shown as SEQ ID NO: 84), tHMGR (polynucleotide sequence shown as SEQ ID NO: 85), promoter TEFp, EXPp, GDPp, terminator PEXt, XPRt, LIPt and the like used in the invention are all derived from yarrowia lipolytica MYA-2613 and obtained by a PCR method.

EXAMPLE 1 construction of yarrowia lipolytica genetically engineered bacterium MC-CTX4

The yarrowia lipolytica MC209 is taken as an initial strain, the yarrowia lipolytica MC209 is an MC-3RP-GH-FOA strain recorded in China patent application No. CNC 202210057473.X, the strain is obtained by taking yarrowia lipolytica MYA-2613 purchased from ATCC as an initial strain, introducing copied beta-carotene synthesis genes carB and carRP into the initial strain, enhancing expression of a geranylgeranyl pyrophosphate (GGPP) synthase gene GGS1 and a truncated HMG-CoA reductase gene tHMGR of the yarrowia lipolytica, and the yarrowia lipolytica MC209 is preserved in China general microbiological culture Collection center with the preservation address of North Korea of Beijing, the North Korea, the accession number of North Korea, the year 2021, the month 08, and the preservation number of CC CGMCC No.23758.

Firstly, respectively introducing two different BSW recombinant plasmids into yarrowia lipolyticaYarrowia lipolytica) And (3) in the MC209 cells, integrating the cells on a yarrowia lipolytica MC209 chromosome through homologous recombination of a homology arm, and then sequentially transferring the recombinant plasmids pMD-H7BRP and pMD-H8GH into the strain to obtain the yarrowia lipolytica genetic engineering strain. The method comprises the following specific steps:

preparation of S1 recombinant plasmid pMD-H1BRP

The method comprises the steps of using a yarrowia lipolytica MYA-2613 chromosome as a template, amplifying homologous arm sequences on the chromosome by using primers H1U-F/R (SEQ ID NO: 1-2) and H1D-F/R (SEQ ID NO: 3-4), and connecting the two by overlapping PCR (polymerase chain reaction) and inserting the two into a pMD19-T simple vector to obtain a pMD-H1 plasmid;

using yarrowia lipolytica MYA-2613 chromosome as a template, and respectively amplifying a promoter TEFp and a terminator PEXt by using primers TEF1-F/R (SEQ ID NO: 5-6) and PEX1-F/R (SEQ ID NO: 9-10);

using pUC-CB as a template and using a primer CB1-F/R (SEQ ID NO: 7-8) to amplify a carB gene; then constructing a TEFp-carB-PEXt module by overlapping PCR;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, and the primers EXP1-F/R (SEQ ID NO: 11-12) and XPR1-F/R (SEQ ID NO: 15-16) are used for respectively amplifying the promoter EXPp and the terminator XPRt;

using pUC-CRP as a template, and amplifying the carRP gene by using a primer CRP1-F/R (SEQ ID NO: 13-14); then constructing an EXPp-carRP-XPRt module through overlapping PCR;

using pUC-LEU as a template, and amplifying the screening mark LEU by using a primer LEU-F/R (SEQ ID NO: 17-18);

and integrating the TEFp-carB-PEXt module, the EXPp-carB-XPRt module and the screening marker leu between homologous arms of the pMD-H1 plasmid by using a One Step Fusion Cloning Mix kit to construct the pMD-H1BRP.

Preparation of S2 recombinant plasmid pMD-H2BRPG

The yarrowia lipolytica MYA-2613 chromosome is used as a template, the primers H2U-F/R (SEQ ID NO: 19-20) and H2D-F/R (SEQ ID NO: 21-22) are used for amplifying homologous arm sequences on the chromosome, and the two are connected through overlapping PCR and inserted into a pMD19-T simple vector to obtain a pMD-H2 plasmid;

inserting a recoverable screening marker hisG-URA3-hisG on pUC-HUH into pMD-H2 by enzyme digestion to obtain pMD-H2-HUH;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, and the primers EXP2-F/R (SEQ ID NO: 23-24) and XPR2-F/R (SEQ ID NO: 27-28) are used for respectively amplifying the promoter EXPp and the terminator XPRt;

using pUC-CB as a template and using a primer CB2-F/R (SEQ ID NO: 25-26) to amplify a carB gene; then constructing an EXPp-carB-XPRt module through overlapping PCR;

using yarrowia lipolytica MYA-2613 chromosome as a template, and respectively amplifying a promoter TEFp and a terminator PEXt by using primers TEF2-F/R (SEQ ID NO: 29-30) and PEX2-F/R (SEQ ID NO: 33-34);

using pUC-CRP as a template, and amplifying the carRP gene by using a primer CRP2-F/R (SEQ ID NO: 31-32); then constructing a TEFp-carRP-PEXt module through overlapping PCR;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, and a primer GPD-F/R (SEQ ID NO: 35-36), GGS-F/R (SEQ ID NO: 37-38) and LIP-F/R (SEQ ID NO: 39-40) are used for respectively amplifying a promoter GPDp, a gene GGS1 and a terminator LIPt, and then a GPDp-GGS1-LIPt module is constructed through overlapping PCR;

the EXPp-carB-XPRt module, the TEFp-carRP-PEXt module, and the GPDp-GGS1-LIPt module were integrated into the pMD-H2-HUH plasmid using the One Step Fusion Cloning Mix kit to obtain pMD-H2BRPG.

Preparation of S3 recombinant plasmid pMD-H4BRP

The yarrowia lipolytica MYA-2613 chromosome is used as a template, the primers H3U-F/R (SEQ ID NO: 41-42) and H3D-F/R (SEQ ID NO: 43-44) are used for amplifying homologous arm sequences on the chromosome, and the two are connected through overlapping PCR and inserted into a pMD19-T simple vector to obtain a pMD-H3 plasmid;

inserting a recoverable screening marker hisG-URA3-hisG on pUC-HUH into pMD-H3 by enzyme digestion to obtain pMD-H3-HUH;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer TEF3-F/R (SEQ ID NO: 45-46), an HMGR-F/R (SEQ ID NO: 47-48) and a PEX3-F/R (SEQ ID NO: 49-50) are used for respectively amplifying a promoter TEFp, a gene tHMGR and a terminator PEXt, and then a TEFp-tHMGR-PEXt module is constructed through overlapping PCR;

the TEFp-tHMGR-PEXt module was integrated into the pMD-H3-HUH plasmid using the One Step Fusion Cloning Mix kit to obtain pMD-H3HM.

Preparation of S4 recombinant plasmid pMD-H4BRP

The yarrowia lipolytica MYA-2613 chromosome is used as a template, the primers H4U-F/R (SEQ ID NO: 51-52) and H4D-F/R (SEQ ID NO: 53-54) are used for amplifying homologous arm sequences on the chromosome, and the two are connected through overlapping PCR and inserted into a pMD19-T simple vector to obtain a pMD-H4 plasmid;

inserting a recoverable screening marker hisG-URA3-hisG on pUC-HUH into pMD-H4 by enzyme digestion to obtain pMD-H4-HUH;

amplifying a TEFp-carB-PEXt+EXPp-carbP-XPRt module by using a recombinant plasmid pMD-H1BRP as a template and a primer BRP-F/R (SEQ ID NO: 55-56);

the TEFp-carB-PEXt+EXPp-carRP-XPRt module was integrated into the pMD-H4-HUH plasmid using the One Step Fusion Cloning Mix kit to obtain pMD-H4BRP.

Preparation of S5 recombinant plasmid pMD-H5BSW

The method comprises the steps of respectively amplifying homologous arm sequences on a chromosome by using yarrowia lipolytica MYA-2613 chromosome as a template and using primers H5U-F/R (SEQ ID NO: 57-58) and H5D-F/R (SEQ ID NO: 59-60), and connecting the two by overlapping PCR and inserting the two into a pMD19-T simple vector to obtain a pMD-H5 plasmid;

inserting a recoverable screening marker hisG-URA3-hisG on pUC-HUH into pMD-H5 by enzyme digestion to obtain pMD-H5-HUH;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, and the primers EXP3-F/R (SEQ ID NO: 61-62) and XPR3-F (SEQ ID NO: 65)/BRP-R (SEQ ID NO: 56) are used for respectively amplifying the promoter EXPp and the terminator XPRt;

using pUC-BSW as a template, amplifying a BSW gene by using a primer BSW-F/R (SEQ ID NO: 63-64), and then constructing an EXPp-BSW-XPRt module by overlapping PCR;

the EXPp-BSW-XPRt module was integrated into the pMD-H5-HUH plasmid using the One Step Fusion Cloning Mix kit to obtain pMD-H5BSW.

Preparation of S6 recombinant plasmid pMD-H6BSW

The yarrowia lipolytica MYA-2613 chromosome is used as a template, the primers H6U-F/R (SEQ ID NO: 66-67) and H6D-F/R (SEQ ID NO: 68-69) are used for amplifying homologous arm sequences on the chromosome, and the two are connected through overlapping PCR and inserted into a pMD19-T simple vector to obtain a pMD-H6 plasmid;

inserting a recoverable screening marker hisG-URA3-hisG on pUC-HUH into pMD-H6 by enzyme digestion to obtain pMD-H6-HUH;

amplifying the EXPp-BSW-XPRt module by using the pMD-H5BSW as a template and using a primer EWX-F (SEQ ID NO: 70)/BRP-R (SEQ ID NO: 56);

the EXPp-BSW-XPRt module was integrated into the pMD-H6-HUH plasmid using the One Step Fusion Cloning Mix kit to obtain pMD-H6BSW.

Preparation of S7 recombinant plasmid pMD-H7BRP

The yarrowia lipolytica MYA-2613 chromosome is used as a template, the primers H7U-F/R (SEQ ID NO: 71-72) and H7D-F/R (SEQ ID NO: 73-74) are used for amplifying homologous arm sequences on the chromosome, and the two are connected through overlapping PCR and inserted into a pMD19-T simple vector to obtain a pMD-H7 plasmid;

inserting a recoverable screening marker hisG-URA3-hisG on pUC-HUH into pMD-H7 by enzyme digestion to obtain pMD-H7-HUH;

amplifying the TEFp-carB-PEXt+EXPp-carRP-XPRt module by using the recombinant plasmid pMD-H4BRP as a template and using a primer BRP2-F (SEQ ID NO: 75)/BRP-R (SEQ ID NO: 56);

the TEFp-carB-PEXt+EXPp-carRP-XPRt module was integrated into the pMD-H7-HUH plasmid using the One Step Fusion Cloning Mix kit to obtain pMD-H7BRP.

Preparation of S8 recombinant plasmid pMD-H8GH

The yarrowia lipolytica MYA-2613 chromosome is used as a template, the primers H8U-F/R (SEQ ID NO: 76-77) and H8D-F/R (SEQ ID NO: 78-79) are used for amplifying homologous arm sequences on the chromosome, and the two are connected through overlapping PCR and inserted into a pMD19-T simple vector to obtain a pMD-H8 plasmid;

inserting a recoverable screening marker hisG-URA3-hisG on pUC-HUH into pMD-H8 by enzyme digestion to obtain pMD-H8-HUH;

amplifying the GPDp-GGS1-LIPt module by using a recombinant plasmid pMD-H2BRPG as a template and a primer GGL-F/R (SEQ ID NO: 80-81);

using recombinant plasmid pMD-H3HM as template, using primer THP-F (SEQ ID NO: 82)/PEX 3-R (SEQ ID NO: 50) to amplify TEFp-tHMGR-PEXt module;

the GPDp-GGS1-LIPt, TEFp-tHMGR-PEXt module was integrated into the pMD-H8-HUH plasmid using One Step Fusion Cloning Mix kit to obtain pMD-H8GH.

Introduction of S9 beta-carotene ketolase Gene

The constructed recombinant plasmids pMD-H5BSW and pMD-H6BSW were digested and linearized by SwaI, transferred into yarrowia lipolytica MC209 cells by using Frozen-EZ Yeast Transformation II Kit (ZYMO RESEARCH Co.), and plated on SD medium plates (glucose 20g/L, yeast basal nitrogen source 6.7g/L, agar powder 20g/L, and the balance water) for screening.

Enhancement of S10 cantharis yellow synthesis effect

And (3) tangentially transforming the constructed recombinant plasmids pMD-H7BRP and pMD-H8GH into cells by SwaI enzyme to obtain yarrowia lipolytica genetic engineering bacteria MC-CTX4.

EXAMPLE 2 shake flask fermentation

Shake flask fermentation was performed using the yarrowia lipolytica genetically engineered bacteria obtained finally in example 1, with the following medium combinations:

slant medium (1): 170/L of malt extract powder, 15g/L of agar and 0.05g/L of chloramphenicol;

seed medium (1): glucose 8g/L, soybean peptone 17g/L, potassium nitrate 6g/L, ammonium sulfate 10g/L, and potassium dihydrogen phosphate 8g/L;

fermentation medium (1): 150g/L of glucose, 20g/L of peptone, 20g/L of corn steep liquor, 9g/L of ammonium sulfate, 5g/L of magnesium sulfate, 5g/L of manganese sulfate and 100g/L of rapeseed oil.

The fermentation method comprises the following steps:

(1) Inoculating yarrowia lipolytica into a slant culture medium (1), culturing in an incubator at 24-28 ℃ and 60-80% humidity for 3 days to obtain first generation slant seeds, selecting a ring of first generation slant seeds, streaking on a fresh blank slant, and culturing in the incubator at 24-28 ℃ and 60-80% humidity for 3 days to obtain second generation slant seeds;

(2) Inoculating the second generation inclined plane into a 250mL triangular shake flask containing 25mL of seed culture medium (1) by adopting a digging method, culturing for 36h under the conditions of 26 ℃ and a ventilation ratio of 1vvm, and obtaining mature seed liquid at a rotation speed of 200r/min of a shaking table;

(3) Transferring the seed liquid into a 250mL triangular shake flask containing 25mL of fermentation medium (1) according to the inoculation amount of 3% -15%, regulating the pH value to 5.0-5.3, culturing for 120h at 26 ℃, and obtaining the fermentation liquid at the rotating speed of a shaking table of 240 r/min.

The fermentation yield was about 1.8-2.2g/L when the bottles were placed, and the specific fermentation results are shown in Table 1 below.

TABLE 1 shaking flask fermentation results (unit: g/L)

EXAMPLE 3 5L glass jar fermentation

The yarrowia lipolytica genetically engineered bacteria obtained in example 1 were used for 5L glass jar fermentation with the following combinations of media:

slant medium (2): 150g/L of malt extract powder, 18g/L of agar and 0.15g/L of chloramphenicol;

seed medium (2): 10g/L of glucose, 8g/L of soybean peptone, 3g/L of potassium nitrate, 12g/L of ammonium sulfate and 3g/L of monopotassium phosphate;

fermentation medium (2): 160g/L glucose (the fermentation medium contains 25% glucose at the beginning of fermentation, and the rest glucose is added in the fermentation process), 45g/L peptone, 10g/L corn steep liquor, 3g/L ammonium sulfate, 2g/L magnesium sulfate, 6g/L manganese sulfate and 30g/L rapeseed oil.

The fermentation method comprises the following steps:

(1) Inoculating yarrowia lipolytica on a slant culture medium (2), culturing in an incubator at 24-28 ℃ and 60-80% humidity for 3 days to obtain first generation slant seeds, selecting a ring of first generation slant seeds, streaking on a fresh blank slant, and culturing in the incubator at 24-28 ℃ and 60-80% humidity for 3 days to obtain second generation slant seeds;

(2) Inoculating the second generation inclined plane strain into a 5L glass fermentation tank containing 3L seed culture medium (2), culturing for 36h at 26 ℃ under the condition of aeration ratio of 1vvm, and obtaining mature seed liquid at the rotation speed of a shaking table of 200 r/min;

(3) Transferring the seed liquid into a 5L glass fermentation tank containing 3L fermentation medium (2) according to 10% of inoculation amount, regulating pH to 5.0-5.3, culturing for 120h at 26 ℃ under the condition that the highest aeration ratio is 2.5vvm, and supplementing the rest 75% of glucose in the fermentation process at the speed of 4-8.5 mL/(h.L) to obtain fermentation liquid.

The highest yield can reach 4.92g/L, and the specific fermentation results are shown in the following table 2.

TABLE 2 fermentation results in 5L glass jar (unit: g/L)

EXAMPLE 4 100L fermenter fermentation

The yarrowia lipolytica genetically engineered bacteria obtained in example 1 were used for fermentation in a 100L fermenter with the following combinations of media:

slant medium (3): 130g/L of malt extract powder, 20g/L of agar and 0.1g/L of chloramphenicol;

seed medium (3): 15g/L of glucose, 10g/L of soybean peptone, 2g/L of potassium nitrate, 5g/L of ammonium sulfate and 3g/L of monopotassium phosphate;

fermentation medium (3): 210-290 g/L of glucose (10 g/L of glucose is contained in a fermentation culture medium at the beginning of fermentation, the rest glucose is added in the fermentation process), 30g/L of peptone, 10g/L of corn steep liquor, 5g/L of ammonium sulfate, 2.5g/L of magnesium sulfate, 2.5g/L of manganese sulfate and 100g/L of rapeseed oil (70 g/L of rapeseed oil is contained in the fermentation culture medium at the beginning of fermentation, and the rest rapeseed oil is added in the fermentation process).

The fermentation method comprises the following steps:

(1) Inoculating yarrowia lipolytica on a slant culture medium (3), culturing in an incubator at 24-28 ℃ and 60-80% humidity for 3 days to obtain first generation slant seeds, selecting a ring of first generation slant seeds, streaking on a fresh blank slant, and culturing in the incubator at 24-28 ℃ and 60-80% humidity for 3 days to obtain second generation slant seeds;

(2) Inoculating a bacterial suspension prepared by the second-generation yarrowia lipolytica inclined plane into 20L of seed culture medium (3), and culturing for 36h at 26 ℃ under the condition of a ventilation ratio of 1vvm, wherein the rotation speed of a shaking table is 300r/m, so as to obtain mature seed liquid;

(3) Transferring the seed liquid into a 100L fermentation tank containing 50L of fermentation medium (3) according to 10% of inoculation amount, regulating pH to 5.0-5.3, culturing for 120h at 26 ℃ under the condition of ventilation ratio of 2vvm, and adding glucose and rapeseed oil in the fermentation process at the rotation speed of 500r/m, wherein the sugar adding rate is 6-8 mL/(h.L) and the oil adding rate is 1-2 mL/(h.L), thus obtaining the fermentation liquid.

The highest yield can reach 5.05g/L, and the specific fermentation results are shown in the following table 3.

TABLE 3 fermentation results in 100L fermentors

Comparative example 1

slant medium (4): glucose 20g/L, peptone 20g/L, yeast powder 10g/L and agar powder 20g/L;

seed medium (4): glucose 20g/L, peptone 20g/L, yeast powder 10g/L;

fermentation medium (4): 540g/L glucose (40 g/L glucose is contained in a fermentation medium at the beginning of fermentation, and the rest glucose is added in the fermentation process), 15g/L peptone, 20g/L yeast powder, 4g/L potassium dihydrogen phosphate and 1g/L ferrous sulfate.

The fermentation method comprises the following steps:

(1) Inoculating yarrowia lipolytica on the slant culture medium (4), and culturing at 28deg.C for 3 days;

(2) Digging blocks, inoculating the blocks into a 500mL shaking flask containing 50mL of seed culture medium (4), and culturing the blocks for 24 hours at 28 ℃ at the rotating speed of a shaking table of 220r/m to obtain mature seed liquid;

(3) Transferring the seed liquid into a 5L fermentation tank containing 3L fermentation medium (4) according to 10% of inoculation amount, wherein the fermentation temperature is 28 ℃, and the ventilation rate is 1:1 (vol: vol), the rotating speed of the shaking table is 400-600r/m, glucose is added in the fermentation process, the sugar adding speed is 6-8 mL/(h.L), and the oil adding speed is 10 mL/(h.L), so that fermentation liquor is obtained. The cantharidin yellow yield at 160h is 3.37g/L, 3.58g/L and 3.54g/L respectively, and the average cantharidin yellow yield is 3.5g/L.

Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.

SEQUENCE LISTING

<110> Shandong micro-research Biotech Co., ltd

<120> a medium composition and fermentation process for high-yield canthaxanthin using yarrowia lipolytica

<130> 2022

<160> 85

<170> PatentIn version 3.5

<210> 1

<211> 33

<212> DNA

<213> artificial sequence

<400> 1

gtttaaactt gatgtttaga gtgtccagat ccg 33

<210> 2

<211> 45

<212> DNA

<213> artificial sequence

<400> 2

tcggtaatgc cactgtagaa agctttaacg ccggtgtaat cttgc 45

<210> 3

<211> 45

<212> DNA

<213> artificial sequence

<400> 3

caagattaca ccggcgttaa agctttctac agtggcatta ccgag 45

<210> 4

<211> 28

<212> DNA

<213> artificial sequence

<400> 4

gtttaaacgc aggtattcca ttgccctt 28

<210> 5

<211> 45

<212> DNA

<213> artificial sequence

<400> 5

cgccgcaaga ttacaccggc gttaataaac tttggcaaag aggct 45

<210> 6

<211> 49

<212> DNA

<213> artificial sequence

<400> 6

ccgataatga caatgtgttt cttggacatt ttgaatgatt cttatactc 49

<210> 7

<211> 47

<212> DNA

<213> artificial sequence

<400> 7

ccttctgagt ataagaatca ttcaaaatgt ccaagaaaca cattgtc 47

<210> 8

<211> 45

<212> DNA

<213> artificial sequence

<400> 8

cactcacttc cccatccaca cttccttaaa tgacattaga gttat 45

<210> 9

<211> 48

<212> DNA

<213> artificial sequence

<400> 9

cgttcataac tctaatgtca tttaaggaag tgtggatggg gaagtgag 48

<210> 10

<211> 45

<212> DNA

<213> artificial sequence

<400> 10

ctccttatat atacctccta cagtcaccag atgcatgagc agacc 45

<210> 11

<211> 47

<212> DNA

<213> artificial sequence

<400> 11

ggcatggtct gctcatgcat ctggtgactg taggaggtat atataag 47

<210> 12

<211> 45

<212> DNA

<213> artificial sequence

<400> 12

ggacttccat gtaggtgagc agcattgctg tagatatgtc ttgtg 45

<210> 13

<211> 47

<212> DNA

<213> artificial sequence

<400> 13

ttacacacaa gacatatcta cagcaatgct gctcacctac atggaag 47

<210> 14

<211> 45

<212> DNA

<213> artificial sequence

<400> 14

caccggcaaa ctatctgtta attgcttaaa tggtatttag atttc 45

<210> 15

<211> 56

<212> DNA

<213> artificial sequence

<400> 15

gggaaaaatg agaaatctaa ataccattta agcaattaac agatagtttg ccggtg 56

<210> 16

<211> 45

<212> DNA

<213> artificial sequence

<400> 16

ggaaaagtac tatgtgctca aggttgagca gtagttgctg ttagc 45

<210> 17

<211> 46

<212> DNA

<213> artificial sequence

<400> 17

tagcagctaa cagcaactac tgctcaacct tgagcacata gtactt 46

<210> 18

<211> 46

<212> DNA

<213> artificial sequence

<400> 18

ctcggtaatg ccactgtaga aagctactaa ggtattgaaa tcccac 46

<210> 19

<211> 31

<212> DNA

<213> artificial sequence

<400> 19

atttaaatgg gcgtggaaag tttcctgaat g 31

<210> 20

<211> 45

<212> DNA

<213> artificial sequence

<400> 20

agcccgtgtt gaattcgcta agctttgtaa ttcttcctac agacg 45

<210> 21

<211> 45

<212> DNA

<213> artificial sequence

<400> 21

aagaattaca aagcttagcg aattcaacac gggctgtgat tatgt 45

<210> 22

<211> 28

<212> DNA

<213> artificial sequence

<400> 22

atttaaatct ggctgtgctg catcatac 28

<210> 23

<211> 47

<212> DNA

<213> artificial sequence

<400> 23

cgtccgtctg taggaagaat tacaagactg taggaggtat atataag 47

<210> 24

<211> 45

<212> DNA

<213> artificial sequence

<400> 24

taatgacaat gtgtttcttg gacattgctg tagatatgtc ttgtg 45

<210> 25

<211> 45

<212> DNA

<213> artificial sequence

<400> 25

ttacacacaa gacatatcta cagcaatgtc caagaaacac attgt 45

<210> 26

<211> 45

<212> DNA

<213> artificial sequence

<400> 26

caccggcaaa ctatctgtta attgcttaaa tgacattaga gttat 45

<210> 27

<211> 48

<212> DNA

<213> artificial sequence

<400> 27

cgttcataac tctaatgtca tttaagcaat taacagatag tttgccgg 48

<210> 28

<211> 45

<212> DNA

<213> artificial sequence

<400> 28

tttgcagcct ctttgccaaa gtttagagca gtagttgctg ttagc 45

<210> 29

<211> 45

<212> DNA

<213> artificial sequence

<400> 29

tagcagctaa cagcaactac tgctctaaac tttggcaaag aggct 45

<210> 30

<211> 45

<212> DNA

<213> artificial sequence

<400> 30

ggacttccat gtaggtgagc agcattttga atgattctta tactc 45

<210> 31

<211> 47

<212> DNA

<213> artificial sequence

<400> 31

cttctgagta taagaatcat tcaaaatgct gctcacctac atggaag 47

<210> 32

<211> 45

<212> DNA

<213> artificial sequence

<400> 32

cactcacttc cccatccaca cttccttaaa tggtatttag atttc 45

<210> 33

<211> 48

<212> DNA

<213> artificial sequence

<400> 33

aatgagaaat ctaaatacca tttaaggaag tgtggatggg gaagtgag 48

<210> 34

<211> 42

<212> DNA

<213> artificial sequence

<400> 34

ggacatccta ctgcgtccgg tttataccag atgcatgagc ag 42

<210> 35

<211> 43

<212> DNA

<213> artificial sequence

<400> 35

ggcatggtct gctcatgcat ctggtataaa ccggacgcag tag 43

<210> 36

<211> 48

<212> DNA

<213> artificial sequence

<400> 36

ctccttgaaa tccgcgctgt tataatccat tgttgatgtg tgtttaat 48

<210> 37

<211> 49

<212> DNA

<213> artificial sequence

<400> 37

ttcttgaatt aaacacacat caacaatgga ttataacagc gcggatttc 49

<210> 38

<211> 45

<212> DNA

<213> artificial sequence

<400> 38

gaagttgtaa agagtgataa atagctcact gcgcatcctc aaagt 45

<210> 39

<211> 50

<212> DNA

<213> artificial sequence

<400> 39

aaagtacttt gaggatgcgc agtgagctat ttatcactct ttacaacttc 50

<210> 40

<211> 45

<212> DNA

<213> artificial sequence

<400> 40

cgtacgacgc cattttaagt aagctggctg cttcttaagt ttgtg 45

<210> 41

<211> 30

<212> DNA

<213> artificial sequence

<400> 41

atttaaatcg ggattccaga gactatggtg 30

<210> 42

<211> 45

<212> DNA

<213> artificial sequence

<400> 42

gctgctacgg aattcattca agcttctaca catgttgcgg ctgag 45

<210> 43

<211> 45

<212> DNA

<213> artificial sequence

<400> 43

catgtgtaga agcttgaatg aattccgtag cagcgctctt atcat 45

<210> 44

<211> 28

<212> DNA

<213> artificial sequence

<400> 44

atttaaatgc cttcgaccag atttgaac 28

<210> 45

<211> 45

<212> DNA

<213> artificial sequence

<400> 45

gcggctcagc cgcaacatgt gtagataaac tttggcaaag aggct 45

<210> 46

<211> 45

<212> DNA

<213> artificial sequence

<400> 46

caaccacctt cacagactgg gtcattttga atgattctta tactc 45

<210> 47

<211> 50

<212> DNA

<213> artificial sequence

<400> 47

cttctgagta taagaatcat tcaaaatgac ccagtctgtg aaggtggttg 50

<210> 48

<211> 45

<212> DNA

<213> artificial sequence

<400> 48

cactcacttc cccatccaca cttccctatg accgtatgca aatat 45

<210> 49

<211> 46

<212> DNA

<213> artificial sequence

<400> 49

ttcgaatatt tgcatacggt catagggaag tgtggatggg gaagtg 46

<210> 50

<211> 45

<212> DNA

<213> artificial sequence

<400> 50

cgtacgacgc cattttaagt aagctaccag atgcatgagc agacc 45

<210> 51

<211> 32

<212> DNA

<213> artificial sequence

<400> 51

atttaaatga ctcttctcct tgtcgagatt cg 32

<210> 52

<211> 45

<212> DNA

<213> artificial sequence

<400> 52

cgaatccgac tgaattccta agcttattac tttgccaagg ttaac 45

<210> 53

<211> 45

<212> DNA

<213> artificial sequence

<400> 53

ggcaaagtaa taagcttagg aattcagtcg gattcgggtg cccat 45

<210> 54

<211> 28

<212> DNA

<213> artificial sequence

<400> 54

atttaaatag accgacccgg acacagtg 28

<210> 55

<211> 47

<212> DNA

<213> artificial sequence

<400> 55

gctggttaac cttggcaaag taatataaac tttggcaaag aggctgc 47

<210> 56

<211> 45

<212> DNA

<213> artificial sequence

<400> 56

cgtacgacgc cattttaagt aagctgagca gtagttgctg ttagc 45

<210> 57

<211> 31

<212> DNA

<213> artificial sequence

<400> 57

atttaaatca gaagaggtgg agactctggc c 31

<210> 58

<211> 45

<212> DNA

<213> artificial sequence

<400> 58

gaatccaatg aattcacaga agctttggcc tccttctcgg aatcc 45

<210> 59

<211> 46

<212> DNA

<213> artificial sequence

<400> 59

aggaggccaa agcttctgtg aattcattgg attcaccgac gccttc 46

<210> 60

<211> 28

<212> DNA

<213> artificial sequence

<400> 60

atttaaatag gaggaatggc tggtggcg 28

<210> 61

<211> 47

<212> DNA

<213> artificial sequence

<400> 61

ccaaggattc cgagaaggag gccaagactg taggaggtat atataag 47

<210> 62

<211> 40

<212> DNA

<213> artificial sequence

<400> 62

ttggcggtgg cggcgtgcat tgctgtagat atgtcttgtg 40

<210> 63

<211> 41

<212> DNA

<213> artificial sequence

<400> 63

cacaagacat atctacagca atgcacgccg ccaccgccaa g 41

<210> 64

<211> 42

<212> DNA

<213> artificial sequence

<400> 64

ggcaaactat ctgttaattg cttagtctcg tcgctccagg gc 42

<210> 65

<211> 45

<212> DNA

<213> artificial sequence

<400> 65

acgagccctg gagcgacgag actaagcaat taacagatag tttgc 45

<210> 66

<211> 30

<212> DNA

<213> artificial sequence

<400> 66

atttaaatca ggctgcaatg atgttgatcg 30

<210> 67

<211> 45

<212> DNA

<213> artificial sequence

<400> 67

ctagctccag gaattcatca agcttagaat aaaacgagat cggtt 45

<210> 68

<211> 49

<212> DNA

<213> artificial sequence

<400> 68

gttttattct aagcttgatg aattcctgga gctagttgat gctgtctcg 49

<210> 69

<211> 28

<212> DNA

<213> artificial sequence

<400> 69

atttaaattg atgcgaacat gggctgct 28

<210> 70

<211> 47

<212> DNA

<213> artificial sequence

<400> 70

cgagaaccga tctcgtttta ttctagactg taggaggtat atataag 47

<210> 71

<211> 29

<212> DNA

<213> artificial sequence

<400> 71

atttaaatcg aatggtgttc cgaacgatc 29

<210> 72

<211> 45

<212> DNA

<213> artificial sequence

<400> 72

gcagaccagg aattctgata agcttgtatg ataccttgca aacac 45

<210> 73

<211> 45

<212> DNA

<213> artificial sequence

<400> 73

gtatcataca agcttatcag aattcctggt ctgcctcggc aagtc 45

<210> 74

<211> 28

<212> DNA

<213> artificial sequence

<400> 74

atttaaatta cgacgacccc tgacccag 28

<210> 75

<211> 45

<212> DNA

<213> artificial sequence

<400> 75

caatgtgttt gcaaggtatc atacataaac tttggcaaag aggct 45

<210> 76

<211> 29

<212> DNA

<213> artificial sequence

<400> 76

atttaaatct tccttgtgtt tgtcaccgc 29

<210> 77

<211> 45

<212> DNA

<213> artificial sequence

<400> 77

ttgtctagga gaattcacta agcttcacag gaagatttgt agcgc 45

<210> 78

<211> 45

<212> DNA

<213> artificial sequence

<400> 78

tcttcctgtg aagcttagtg aattctccta gacaagtctc agttt 45

<210> 79

<211> 28

<212> DNA

<213> artificial sequence

<400> 79

atttaaatgc tggccaaagt gttcaagg 28

<210> 80

<211> 46

<212> DNA

<213> artificial sequence

<400> 80

caaggcgcta caaatcttcc tgtgaataaa ccggacgcag taggat 46

<210> 81

<211> 45

<212> DNA

<213> artificial sequence

<400> 81

tttgcagcct ctttgccaaa gtttaggctg cttcttaagt ttgtg 45

<210> 82

<211> 47

<212> DNA

<213> artificial sequence

<400> 82

gatgtcacaa acttaagaag cagcctaaac tttggcaaag aggctgc 47

<210> 83

<211> 777

<212> DNA

<213> peanut rhizobia (Bradyrhizobium sp.)

<400> 83

atgcacgccg ccaccgccaa ggccaccgag ttcggcgcct ctcgacgaga cgacgcccga 60

cagcgacgag tgggcctgac cctggccgcc gtgattatcg ccgcctggct ggtgctgcac 120

gtgggcctga tgttcttctg gcccctgacc ctgcactctc tgctgcccgc cctgcccctg 180

gtggtgctgc agacctggct gtacgtgggc ctgttcatca tcgcccacga ctgtatgcac 240

ggctctctgg tgcccttcaa gccccaagtg aaccgacgaa tcggacagct gtgtctgttc 300

ctgtacgccg gcttctcttt cgacgccctg aacgtagagc accacaagca ccaccgacac 360

cccggcacag ccgaagaccc ggatttcgac gaggtgcccc cccacggctt ctggcactgg 420

ttcgcctctt tcttcctgca ctacttcggc tggaagcaag tggccataat cgccgccgtg 480

tctctggtgt atcagctggt gttcgccgtg cccctgcaga acatcctgct gttctgggcc 540

ctgcccggcc tgctgtctgc cctgcagctg ttcaccttcg gcacctacct gccccacaag 600

cccgccactc agcccttcgc cgaccgacac aacgcccgaa cctctgagtt ccccgcctgg 660

ctgtctctgc tgacctgttt ccacttcggc ttccatcatg agcaccactt gcacccggat 720

gccccctggt ggcgactgcc cgagatcaag cgacgagccc tggagcgacg agactaa 777

<210> 84

<211> 984

<212> DNA

<213> yarrowia lipolytica (Yarrowia lipolytica)

<400> 84

atggattata acagcgcgga tttcaaggag atatggggca aggccgccga caccgcgctg 60

ctgggaccgt acaactacct cgccaacaac cggggccaca acatcagaga acacttgatc 120

gcagcgttcg gagcggttat caaggtggac aagagcgatc tcgagaccat ttcgcacatc 180

accaagattt tgcataactc gtcgctgctt gttgatgacg tggaagacaa ctcgatgctc 240

cgacgaggcc tgccggcagc ccattgtctg tttggagtcc cccaaaccat caactccgcc 300

aactacatgt actttgtggc tctgcaggag gtgctcaagc tcaagtctta tgatgccgtc 360

tccattttca ccgaggaaat gatcaacttg catagaggtc agggtatgga tctctactgg 420

agagaaacac tcacttgccc ctcggaagac gagtatctgg agatggtggt gcacaagacc 480

ggtggactgt ttcggctggc tctgagactt atgctgtcgg tggcatcgaa acaggaggac 540

catgaaaaga tcaactttga tctcacacac cttaccgaca cactgggagt catttaccag 600

attctggatg attacctcaa cctgcagtcc acggaattga ccgagaacaa gggattctgc 660

gaagatatca gcgaaggaaa gttttcgttt ccgctgattc acagcatacg caccaacccg 720

gataaccacg agattctcaa cattctcaaa cagcgaacaa gcgacgcttc actcaaaaag 780

tacgccgtgg actacatgag aacagaaacc aagagtttcg actactgcct caagaggata 840

caggccatgt cactcaaggc aagttcgtac attgatgatc tagcagcagc tggccacgat 900

gtctccaagc tacgagccat tttgcattat tttgtgtcca cctctgactg tgaggagaga 960

aagtactttg aggatgcgca gtga 984

<210> 85

<211> 1503

<212> DNA

<213> yarrowia lipolytica (Yarrowia lipolytica)

<400> 85

atgacccagt ctgtgaaggt ggttgagaag cacgttccta tcgtcattga gaagcccagc 60

gagaaggagg aggacacctc ttctgaagac tccattgagc tgactgtcgg aaagcagccc 120

aagcccgtga ccgagacccg ttctctggac gacctagagg ctatcatgaa ggcaggtaag 180

accaagcttc tggaggacca cgaggttgtc aagctctctc tcgagggcaa gcttcctttg 240

tatgctcttg agaagcagct tggtgacaac acccgagctg ttggcatccg acgatctatc 300

atctcccagc agtctaatac caagacttta gagacctcaa agcttcctta cctgcactac 360

gactacgacc gtgtttttgg agcctgttgc gagaacgtta ttggttacat gcctctcccc 420

gttggtgttg ctggccccat gaacattgat ggcaagaact accacattcc tatggccacc 480

actgagggtt gtcttgttgc ctcaaccatg cgaggttgca aggccatcaa cgccggtggc 540

ggtgttacca ctgtgcttac tcaggacggt atgacacgag gtccttgtgt ttccttcccc 600

tctctcaagc gggctggagc cgctaagatc tggcttgatt ccgaggaggg tctcaagtcc 660

atgcgaaagg ccttcaactc cacctctcga tttgctcgtc tccagtctct tcactctacc 720

cttgctggta acctgctgtt tattcgattc cgaaccacca ctggtgatgc catgggcatg 780

aacatgatct ccaagggcgt cgaacactct ctggccgtca tggtcaagga gtacggcttc 840

cctgatatgg acattgtgtc tgtctcgggt aactactgca ctgacaagaa gcccgcagcg 900

atcaactgga tcgaaggccg aggcaagagt gttgttgccg aagccaccat ccctgctcac 960

attgtcaagt ctgttctcaa aagtgaggtt gacgctcttg ttgagctcaa catcagcaag 1020

aatctgatcg gtagtgccat ggctggctct gtgggaggtt tcaatgcaca cgccgcaaac 1080

ctggtgaccg ccatctacct tgccactggc caggatcctg ctcagaatgt cgagtcttcc 1140

aactgcatca cgctgatgag caacgtcgac ggtaacctgc tcatctccgt ttccatgcct 1200

tctatcgagg tcggtaccat tggtggaggt actattttgg agccccaggg ggctatgctg 1260

gagatgcttg gcgtgcgagg tcctcacatc gagacccccg gtgccaacgc ccaacagctt 1320

gctcgcatca ttgcttctgg agttcttgca gcggagcttt cgctgtgttc tgctcttgct 1380

gccggccatc ttgtgcaaag tcatatgacc cacaaccggt cccaggctcc tactccggcc 1440

aagcagtctc aggccgatct gcagcgtcta caaaacggtt cgaatatttg catacggtca 1500

tag 1503

Claims

1. A fermentation process, comprising the steps of:

(1) Inoculating the frozen yarrowia lipolytica genetic engineering bacteria into a slant culture medium for activation, wherein the slant culture medium comprises 100-190g/L malt extract powder, 15-20g/L agar and 0.05-0.2g/L chloramphenicol;

(2) Inoculating the activated strain into a seed culture medium, and culturing at 24-28 ℃ under the conditions of a ventilation ratio of 1vvm and a rotation speed of 200-300r/min to obtain mature seed liquid, wherein the seed culture medium comprises 5-15g/L glucose, 5-20g/L soybean peptone, 1-10g/L potassium nitrate, 2-12g/L ammonium sulfate and 2-10g/L potassium dihydrogen phosphate;

(3) Inoculating 3% -15% of seed solution into a fermentation culture medium, regulating pH to 5.0-5.3, fermenting and culturing at 24-28deg.C under the conditions of aeration ratio of 1-2.5vvm and rotation speed of 200-800r/min to obtain fermentation broth, wherein the fermentation culture medium comprises 120-300g/L glucose, 15-45g/L peptone, 5-20g/L corn steep liquor, 2-12g/L ammonium sulfate, 1-6g/L magnesium sulfate, 1-6g/L manganese sulfate and 20-110g/L rapeseed oil;

the construction steps of the yarrowia lipolytica genetically engineered bacterium are as follows:

preparation of S1 recombinant plasmid pMD-H1BRP

The yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer H1U-F/R, H1D-F/R is used for amplifying a homologous arm sequence on the chromosome, and the two are connected through overlapping PCR and inserted into a pMD19-T simple vector to obtain a pMD-H1 plasmid;

using yarrowia lipolytica MYA-2613 chromosome as template, using primer TEF1-F/R, PEX1-F/R to amplify promoter TEFp and terminator PEXt;

amplifying the carB gene by using the primers with pUC-CB as a template; then constructing a TEFp-carB-PEXt module by overlapping PCR;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, and the primer EXP1-F/R, XPR1-F/R is used for respectively amplifying the promoter EXPp and the terminator XPRt;

amplifying the carRP gene by using the primers CRP1-F/R by using pUC-CRP as a template; then constructing an EXPp-carRP-XPRt module through overlapping PCR;

amplifying the screening mark LEU by using the primer LEU-F/R by using pUC-LEU as a template;

integrating the TEFp-carB-PEXt module, the EXPp-carB-XPRt module and the screening marker leu between homologous arms of the pMD-H1 plasmid by using a One Step Fusion Cloning Mix kit to obtain pMD-H1BRP;

preparation of S2 recombinant plasmid pMD-H2BRPG

The yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer H2U-F/R, H2D-F/R is used for amplifying a homologous arm sequence on the chromosome, and the two are connected through overlapping PCR and inserted into a pMD19-T simple vector to obtain a pMD-H2 plasmid;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, and the primer EXP2-F/R, XPR2-F/R is used for respectively amplifying the promoter EXPp and the terminator XPRt;

amplifying the carB gene by using a pUC-CB as a template and using a primer CB 2-F/R; then constructing an EXPp-carB-XPRt module through overlapping PCR;

using yarrowia lipolytica MYA-2613 chromosome as template, using primer TEF2-F/R, PEX-F/R to amplify promoter TEFp and terminator PEXt;

amplifying the carRP gene by using the primer CRP2-F/R by taking pUC-CRP as a template; then constructing a TEFp-carRP-PEXt module through overlapping PCR;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer GPD-F/R, GGS-F/R, LIP-F/R is used for respectively amplifying a promoter GPDp, a gene GGS1 and a terminator LIPt, and then a GPDp-GGS1-LIPt module is constructed through overlapping PCR;

integrating the EXPp-carB-XPRt module, the TEFp-carRP-PEXt module and the GPDp-GGS1-LIPt module into a pMD-H2-HUH plasmid by using a One Step Fusion Cloning Mix kit to obtain pMD-H2BRPG;

preparation of S3 recombinant plasmid pMD-H4BRP

The yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer H3U-F/R, H3D-F/R is used for amplifying a homologous arm sequence on the chromosome, and the two are connected through overlapping PCR and inserted into a pMD19-T simple vector to obtain a pMD-H3 plasmid;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer TEF3-F/R, HMGR-F/R, PEX-F/R is used for respectively amplifying a promoter TEFp, a gene tHMGR and a terminator PEXt, and then a TEFp-tHMGR-PEXt module is constructed through overlapping PCR;

integrating the TEFp-tHMGR-PEXt module into the pMD-H3-HUH plasmid using a One Step Fusion Cloning Mix kit to obtain pMD-H3HM;

preparation of S4 recombinant plasmid pMD-H4BRP

The yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer H4U-F/R, H D-F/R is used for amplifying a homologous arm sequence on the chromosome, and the two are connected and inserted into a pMD19-T simple vector by overlapping PCR to obtain a pMD-H4 plasmid;

amplifying the TEFp-carB-PEXt+EXPp-carbP-XPRt module by using the recombinant plasmid pMD-H1BRP as a template and using a primer BRP-F/R;

integrating the TEFp-carB-PEXt+EXPp-carRP-XPRt module into the pMD-H4-HUH plasmid by using a One Step Fusion Cloning Mix kit to obtain pMD-H4BRP;

preparation of S5 recombinant plasmid pMD-H5BSW

The yarrowia lipolytica MYA-2613 chromosome is used as a template, the homologous arm sequences on the chromosome are respectively amplified by using a primer H5U-F/R, H D-F/R, and the two are connected and inserted into a pMD19-T simple vector by overlapping PCR to obtain a pMD-H5 plasmid;

the yarrowia lipolytica MYA-2613 chromosome is used as a template, and the primer EXP3-F/R, XPR3-F/BRP-R is used for respectively amplifying the promoter EXPp and the terminator XPRt;

using pUC-BSW as a template, amplifying a BSW gene by using a primer BSW-F/R, and then constructing an EXPp-BSW-XPRt module by overlapping PCR;

integrating the EXPp-BSW-XPRt module into the pMD-H5-HUH plasmid by using a One Step Fusion Cloning Mix kit to obtain pMD-H5BSW;

preparation of S6 recombinant plasmid pMD-H6BSW

The yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer H6U-F/R, H D-F/R is used for amplifying a homologous arm sequence on the chromosome, and the two are connected and inserted into a pMD19-T simple vector by overlapping PCR to obtain a pMD-H6 plasmid;

amplifying the EXPp-BSW-XPRt module by using the pMD-H5BSW as a template and using a primer EWX-F/BRP-R;

integrating the EXPp-BSW-XPRt module into the pMD-H6-HUH plasmid by using a One Step Fusion Cloning Mix kit to obtain pMD-H6BSW;

preparation of S7 recombinant plasmid pMD-H7BRP

The yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer H7U-F/R, H D-F/R is used for amplifying a homologous arm sequence on the chromosome, and the two are connected and inserted into a pMD19-T simple vector by overlapping PCR to obtain a pMD-H7 plasmid;

amplifying a TEFp-carB-PEXt+EXPp-carbP-XPRt module by using a recombinant plasmid pMD-H4BRP as a template and using a primer BRP 2-F/BRP-R;

integrating the TEFp-carB-PEXt+EXPp-carRP-XPRt module into the pMD-H7-HUH plasmid by using a One Step Fusion Cloning Mix kit to obtain pMD-H7BRP;

preparation of S8 recombinant plasmid pMD-H8GH

The yarrowia lipolytica MYA-2613 chromosome is used as a template, a primer H8U-F/R, H D-F/R is used for amplifying a homologous arm sequence on the chromosome, and the two are connected and inserted into a pMD19-T simple vector by overlapping PCR to obtain a pMD-H8 plasmid;

amplifying the GPDp-GGS1-LIPt module by using a recombinant plasmid pMD-H2BRPG as a template and a primer GGL-F/R;

using recombinant plasmid pMD-H3HM as a template, and amplifying a TEFp-tHMGR-PEXt module by using a primer THP-F/PEX 3-R;

integrating GPDp-GGS1-LIPt and TEFp-tHMGR-PEXt modules into a pMD-H8-HUH plasmid by using a One Step Fusion Cloning Mix kit to obtain pMD-H8GH;

introduction of S9 beta-carotene ketolase Gene

The constructed recombinant plasmids pMD-H5BSW and pMD-H6BSW are subjected to enzyme digestion linearization by SwaI, transferred into yarrowia lipolytica MC209 cells by using Frozen-EZ Yeast Transformation II Kit, and coated on a SD culture medium plate for screening;

enhancement of S10 cantharis yellow synthesis effect

The constructed recombinant plasmids pMD-H7BRP and pMD-H8GH are tangentially transformed into cells by SwaI enzyme to obtain yarrowia lipolytica genetic engineering bacteria MC-CTX4;

wherein the preservation number of the yarrowia lipolytica MC209 is CGMCC No.23758, and the yarrowia lipolytica MYA-2613 is purchased from ATCC;

the genebank accession number of the carB gene is AJ238028, the genebank accession number of the carRP gene is AJ250827, the genebank accession number of the leu screening marker is M37309, the genebank accession number of URA3 is AJ306421, and the genebank accession number of the hisG is AF324729;

the GGS1 gene, the tHMGR promoter TEFp, EXPp, GDPp and the terminator PEXt, XPRt, LIPt are all derived from yarrowia lipolytica MYA-2613 and are obtained by a PCR method;

the sequence of H1U-F is shown in SEQ ID NO. 1, the sequence of H1U-R is shown in SEQ ID NO.2, the sequence of H1D-F is shown in SEQ ID NO. 3, the sequence of H1D-R is shown in SEQ ID NO. 4, the sequence of TEF1-F is shown in SEQ ID NO. 5, the sequence of TEF1-R is shown in SEQ ID NO. 6, the sequence of CB1-F is shown in SEQ ID NO. 7, the sequence of CB1-R is shown in SEQ ID NO. 8, the sequence of PEX1-F is shown in SEQ ID NO. 9, the sequence of PEX1-R is shown in SEQ ID NO. 10, the sequence of EXP1-F is shown in SEQ ID NO. 11, the sequence of EXP1-R is shown in SEQ ID NO. 12, the sequence of CRP1-F is shown in SEQ ID NO. 13, the sequence of CRP1-R is shown in SEQ ID NO. 14, the sequence of XPR1-F is shown in SEQ ID NO. 15, the sequence of CB1-R is shown in SEQ ID NO. 16, the sequence of SEQ ID NO. 16 is shown in SEQ ID NO.2, the sequence of EXP1-R is shown in SEQ ID NO.2, the sequence of SEQ ID NO.2 is shown in SEQ ID NO.2, and the sequence of SEQ ID NO.2 is shown in SEQ ID NO. 18, the sequence of SEQ ID NO.2 is shown in SEQ NO.2 is shown in SEQ NO. 2. XPR2-R has a sequence shown in SEQ ID NO. 28, TEF2-F has a sequence shown in SEQ ID NO. 29, TEF2-R has a sequence shown in SEQ ID NO. 30, CRP2-F has a sequence shown in SEQ ID NO. 31, CRP2-R has a sequence shown in SEQ ID NO. 32, PEX2-F has a sequence shown in SEQ ID NO. 33, PEX2-R has a sequence shown in SEQ ID NO. 34, GPD-F has a sequence shown in SEQ ID NO. 35, GPD-R has a sequence shown in SEQ ID NO. 36, GGS-F has a sequence shown in SEQ ID NO. 37, GGS-R has a sequence shown in SEQ ID NO. 38, LIP-F has a sequence shown in SEQ ID NO. 39, LIP-R has a sequence shown in SEQ ID NO. 40, H3U-F has a sequence shown in SEQ ID NO. 41, H3U-R has a sequence shown in SEQ ID NO. 42, H3D-F has a sequence shown in SEQ ID NO. 43, GPD-F has a sequence shown in SEQ ID NO. 4, GGS-F has a sequence shown in SEQ ID NO. 37, and HMP-R has a sequence shown in SEQ ID NO. 4, and HMP-F has a sequence shown in SEQ ID NO. 4, and SEQ ID NO. 46, SEQ ID NO. 4-F has a sequence shown in SEQ ID NO. 40, and SEQ ID NO. 4-F has a sequence shown in SEQ ID NO. 52, SEQ ID NO. 4 and SEQ ID NO. 4-F has a sequence shown in SEQ ID NO. 50, BRP-F has a sequence shown in SEQ ID NO. 55, BRP-R has a sequence shown in SEQ ID NO. 56, H5U-F has a sequence shown in SEQ ID NO. 57, H5U-R has a sequence shown in SEQ ID NO. 58, H5D-F has a sequence shown in SEQ ID NO. 59, H5D-R has a sequence shown in SEQ ID NO. 60, EXP3-F has a sequence shown in SEQ ID NO. 61, EXP3-R has a sequence shown in SEQ ID NO. 62, BSW-F has a sequence shown in SEQ ID NO. 63, BSW-R has a sequence shown in SEQ ID NO. 64, XPR3-F has a sequence shown in SEQ ID NO. 65, H6U-F has a sequence shown in SEQ ID NO. 66, H6D-F has a sequence shown in SEQ ID NO. 68, H6D-R has a sequence shown in SEQ ID NO. 69, and EWX-F has a sequence shown in SEQ ID NO. 61, and has a sequence shown in SEQ ID NO. 7, and SEQ ID NO. 7, 75, SEQ ID NO. 7-F has a sequence shown in SEQ ID NO. 7, and SEQ ID NO. 7, 75, and SEQ ID NO. 7-F has a sequence shown in SEQ ID NO. 7. The THP-F has the sequence shown in SEQ ID NO. 82, the BSW has the sequence shown in SEQ ID NO. 83, the GGS1 has the sequence shown in SEQ ID NO. 84, and the tHMGR has the sequence shown in SEQ ID NO. 85.

2. The fermentation process of claim 1, wherein the slant medium comprises malt extract powder 130g/L, agar 20g/L, chloramphenicol 0.1g/L;

3. The fermentation process of claim 1, wherein in step (3) a portion of the amount of glucose and/or canola oil is added to the medium at the start of the fermentation and the remainder of the amount of glucose and/or canola oil is added during the fermentation.