CN113151027B

CN113151027B - Recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and construction method thereof

Info

Publication number: CN113151027B
Application number: CN202110319820.7A
Authority: CN
Inventors: 元英进; 郭晓静; 王颖; 肖文海; 姚明东
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2023-05-26
Anticipated expiration: 2041-03-25
Also published as: CN113151027A

Abstract

The invention relates to the field of microbial metabolism, in particular to a saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and a construction method thereof. The invention discloses two recombinant saccharomyces cerevisiae strains (Saccharomyces cerevisiae) for producing 7-dehydrocholesterol, which are named as saccharomyces cerevisiae (Saccharomyces cerevisiae) SyBE_Sc01250043 and saccharomyces cerevisiae (Saccharomyces cerevisiae) SyBE_Sc01250045, and are abbreviated as SyBE_Sc01250043 and SyBE_Sc01250045 and a construction method thereof. Compared with the prior reported strain, the recombinant saccharomyces cerevisiae strain provided by the invention obviously improves the yield of 7-dehydrocholesterol.

Description

Recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and construction method thereof

Technical Field

The invention relates to the field of microbial metabolism, in particular to a saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and a construction method thereof.

Background

7-dehydrocholesterol is a steroid with high added value and wide application in industry and medicine, and can be used in the fields of liquid crystal manufacture, electromagnetic detection, biomedicine and the like. The 7-dehydrocholesterol can be converted into vitamin D3 by ultraviolet irradiation, and the 7-dehydrocholesterol in human skin can be converted into vitamin D by sunlight ultraviolet irradiation. Vitamin D can be used for treating vitamin D3 deficiency, rickets, familial hypophosphatemia, hypoparathyroidism, etc., and vitamin D deficiency may lead to cardiovascular diseases, metabolic syndrome, cancer, autoimmune diseases, etc. The current vitamin D3 obtaining way in the market is mainly to extract from tuna liver oil or synthesize by photochemical reaction of 7-dehydrocholesterol, the conversion rate of the photochemical reaction can reach 96%, so the synthesis of 7-dehydrocholesterol becomes a key step for synthesizing vitamin D3.

Currently common chemical methods for synthesizing 7-dehydrocholesterol generally use cholesterol as a raw material to derive bromination/dehydrobromination, oxidation/reduction/elimination, thermal decomposition, and conversion of cholesterol into 7-dehydrocholesterol by bioconversion. The traditional 7-dehydrogenation synthetic method is a chemical synthesis method and has the defects of complicated reaction steps, low yield, more byproducts, high energy consumption, heavy pollution and the like. The Christine Lang group of the university of Berlin industry in 2006 discloses a method for its synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae. They overexpressed a truncated form of HMG-CoA reductase tHMG1 in Saccharomyces cerevisiae chassis, knocked out the C22 sterol dehydrogenase gene (ERG 5) and sterol C24 methyltransferase gene (ERG 6), and introduced expression plasmids of sterol C24 reductase (DHCR 24) and C8 isomerase (ERG 2) and C5 sterol desaturase (ERG 3) inserted into human or mouse, thereby synthesizing 7-dehydrocholesterol. In addition, hohmann Hans-Peter Netherlands scientist also constructed yeast strains capable of synthesizing 7-dehydrocholesterol by similar methods. Furthermore, in previous experiments, our laboratory also conducted many studies on the de novo synthesis of 7-dehydrocholesterol by Saccharomyces cerevisiae, greatly increasing the level of 7-dehydrocholesterol synthesis.

However, no studies have been made so far concerning the improvement of 7-dehydrocholesterol production by rearranging the distribution of enzymes in the 7-dehydrocholesterol synthesis pathway among different organelles.

Disclosure of Invention

In view of this, the present invention provides a strain of Saccharomyces cerevisiae that produces 7-dehydrocholesterol and a method of constructing the same. The invention optimizes the imbalance of metabolic preconditions among organelles better through rearrangement protein positioning, thereby achieving the improvement of the yield of 7-dehydrocholesterol.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides the use of overexpression of 7-dehydrocholesterol pathway enzymes and/or rearrangement thereof in organelles for the synthesis of 7-dehydrocholesterol, for increasing the yield of 7-dehydrocholesterol and/or for reducing intermediates or byproducts of 7-dehydrocholesterol.

In some embodiments of the invention, the 7-dehydrocholesterol pathway enzyme comprises one or more of DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, ERG 27.

In some embodiments of the invention, the organelle comprises an endoplasmic reticulum and/or a liposome.

More importantly, the invention also provides recombinant strains of Saccharomyces cerevisiae that overexpress 7-dehydrocholesterol pathway enzymes and/or rearrange the 7-dehydrocholesterol pathway enzymes in organelles.

In some embodiments of the invention, the 7-dehydrocholesterol pathway enzyme in the recombinant strain of saccharomyces cerevisiae comprises one or more of DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, ERG 27.

In some embodiments of the invention, the recombinant strain of saccharomyces cerevisiae comprises the following modules:

module one: ERG2-ERG3;

and a second module: DHCR24;

wherein ERG2 and DHCR24 are located with AAM-B; ERG3 is positioned by oleosin;

AAM-B-ERG2, oleosin-ERG3 (or ERG2, ERG 3) integrated at the HO site, AAM-B-DHCR24 or DHCR24 integrated at the ERG6 site, while knocking out ERG6; or (b)

And a third module: ERG1-ERG11-ERG24;

and a fourth module: ERG25-ERG26-ERG27;

the first two genes of the three genes in the third and fourth modules are fused, and an Oleosin sequence is arranged between the two genes for positioning; the third gene is positioned by AAM-B sequence, and two modules are integrated at delta22 site and delta15 site respectively;

or the introduction of module one into the tau3 site.

In some embodiments of the invention, the s.cerevisiae recombinant strain:

DHCR24, ERG2 and/or ERG3 localization to liposomes; or (b)

DHCR24, ERG2 and/or ERG3 are overexpressed in the endoplasmic reticulum; or (b)

ERG2, ERG3 localization to liposomes and DHCR24 overexpression to endoplasmic reticulum; or (b)

ERG2, ERG3 over-expressed in the endoplasmic reticulum and DHCR24 localized to the liposomes; or (b)

DHCR24, ERG2 and/or ERG3 localization to liposomes and ERG2 and/or ERG3 overexpression to the endoplasmic reticulum; or (b)

DHCR24, ERG2 and/or ERG3 localization to liposomes and ERG25, ERG26 and/or ERG27 overexpression to the endoplasmic reticulum; or (b)

DHCR24, ERG2 and/or ERG3 localization to liposomes and ERG25, ERG26 and/or ERG27 localization to liposomes; or (b)

DHCR24, ERG2 and/or ERG3 localization to liposomes and ERG1, ERG11 and/or ERG24 overexpression to the endoplasmic reticulum; or (b)

DHCR24, ERG2 and/or ERG3 localization to liposomes and ERG1, ERG11 and/or ERG24 localization to liposomes; or (b)

DHCR24, ERG2 and/or ERG3, and ERG25, ERG26 and/or ERG27, and ERG1, ERG11 and/or ERG24 over-expressed in the endoplasmic reticulum; or (b)

DHCR24, ERG2 and/or ERG3, ERG25, ERG26 and/or ERG27, ERG1, ERG11 and/or ERG24;

DHCR24, ERG2 and/or ERG3 are localized to the liposome, ERG25, ERG26 and/or ERG27 are localized to the liposome and ERG1, ERG11 and/or ERG24 are overexpressed to the endoplasmic reticulum and ERG2 and/or ERG3 are overexpressed to the endoplasmic reticulum.

The invention also provides a construction method of the saccharomyces cerevisiae recombinant strain, and the module is imported into an original strain;

the module comprises:

module one: ERG2-ERG3;

and a second module: DHCR24;

wherein ERG2 and DHCR24 are located with AAM-B; ERG3 is positioned by oleosin;

And a third module: ERG1-ERG11-ERG24;

and a fourth module: ERG25-ERG26-ERG27;

or introducing module one into tau3 site;

in some embodiments of the invention, the construction method comprises:

DHCR24, ERG2 and/or ERG3 localization to liposomes; or (b)

DHCR24, ERG2 and/or ERG3 are overexpressed in the endoplasmic reticulum; or (b)

DHCR24, ERG2 and/or ERG3, ERG25, ERG26 and/or ERG27, ERG1, ERG11 and/or ERG24;

The invention also provides application of the saccharomyces cerevisiae recombinant strain in synthesizing 7-dehydrocholesterol, improving the yield of 7-dehydrocholesterol and/or reducing the intermediate or byproducts of 7-dehydrocholesterol.

The invention also provides a method for synthesizing 7-dehydrocholesterol by using the saccharomyces cerevisiae recombinant strain, which comprises the steps of inoculating, culturing, collecting culture solution, separating and purifying.

In the experiment of constructing the high-yield 7-dehydrocholesterol strain, 7-dehydrocholesterol pathway enzymes originally distributed on endoplasmic reticulum and liposome are rearranged, so that the problem that metabolic flows are unbalanced between two organelles caused by an original arrangement mode is solved, the total amount of the metabolic flows is pulled, the arrangement is more favorable for the production of 7-dehydrocholesterol, and the yield of 7-dehydrocholesterol is greatly improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a pathway diagram for the synthesis of 7-dehydrocholesterol using recombinant Saccharomyces cerevisiae;

FIG. 2 shows a diagram of a yeast sterol downstream repositioning gene expression cassette plasmid construction process;

FIG. 3 shows a diagram of the integration of the yeast sterol downstream repositioning genes ERG2-ERG3 into the genome;

FIG. 4 shows a diagram of the integration of the yeast sterol downstream repositioning gene DHCR24 into the genome;

FIG. 5 shows a diagram of the process of plasmid construction and integration of the yeast sterol upstream repositioning gene ERG25-26-27 into the genome;

FIG. 6 shows a diagram of the process of plasmid construction and integration of the yeast sterol upstream repositioning gene ERG1-11-24 into the genome;

FIG. 7 shows a diagram of the integration of the yeast sterol downstream gene ERG2-ERG3 into the genome of the 7-dehydrocholesterol high-producing strain SyBE_Sc 01250043;

FIG. 8 shows a graph of 7-dehydrocholesterol content, total sterol content, and 7-dehydrocholesterol ratio of each strain produced after rearrangement of genes located downstream of yeast sterols;

FIG. 9 shows a graph of 7-dehydrocholesterol content, total sterol content, and 7-dehydrocholesterol ratio of each strain produced after rearrangement of genes located upstream of yeast sterols;

FIG. 10 shows a graph of the 7-dehydrocholesterol content, total sterol content, and 7-dehydrocholesterol duty cycle of the strain SyBE_Sc01250045 obtained after introducing the downstream module endoplasmic reticulum-expressed ERG2-ERG3 into the highest yield strain SyBE_Sc01250043 among the strains produced after rearrangement of the located yeast sterol upstream genes.

Detailed Description

The invention discloses a saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and a construction method thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

The invention aims to overcome the defects in the prior art and provides a construction method of a recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol.

The second object of the present invention is to provide a recombinant strain of Saccharomyces cerevisiae that produces 7-dehydrocholesterol in high yield.

The technical scheme of the invention is summarized as follows:

a construction method of a recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol comprises the following steps:

(1) Recombinant Saccharomyces cerevisiae with high yield of 7-dehydrocholesterol is taken as chassis strain, and three enzymes DHCR24, ERG2 and ERG3 downstream of the yeast sterol in the postsqualene pathway are divided into two modules: DHCR24 alone is a module and ERG2-ERG3 is a module. The two modules are respectively over-expressed in the original positioning endoplasmic reticulum or the repositioning liposome, and different modules and different positioning modes are arranged and combined to obtain the distribution mode of various enzymes in the organelle, so that a plurality of strains are obtained. The optimal distribution mode of the enzyme is found out through the measurement of the yield of different strains. (2) The highest yielding strain obtained in the first step was used as the starting strain, and enzymes in the post squalene pathway that were originally located in the endoplasmic reticulum upstream of the yeast sterol were divided into two groups. The first group contains ERG1, ERG11, ERG24, and the second group contains ERG25, ERG26, ERG27, respectively constructing two modules. The two modules are respectively over-expressed in the original positioning endoplasmic reticulum or the repositioning liposome, and different modules and different positioning modes are arranged and combined to obtain the distribution mode of various enzymes in the organelle. Thus obtaining a plurality of strains. And (3) carrying out shake flask fermentation on the obtained strain, and screening to obtain the strain which is most beneficial to high-yield 7-dehydrocholesterol and an organelle arrangement mode of enzymes.

The invention has the advantages that:

In the saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and the construction method thereof, the raw materials and the reagents used in the method can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 rearrangement of organelle distribution of enzymes downstream of yeast sterols to increase 7-dehydrocholesterol production the preliminary construction of the recombinant s.cerevisiae strain for 7-dehydrocholesterol production of the present invention was as follows:

1. the acquisition of high-yielding 7-dehydrocholesterol s.cerevisiae was provided by the metaenglish-entry group, strain No. sybe_sc0125X001 (Guo, x.j., xiao, w.h., wang, y., yao, m.d., zeng, b.x., liu, h., zhao, g.r., and Yuan, y.j. (2018), metabolic engineering of Saccharomyces cerevisiae for-dehydrocholesterol overproduction.

2. Acquisition of exogenous functional Gene elements

The exogenous gene is a C24 site reductase gene DHCR24 (shown as SEQ ID No. 1) for synthesizing 7-dehydrocholesterol, the source is chicken source (bacillus), and after the genes are all subjected to Saccharomyces cerevisiae codon optimization and suitable avoidance of common restriction enzyme cutting sites, 5' ends gcggccgcggtctcca (shown as SEQ ID No. 2) are additionally added at two ends of the genes; the 3' taaaggagaccgcggccgc (shown as SEQ ID No. 3) is obtained through artificial synthesis.

3. Construction of a Modular integration plasmid

Building a first module: first, the authors designed simultaneous repositioning of the ERG2 (Accession: KZV 09055.1), ERG3 (Accession: CAA 97586.1) and DHCR24 genes of chicken origin to liposomes using the liposome targeting sequences AAM-B (shown as SEQ ID No. 4), oleosin (shown as SEQ ID No. 5) and AAM-B, respectively. Specifically, ERG2 and DHCR24 are located with AAM-B; ERG3 was localized with oleosin. To enable stable presence of the module, a method of integrating the module into the genome is selected. Since homologous recombination of repetitive sequences is easily generated when adjacent sequences in Saccharomyces cerevisiae are repeated and is lost from genome, two modules comprising AAM-B sequences must be avoided from being integrated at the same site, and thus the test is designed to integrate three gene modules in two steps: AAM-B-ERG2, oleosin-ERG3 (or ERG2, ERG 3) is integrated at the HO site, AAM-B-DHCR24 (or DHCR 24) is integrated at the ERG6 site, while ERG6 is knocked out. The starting chassis of the two strains are SyBE_Sc0125X001. The specific construction process is that the positioning sequence AAMB or oleosin and the gene sequence are subjected to OE-PCR, and are connected into an expression cassette TDH2t-gal1p-FBA1t or FBA1t-gal7p-PGK1t in a laboratory module library in an enzyme digestion connection mode. Wherein Oleosin-ERG3 (ERG 3) and (AAM-B-DHCR 24) DHCR24 use gal1p expression cassettes, and AAM-B-ERG2 (ERG 2) uses gal7p expression cassettes. Then, the Oleosin-ERG3 (ERG 3) and the AAM-B-ERG2 (ERG 2) are connected in a seamless connection mode to form a large fragment TDH2t-gal1p-Oleosin-ERG3 (ERG 3) -FBA1t-gal 7p-AAM-B-ERG2 (ERG 2) -PGK1t. Meanwhile, a homologous recombination left arm HO_L-URA-TDH2t and a right arm PGK1t-HO_R are constructed by an OE-PCR method and are connected into a Blunt end vector pEAZY-Blunt. The expression module and the homology arm module constructed above are respectively transformed into escherichia coli competent DH 5alpha, colony PCR screening is carried out, and single and double digestion verification and sequencing verification are carried out on plasmid extraction so as to ensure that the target fragment is connected correctly and the base sequence is not mutated.

4. Recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol through modularized integration construction

Firstly, an expression module and homologous recombination left and right arm module plasmids are cut by using a non 1 enzyme cutting site to obtain an integrated fragment, and the module TDH2t-gal1p-Oleosin-ERG3 (or ERG 3) -FBA1t-gal 7p-AAM-B-ERG2 (or ERG 2) -PGK1t and three fragments of homologous recombination left and right arms HO_L-URA-TDH2t and PGK1t-HO_R are firstly transformed into a high-yield 7-dehydrocholesterol yeast strain SyBE_Sc0125X001 by a lithium acetate method, and are integrated on a genome by recombining HO left and right homologous sequences and HO sites on a yeast genome. Strains with HO sites integrated into ERG2 and ERG3 and strains with HO sites integrated into AAMB-ERG2 and Oleosin-ERG3 are obtained respectively. The right arm ERG6_L-LEU-TDH2t and FBA1t-ERG6_R of the homologous recombination group of the module ERG6 locus are obtained from a laboratory module library. It was cut with not1 and transformed into the two chassis strains obtained as described above together with the fragment AAMB-DHCR24 (or DHCR 24). Four high-yield 7-dehydrocholesterol strains are obtained.

After transformation, SD-TRP-LEU-HIS-URA solid plates (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, mixed amino acid powder of tryptophan, leucine, histidine and uracil 2g/L, agar powder of 2%) are adopted for screening, the obtained transformant is subjected to streak purity culture, yeast genome is extracted for PCR verification, and the correct recombinant strain is verified to store glycerol bacteria and is named SyBE_Sc01250034SyBE_Sc01250035, syBE_Sc01250036 and SyBE_Sc01250037 respectively. Wherein, syBE_Sc01250034: syBE_Sc0125X001, Δho:: P _GAL1 -ERG3-P _GAL7 ERG2,ΔERG6::P _GAL1 -DHCR24；SyBE_Sc01250035：SyBE_Sc0125X001，Δho::P _GAL1 -Oleosin-ERG3-P _GAL7 -AAMB-ERG2,ΔERG6::P _GAL1 -AAMB-DHCR24；SyBE_Sc01250036：SyBE_Sc0125X001，Δho::P _GAL1 -Oleosin-ERG3-P _GAL7 AAMB-ERG2,ΔERG6::P _GAL1 -DHCR24；SyBE_Sc01250037：SyBE_Sc0125X001，Δho::P _GAL1 -ERG3-P _GAL7 ERG2,

ΔERG6::P _GAL1 -AAMB-DHCR24；

The delta15 locus in the strain SyBE_Sc01250035 is further integrated with a module TDH2t-gal1p-ERG3-FBA 1t-gal7p-ERG 2-PGK1t to obtain the strain SyBE_Sc01250038.SyBE_Sc01250038：SyBE_Sc0125X001，Δho::P _GAL1 -Oleosin-ERG3-P _GAL7 -AAMB-ERG2,ΔERG6::P _GAL1 -AAMB-DHCR24，Δdelta15::P _GAL1 -ERG3-P _GAL7 ERG2。

The specific construction method of the strain SyBE_Sc01250038 is similar to the strain construction method described above, except that the auxotroph tag is not selected as a screening method, but the genome integration is performed by using a CRISPR method.

5. The 7-dehydrocholesterol shake flask yields of the newly constructed strain SyBE_Sc01250034-SyBE_SyBE_Sc01250038 and the control strain SyBE_Sc0125XJ06 were compared. Genotype of SyBE_Sc0125XJ06 is SyBE_Sc0125XJ06: syBE_Sc0125X001, ΔERG6:: P _GAL1 -DHCR24。

Test materials: the strain SyBE_Sc01250034-SyBE_SyBE_Sc01250038 and the control strain SyBE_Sc0125XJ06 (Guo, X.J., xiao, W.H., wang, Y., yao, M.D., zeng, B.X., liu, H., zhao, G.R., and Yuan, Y.J. (2018) Metabolic engineering of Saccharomyces cerevisiae for-dehydrocholeastercap production. Biotechnology Biofurles 11,192).

The test method comprises the following steps:

seed culture medium: 40g/L glucose, 20g/L peptone and 10g/L yeast extract powder;

fermentation medium: 40g/L glucose, 20g/L peptone, 10g/L yeast extract, 10g/L D-galactose.

Inoculating the strain into 5mL seed culture medium, culturing at 30deg.C and 250rpm for 14-16 hr, and concentrating at initial thallus concentration OD ₆₀₀ Respectively, 50mL of fermentation medium was inoculated with 0.2, galactose was added, and the culture was carried out at 30℃and 250rpm, and the cell density (OD 600) and the 7-dehydrocholesterol yield during the fermentation were monitored.

Method for quantifying 7-dehydrocholesterol: 4ml of fermentation broth, 5000g of the fermentation broth are centrifuged for 2min to collect thalli, 3N HCl is used for resuspension of cells, the thalli is placed in a boiling water bath for boiling for 5min, then the thalli are collected by centrifugation, washed once by water, resuspended by 3M NaOH/methanol solution, and the centrifuge tube is placed in a water bath kettle at 60 ℃ for saponification for 4h. After saponification is completed, 400mL of normal hexane is added, a certain amount of quartz sand is added, vortex vibration is carried out for 20min, and the upper layer solution is collected by centrifugation. The extraction process was repeated three times to ensure that the product was sufficiently extracted. And (3) centrifuging in vacuum for 30min, and evaporating the collected normal hexane phase to dryness to obtain solid powder containing 7-dehydrocholesterol. The powder was derivatized with MSTFA and diluted before being gaseously loaded.

The test results are shown in FIG. 8 and Table 1.

Table 1 figure 8 data

The test results in fig. 8 show that:

the above five strains were classified according to the location of ERG2/ERG 3: the first are SyBE_Sc01250034 and SyBE_Sc01250037; the second class is SyBE_Sc01250036 and SyBE_Sc01250035; the third class is SyBE_Sc01250038. In the first strain, ERG2-3 is over-expressed in endoplasmic reticulum; in the second strain, ERG2-3 is over-expressed in liposomes; the third class of strains comprises the endoplasmic reticulum and the liposomal overexpressed ERG2/ERG3.

The total sterols of the first strain were not significantly changed compared to the control strain, in which only one copy of the liposome DHCR 24-expressing strain sybe_sc01250037 was increased in either 7-dehydrocholesterol production or duty cycle (46.1% to 274.2mg/L increase in production and 40.7% increase in duty cycle). The increase in 7-dehydrocholesterol results from DHCR24 expressed by liposomes, which was also demonstrated when comparing the second strain, sybe_sc01250036, with sybe_sc01250035. The second strain shares a copy of liposome-expressed ERG2-3. The total sterol levels of the second class strains, sybe_sc01250036 and sybe_sc01250035, were increased by 20.5% and 16.8%, respectively, compared to the control strain; the yield of 7-dehydrocholesterol was increased by 51.0% (to 283.4 mg/L) and 64.2% (to 308.2 mg/L), respectively; the 7-dehydrocholesterol ratio was increased by 25.1% and 40.7%, respectively. These results indicate that localization of ERG2/3 to liposomes not only pulls the entire postsqualene pathway metabolic flux, but can also further increase the production of 7-dehydrocholesterol. The third strain possesses both endoplasmic reticulum and liposomal expressed ERG2/ERG3. The total cholesterol amount, the 7-dehydrocholesterol ratio, and the 7-dehydrocholesterol yield were increased by 21.0%,18.0%, and 42.7% (to 267.8 mg/L), respectively, as compared to the control strain, but the 7-dehydrocholesterol yield was decreased as compared to the strain SyBE_Sc01250035.

Of the 5 newly constructed strains, the strain SyBE_Sc01250035, in which ERG2, ERG3 and DHCR24 were all relocated to the liposomes, had the highest 7-dehydrocholesterol production. Indicating that the distribution of all of the ERG2, ERG3, DHCR24 rearranged into liposomes with one copy of DHCR24 already in the chassis is most advantageous for 7-dehydrocholesterol production. Notably, the highest proportion of 7-dehydrocholesterol in the yeast sterol downstream product in strain SyBE_Sc01250034 suggests that overexpression of ERG2-ERG3-DHCR24 in its original position can reduce the levels of other intermediate products downstream of yeast sterols.

Example 2 rearrangement of the organelle distribution of enzymes upstream of yeast sterols to increase 7-dehydrocholesterol production

1. Construction of a Modular integration plasmid

First, the applicant devised to split enzymes present in the endoplasmic reticulum upstream of yeast sterols in the postsqualene pathway into two modules: ERG1-ERG11-ERG24 is module one and ERG25-ERG26-ERG27 is module two. To enable stable presence of the module, a method of integrating the module into the genome is selected. Since homologous recombination of repetitive sequences is easily generated when adjacent sequences in Saccharomyces cerevisiae are repeated and is lost from genome, two modules containing AAM-B sequences must be avoided from being integrated at the same site, the test is designed to fuse the first two genes of three genes in each module and place an Oleosin sequence between the two genes for localization; the third gene was mapped with AAM-B sequence and the two modules were integrated in two steps at the delta22 site and the delta15 site, respectively. The starting chassis of the two strains is SyBE_Sc01250035.

The specific construction process is that the positioning sequence AAMB or oleosin and the gene sequence are subjected to OE-PCR, and are connected into an expression cassette TDH2t-gal1p-FBA1t or FBA1t-gal7p-PGK1t in a laboratory module library in a seamless connection mode. Wherein ERG1-Oleosin-ERG11 (ERG 1-ERG 11) and ERG25-Oleosin-ERG26 (ERG 25-ERG 26) use gal1p expression cassettes, and AAM-B-ERG24 (ERG 24) and AAM-B-ERG27 (ERG 27) use gal7p expression cassettes. Then constructing homologous recombination left arm delta22 (or 15) _L-TDH2t and right arm FBA1t-GAL7p-AAM-B-ERG24 (ERG 24) -PGK1t-delta22 (or 15) _R or FBA1t-GAL7p-AAM-B-ERG27 (ERG 27) -PGK1t-delta22 (or 15) _R by using an OE-PCR method, and connecting into a Blunt end vector pEAZY-Blunt or PRS425K plasmid. In addition, CRISPR plasmids with targets of delta22 and delta15 are also required to be constructed for cutting corresponding sites, and the screening of nutrition labels is replaced. The expression module and the homology arm module constructed above are respectively transformed into escherichia coli competent DH 5alpha, colony PCR screening is carried out, and single and double digestion verification and sequencing verification are carried out on plasmid extraction so as to ensure that the target fragment is connected correctly and the base sequence is not mutated.

2. Recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol through modularized integration construction

Firstly, the expression module and homologous recombination left and right arm module plasmids are cut by using a non 1 restriction enzyme cutting site to obtain an integrated fragment, and three fragments of a module ERG1-Oleosin-ERG11 (ERG 1-ERG 11) or an ERG25-Oleosin-ERG26 (ERG 25-ERG 26) and homologous recombination left and right arms HO_L-TDH2t and FBA1t-GAL7p-AAM-B-ERG24 (ERG 24) -PGK1t-delta22_R or FBA1t-GAL7p-AAM-B-ERG27 (SyG 27) -PGK1t-delta22 are firstly transformed into a high-yield 7-dehydrocholesterol yeast strain BE_Sc01250035 by a lithium acetate method, and are integrated into a genome by recombination of left and right homologous sequences and delta22 sites on a yeast genome. The delta22 site was incorporated into GAL1p-ERG25-ERG26-FBA1t-GAL7p-ERG27-PGK1t/GAL1p-ERG25-Oleosin-ERG26-FBA1t-GAL7p-AAMBERG27-PGK1t and the delta22 site was incorporated into GAL1p-ERG1-ERG11-FBA1t-GAL7p-ERG24-PGK1t/GAL1p-ERG1-Oleosin-ERG11-FBA1t-GAL7p-AAMBERG24-PGK1t, respectively. Plasmid in yeast cells was discarded by serial passage. The strains SyBE_Sc01250039, syBE_Sc01250040, syBE_Sc01250041 and SyBE_Sc01250042 are obtained respectively. Then, introducing ERG25-Oleosin-ERG26-AAMB-ERG27 into SyBE_Sc01250040 strain at delta22 site, continuously integrating GAL1p-ERG1-ERG11-FBA1t-GAL7p-ERG24-PGK1t and GAL1p-ERG1-Oleosin-ERG11-FBA1t-GAL7p-AAMBERG24-PGK1t at delta15 site to obtain SyBE_Sc01250043 and SyBE_Sc01250044 strain respectively. Six different arrangements of recombinant strains were thus obtained in experiments in which the localization of the enzyme organelles upstream of the rearranged yeast sterols.

After transformation, SD-TRP-LEU-HIS-URA solid plates (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, mixed amino acid powder of tryptophan, leucine, histidine and uracil 2g/L, agar powder of 2%) are adopted for screening, the obtained transformant is subjected to streak purity culture, yeast genome is extracted for PCR verification, and the verified correct recombinant strain is used for preserving glycerinum and is named SyBE_Sc01250039 respectively: syBE_Sc01250035, delta22: PGAL1-ERG25-GGGGS-ERG26-TFBA1t-PGAL7-ERG27-TPGK1t SyBE_Sc01250040: syBE_Sc01250035, delta 22:PGAL 1-ERG25-Oleosin-ERG26-TFBA1t-PGAL7-AAMB-ERG27-TPGK1t SyBE_Sc01250041:SyBE_Sc01250035, delta 22:PGAL 1-ERG1-GGGGS-ERG11-TFBA1t-PGAL7-ERG24-TPGK1t:SyBE_Sc01250042:SyBE_Sc01250035, delta 22:PGAL 1-ERG1-Oleosin-ERG11-TFBA1t-PGAL7-AAMB-ERG24-TPGK1t SyBE_Sc01250043:SyBE_Sc01250040, delta 15:PGAL 1-ERG 1-GGS-ERG 11-ERG 1 t-PGGGS 7-ERG 24-GK 1 t-BE_Sc 01250044:SyBE_Sc5648, delta 22:PGAL 1-ERG1-OleOSin-ERG11-TFBA1 t-PGMB-ERG 7-TFBA 1 t-SyBE_Sc 01250043:SyBE_Sc01250040, delta 15:PGAL 1-ERG11-ERG 1-GKBE 9, and Delta 1-QCAMB-ERG 1-ERG2

3. The 7-dehydrocholesterol shake flask yields of the newly constructed strain SyBE_Sc01250039-SyBE_SyBE_Sc01250044 and the control strain SyBE_Sc01250035 were compared.

Test materials: the strain SyBE_Sc01250039-SyBE_SyBE_Sc01250044 and the control strain SyBE_Sc01250035.

The test method comprises the following steps: the same as in the first embodiment.

The test results are shown in FIG. 9 and Table 2.

Table 2 figure 9 data

The test results in fig. 9 show that:

the strains constructed in this section were classified into three groups according to the location of ERG1-11-24 localization. Class I: syBE_Sc01250039, 40; class II: syBE_Sc01250042, 44; class III: syBE_Sc01250041, 43. The first strain comprises an endoplasmic reticulum over-expression or liposome repositioning module ERG25-26-27, and no over-expression of ERG 1-11-24; ERG1-11-24 localization to liposomes in the second strain; ERG1-11-24 is localized to the endoplasmic reticulum in the third strain. Compared with the control strain SyBE_Sc01250035, the total sterol content and the 7-dehydrocholesterol content in the first strain are reduced, and the 7-dehydrocholesterol ratio is not increased. In the second strain, liposome-localized ERG1-11-24 was introduced into the control strain SyBE_Sc01250035 and the first strain SyBE_Sc 01250040. In the second strain, 7-dehydrocholesterol was not significantly changed, total cholesterol content was reduced, and the 7-dehydrocholesterol ratio was increased by 22.9%. Under pulling of endoplasmic reticulum ERG1-11-24, although the total sterol content in the third strain was lower than that of the control strain, the 7-dehydrocholesterol content of the SyBE_Sc01250041 and SyBE_Sc01250043 strains was increased by 10.5% (to 342.2 mg/L) and 16.4% (to 360.6 mg/L), respectively, while the 7-dehydrocholesterol ratio was also increased. Furthermore, in the third strain, the yield and ratio of 7-dehydrocholesterol of SyBE_Sc01250043 was higher than that of SyBE_Sc01250041 due to the introduction of liposome ERG 25-26-27. The applicant therefore speculates that the introduction of liposomal ERG25-26-27 in certain strains could pull the metabolic flux down and increase the yield of 7-dehydrocholesterol, while strain sybe_sc01250043 is also the strain with the highest current shake flask level.

Example 3 binding of organelle distribution of enzymes upstream and downstream of Yeast sterol further increases 7-dehydrocholesterol production

1. Construction of a Modular integration plasmid

To further reduce by-products in the strain, authors introduced the downstream genes ERG2, ERG3 in the post squalene pathway to the tau3 site in the high yield 7-dehydrocholesterol strain. The specific construction process is that a genome is taken as a template to amplify a fragment of tau3 upstream and downstream 700bp and terminators TDH2t and PGK1t. The upstream homology arm tau3_L was obtained by ligating tau3_up700 with TDH2t using OE-PCR, and the downstream homology arm tau3_R was obtained by ligating PGK1t with tau3_Down 700.

Firstly, the constructed expression module TDH2t-gal1p-ERG3-FBA 1t-gal7p-ERG 2-PGK1t is cut by a non 1 enzyme cutting site to obtain an integrated fragment, and the gene expression module, a homologous recombination left and right arm module tau3_L and tau3_R are transformed into a strain SyBE_Sc01250043 which produces 7-dehydrocholesterol at the highest temperature by a lithium acetate method to obtain a strain SyBE_Sc01250045.

SyBE_Sc01250045:SyBE_Sc01250040,Delta15::PGAL1-ERG1-GGGGS-ERG11-TFBA1t-PGAL7-ERG24-TPGK1tΔtau3::P _GAL1 -ERG3-P _GAL7 ERG2。

3. The 7-dehydrocholesterol shake flask yields of the newly constructed strain SyBE_Sc01250045 and the control strain SyBE_Sc01250043 were compared.

Test materials: strain sybe_sc01250045 and control strain sybe_sc01250043.

The test results are shown in FIG. 10 and tables 3 to 4.

TABLE 3 data of FIG. 10

	SyBE_Sc01250043	SyBE_Sc01250045
			5alpha-Cholest-8-en-3beta-ol(mg/L)	268.36±8.8	134.45±1.47
lathosterol(mg/L)	158.96±5.44	87.98±1.86
			7DHC(mg/L)	360.61±10.76	358.2±11.65

TABLE 4 Table 4

	SyBE_Sc01250043	SyBE_Sc01250045
			total sterol(mg/L)	1120.05±30.36	929.85±52.45
7-DHC ratio(％)	32.2±1.23	38.73±0.46

The test results in fig. 10 show that: although the 7-dehydrocholesterol production did not change significantly in strain SyBE_Sc01250045, the content of by-product 5alpha-Cholest-8-en-3beta-ol in strain SyBE_Sc01250045 was reduced by 49.9% compared to the control strain SyBE_Sc01250043, the content of lathosterol was reduced by 44.7%, and the total cholesterol content was reduced by 17.0%, thus the 7-dehydrocholesterol production was improved by 20.9%. It was found that the combined rearrangement of genes upstream and downstream of yeast sterol can reduce the content of by-products in addition to the strain that produces 7-dehydrocholesterol at a high yield, thereby increasing the ratio of 7-dehydrocholesterol to sterols.

Compared with the reported highest yield strain SyBE_Sc0125XJ08, the highest yield strain SyBE_Sc01250043 has the advantage of obviously improving the yield (P is less than 0.05).

TABLE 5

SyBE_Sc0125XJ08	SyBE_Sc01250043
		251.8±5.66	360.61±3.98

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> university of Tianjin

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Claims

Overexpression of 7-dehydrocholesterol pathway enzymes and their use in organelles to synthesize 7-dehydrocholesterol or to increase the yield of 7-dehydrocholesterol;

the 7-dehydrocholesterol pathway enzymes include DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, and ERG27;

during the application process, chassis bacteria saccharomyces cerevisiae is adopted to knock out ERG6; and the above-mentioned pathway enzymes are subjected to the following overexpression or rearrangement:

ERG2, ERG3 are simultaneously over-expressed and localized to the liposome, and DHCR24 is over-expressed and localized to the endoplasmic reticulum or liposome; or (b)

ERG2, ERG3 are simultaneously over-expressed and localized to the endoplasmic reticulum, and DHCR24 is over-expressed and localized to the liposome; or (b)

DHCR24, ERG2 and ERG3 are simultaneously overexpressed and localized to liposomes, and ERG2 and ERG3 are simultaneously overexpressed and localized to the endoplasmic reticulum; or (b)

DHCR24, ERG2 and ERG3 are simultaneously overexpressed and localized to liposomes, and ERG25, ERG26 and ERG27 are simultaneously overexpressed and localized to liposomes; or DHCR24, ERG2 and ERG3 are simultaneously overexpressed and localized to liposomes, and ERG1, ERG11 and ERG24 are simultaneously overexpressed and localized to liposomes or endoplasmic reticulum; or (b)

DHCR24, ERG2 and ERG3 are simultaneously over-expressed and localized to the liposome, and ERG1, ERG11 and ERG24 are simultaneously over-expressed and localized to the liposome, and ERG25, ERG26 and ERG27 are simultaneously over-expressed and localized to the liposome; or (b)

DHCR24, ERG2 and ERG3 are simultaneously over-expressed and localized to the liposome, and ERG1, ERG11 and ERG24 are simultaneously over-expressed and localized to the endoplasmic reticulum, and ERG25, ERG26 and ERG27 are simultaneously over-expressed and localized to the liposome.
Overexpression of 7-dehydrocholesterol pathway enzymes and their use in organelles to reduce 7-dehydrocholesterol intermediates or byproducts;

the 7-dehydrocholesterol pathway enzymes include DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, and ERG27;

during the application process, chassis bacteria saccharomyces cerevisiae is adopted to knock out ERG6; and the above-mentioned pathway enzymes are subjected to the following overexpression or rearrangement:

DHCR24, ERG2 and ERG3 are simultaneously over-expressed and localized to the liposome, and ERG1, ERG11 and ERG24 are simultaneously over-expressed and localized to the endoplasmic reticulum, and ERG25, ERG26 and ERG27 are simultaneously over-expressed and localized to the liposome, and ERG2 and ERG3 are simultaneously over-expressed and localized to the endoplasmic reticulum.
3. A recombinant strain of saccharomyces cerevisiae, characterized in that its 7-dehydrocholesterol pathway enzyme is overexpressed and rearranged in the organelle;

the 7-dehydrocholesterol pathway enzymes include DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, and ERG27;

the ERG6 is knocked out by using chassis fungus saccharomyces cerevisiae; and the above-mentioned pathway enzymes are subjected to the following overexpression or rearrangement:

ERG2, ERG3 are simultaneously over-expressed and localized to the liposome, and DHCR24 is over-expressed and localized to the endoplasmic reticulum or liposome; or (b)

ERG2, ERG3 are simultaneously over-expressed and localized to the endoplasmic reticulum, and DHCR24 is over-expressed and localized to the liposome; or (b)

DHCR24, ERG2 and ERG3 are simultaneously overexpressed and localized to liposomes, and ERG2 and ERG3 are simultaneously overexpressed and localized to the endoplasmic reticulum; or (b)

DHCR24, ERG2 and ERG3 are simultaneously overexpressed and localized to liposomes, and ERG25, ERG26 and ERG27 are simultaneously overexpressed and localized to liposomes; or (b)

DHCR24, ERG2 and ERG3 are simultaneously overexpressed and localized to liposomes, and ERG1, ERG11 and ERG24 are simultaneously overexpressed and localized to liposomes or endoplasmic reticulum; or (b)

DHCR24, ERG2 and ERG3 are simultaneously over-expressed and localized to the liposome, and ERG1, ERG11 and ERG24 are simultaneously over-expressed and localized to the liposome, and ERG25, ERG26 and ERG27 are simultaneously over-expressed and localized to the liposome; or (b)

DHCR24, ERG2 and ERG3 are simultaneously over-expressed and localized to the liposome, and ERG1, ERG11 and ERG24 are simultaneously over-expressed and localized to the endoplasmic reticulum, and ERG25, ERG26 and ERG27 are simultaneously over-expressed and localized to the liposome.
4. A recombinant strain of saccharomyces cerevisiae, characterized in that its 7-dehydrocholesterol pathway enzyme is overexpressed and rearranged in the organelle;

the 7-dehydrocholesterol pathway enzymes include DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, and ERG27;

the strain of the chassis fungus Saccharomyces cerevisiae is adopted to knock out ERG6; and the above-mentioned pathway enzymes are subjected to the following overexpression or rearrangement:

DHCR24, ERG2 and ERG3 are simultaneously over-expressed and localized to the liposome, and ERG1, ERG11 and ERG24 are simultaneously over-expressed and localized to the endoplasmic reticulum, and ERG25, ERG26 and ERG27 are simultaneously over-expressed and localized to the liposome, and ERG2 and ERG3 are simultaneously over-expressed and localized to the endoplasmic reticulum.
5. A method of constructing a recombinant strain of saccharomyces cerevisiae according to claim 3, wherein its 7-dehydrocholesterol pathway enzyme is overexpressed and rearranged in the organelle;

the 7-dehydrocholesterol pathway enzymes include DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, and ERG27;

the ERG6 is knocked out by using chassis fungus saccharomyces cerevisiae; and the above-mentioned pathway enzymes are subjected to the following overexpression or rearrangement:

ERG2, ERG3 are simultaneously over-expressed and localized to the liposome, and DHCR24 is over-expressed and localized to the endoplasmic reticulum or liposome; or (b)

ERG2, ERG3 are simultaneously over-expressed and localized to the endoplasmic reticulum, and DHCR24 is over-expressed and localized to the liposome; or (b)

DHCR24, ERG2 and ERG3 are simultaneously overexpressed and localized to liposomes, and ERG2 and ERG3 are simultaneously overexpressed and localized to the endoplasmic reticulum; or (b)

DHCR24, ERG2 and ERG3 are simultaneously overexpressed and localized to liposomes, and ERG25, ERG26 and ERG27 are simultaneously overexpressed and localized to liposomes; or (b)

DHCR24, ERG2 and ERG3 are simultaneously overexpressed and localized to liposomes, and ERG1, ERG11 and ERG24 are simultaneously overexpressed and localized to liposomes or endoplasmic reticulum; or (b)

DHCR24, ERG2 and ERG3 are simultaneously over-expressed and localized to the liposome, and ERG1, ERG11 and ERG24 are simultaneously over-expressed and localized to the liposome, and ERG25, ERG26 and ERG27 are simultaneously over-expressed and localized to the liposome; or (b)

DHCR24, ERG2 and ERG3 are simultaneously over-expressed and localized to the liposome, and ERG1, ERG11 and ERG24 are simultaneously over-expressed and localized to the endoplasmic reticulum, and ERG25, ERG26 and ERG27 are simultaneously over-expressed and localized to the liposome.
6. The method for constructing a recombinant strain of Saccharomyces cerevisiae according to claim 4, wherein the 7-dehydrocholesterol pathway enzyme is overexpressed and rearranged in the organelle;

the 7-dehydrocholesterol pathway enzymes include DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, and ERG27;

the ERG6 is knocked out by using chassis fungus saccharomyces cerevisiae; and the above-mentioned pathway enzymes are subjected to the following overexpression or rearrangement:

DHCR24, ERG2 and ERG3 are simultaneously over-expressed and localized to the liposome, and ERG1, ERG11 and ERG24 are simultaneously over-expressed and localized to the endoplasmic reticulum, and ERG25, ERG26 and ERG27 are simultaneously over-expressed and localized to the liposome, and ERG2 and ERG3 are simultaneously over-expressed and localized to the endoplasmic reticulum.
7. Use of a recombinant strain of saccharomyces cerevisiae according to claim 3 or 4 for the synthesis of 7-dehydrocholesterol or for increasing the yield of 7-dehydrocholesterol.
8. Use of the recombinant strain of saccharomyces cerevisiae according to claim 4 for reducing intermediates or byproducts of 7-dehydrocholesterol.
9. A method for synthesizing 7-dehydrocholesterol by using the recombinant strain of saccharomyces cerevisiae, which is characterized in that the recombinant strain of saccharomyces cerevisiae according to claim 3 or 4 is inoculated, cultured, the culture solution is collected, separated and purified.