CN113151027A

CN113151027A - Recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and construction method thereof

Info

Publication number: CN113151027A
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: 2021-07-23
Anticipated expiration: 2041-03-25
Also published as: CN113151027B

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 called as SyBE _ Sc01250043 and SyBE _ Sc01250045 for short, and a construction method thereof. Compared with the existing reported strains, the recombinant saccharomyces cerevisiae strain provided by the invention has the advantage that the yield of 7-dehydrocholesterol is remarkably improved.

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 having a high added value and widely used in both industry and medicine, and can be used in the fields of liquid crystal manufacturing, 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 solar ultraviolet irradiation. Vitamin D can be used for treating vitamin D3 deficiency, rickets, familial hypophosphatemia, hypoparathyroidism, etc., and vitamin D deficiency may cause cardiovascular disease, metabolic syndrome, cancer, autoimmune disease, etc. At present, the acquisition route of vitamin D3 in the market is mainly extraction from tuna liver oil or synthesis through photochemical reaction of 7-dehydrocholesterol, and the conversion rate of the photochemical reaction can reach 96 percent, so that the synthesis of the 7-dehydrocholesterol becomes a key step for synthesizing the vitamin D3.

The chemical methods commonly used at present for synthesizing 7-dehydrocholesterol generally derive bromination/dehydrobromination, oxidation/reduction/elimination, thermal decomposition and conversion of cholesterol into 7-dehydrocholesterol by biotransformation from cholesterol as a raw material. The traditional 7-dehydrogenation synthesis 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. In 2006, Christine Lang project group, university of Berlin, Germany, disclosed its method of synthesizing 7-dehydrocholesterol in Saccharomyces cerevisiae. They overexpressed truncated forms of HMG-CoA reductase tmg 1 in the saccharomyces cerevisiae chassis, knocked out the C22 sterol dehydrogenase gene (ERG5) and the sterol C24 methyltransferase gene (ERG6), and introduced expression plasmids inserted with human or mouse sterol C24 reductase (DHCR24), C8 isomerase (ERG2) and C5 sterol desaturase (ERG3) to synthesize 7-dehydrocholesterol. In addition, Hohmann Hans-Peter Dutch scientists have similarly constructed yeast strains capable of synthesizing 7-dehydrocholesterol. In addition, in previous experiments, our laboratory also made many explorations on the de novo synthesis of 7-dehydrocholesterol by Saccharomyces cerevisiae, greatly improving 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 in different organelles.

Disclosure of Invention

In view of the above, the invention provides a saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and a construction method thereof. The invention better optimizes the imbalance of metabolism precondition among organelles by rearranged protein positioning, thereby achieving the purpose of improving 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 a 7-dehydrocholesterol pathway enzyme and/or rearrangement thereof in organelles in the synthesis of 7-dehydrocholesterol, increasing the yield of 7-dehydrocholesterol and/or reducing intermediates or by-products 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 organelles include endoplasmic reticulum and/or liposomes.

More importantly, the invention also provides a saccharomyces cerevisiae recombinant strain, wherein the 7-dehydrocholesterol pathway enzyme is over-expressed and/or the 7-dehydrocholesterol pathway enzyme is rearranged in an organelle.

In some embodiments of the invention, the 7-dehydrocholesterol pathway enzymes in the recombinant strain of saccharomyces cerevisiae include 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:

a first module: ERG2-ERG 3;

and a second module: DHCR 24;

wherein ERG2 and DHCR24 are located with AAM-B; ERG3 was located with oleosin;

AAM-B-ERG2, Oleosin-ERG3 (or ERG2, ERG3) are integrated at HO site, AAM-B-DHCR24 or DHCR24 are integrated at ERG6 site, and ERG6 is knocked out; or

And a third module: ERG1-ERG11-ERG 24;

and a module IV: ERG25-ERG26-ERG 27;

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

or introduce module one at the tau3 site.

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

DHCR24, ERG2, and/or ERG3 are localized to the liposomes; or

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

ERG2, ERG3 localized to liposomes and DHCR24 was overexpressed in the endoplasmic reticulum; or

ERG2, ERG3 were overexpressed in the endoplasmic reticulum and DHCR24 localized to the liposomes; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG2 and/or ERG3 are overexpressed in the endoplasmic reticulum; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG25, ERG26 and/or ERG27 are overexpressed in the endoplasmic reticulum; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG25, ERG26 and/or ERG27 are localized to the liposomes; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG1, ERG11 and/or ERG24 are overexpressed in the endoplasmic reticulum; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG1, ERG11 and/or ERG24 are localized to the liposomes; or

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

DHCR24, ERG2 and/or ERG3 are localized to liposomes, ERG25, ERG26 and/or ERG27 are localized to liposomes and ERG1, ERG11 and/or ERG24 are localized to liposomes;

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

The invention also provides a construction method of the saccharomyces cerevisiae recombinant strain, which is characterized in that a module is introduced into an original strain;

the module comprises:

a first module: ERG2-ERG 3;

and a second module: DHCR 24;

wherein ERG2 and DHCR24 are located with AAM-B; ERG3 was located with oleosin;

And a third module: ERG1-ERG11-ERG 24;

and a module IV: ERG25-ERG26-ERG 27;

or introducing module one into the tau3 site;

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

DHCR24, ERG2, and/or ERG3 are localized to the liposomes; or

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

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

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

In the test for constructing the high-yield 7-dehydrocholesterol strain, the 7-dehydrocholesterol pathway enzymes originally distributed on the endoplasmic reticulum and the liposome are rearranged, so that the problem that metabolic flow is unbalanced between two organelles due to the original arrangement mode is solved, the total amount of the metabolic flow is pulled, the arrangement is more favorable for the production of the 7-dehydrocholesterol, and the yield of the 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 scheme for the synthesis of 7-dehydrocholesterol using recombinant Saccharomyces cerevisiae;

FIG. 2 shows a process diagram of construction of a plasmid for a gene expression cassette for a downstream translocation of zymosterol;

FIG. 3 shows a diagram of the integration of the zymosterol downstream translocation genes ERG2-ERG3 into the genome;

FIG. 4 shows a diagram of the process of integration of the zymosterol downstream translocation gene DHCR24 into the genome;

FIG. 5 shows a process diagram of plasmid construction and integration of the expression cassette for the zymosterol upstream relocating gene ERG25-26-27 into the genome;

FIG. 6 shows a process diagram of plasmid construction and integration of the expression cassette for the zymosterol upstream relocating gene ERG1-11-24 into the genome;

FIG. 7 shows a diagram of the process of integration of the downstream gene of zymosterol ERG2-ERG3 into the genome of 7-dehydrocholesterol high producing strain SyBE _ Sc 01250043;

FIG. 8 is a graph showing the 7-dehydrocholesterol content, total cholesterol content, and 7-dehydrocholesterol ratio of each strain produced after rearrangement of genes located downstream of zymosterol;

FIG. 9 is a graph showing the 7-dehydrocholesterol content, total cholesterol content, and 7-dehydrocholesterol ratio of each strain produced after rearrangement of the gene upstream of zymosterol;

FIG. 10 shows the 7-dehydrocholesterol content, total cholesterol content, 7-dehydrocholesterol ratio of the strain SyBE _ Sc01250045 obtained after introducing ERG2-ERG3 expressed in the downstream module endoplasmic reticulum into the highest-yielding strain SyBE _ Sc01250043 among the strains produced after rearrangement localization of the zymosterol upstream gene.

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 appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and 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 purpose of the invention is to provide a recombinant saccharomyces cerevisiae strain with high yield of 7-dehydrocholesterol.

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) a recombinant saccharomyces cerevisiae capable of producing 7-dehydrocholesterol at high yield is taken as a chassis strain, and three enzymes DHCR24, ERG2 and ERG3 downstream of the yeast sterol in a post-squalene pathway are divided into two modules: the DHCR24 is a module alone, and the ERG2-ERG3 are modules. The two modules are respectively over-expressed in the original location endoplasmic reticulum or relocated in the liposome, and different modules and different location modes are arranged and combined to obtain the distribution mode of various enzymes in organelles, thereby obtaining a plurality of strains. The optimal distribution pattern of the enzymes was found by measuring the yields of the different strains. (2) The highest producing strain from the first step was used as the starting strain, and the enzymes that are prototyped in the endoplasmic reticulum upstream of the zymosterol in the post-squalene pathway were divided into two groups. The first group comprised ERG1, ERG11, ERG24, and the second group comprised ERG25, ERG26, ERG27, building two modules, respectively. The two modules are respectively over-expressed in the original location endoplasmic reticulum or relocated in the liposome, and different modules and different location modes are arranged and combined to obtain the distribution mode of various enzymes in organelles. Thereby obtaining a plurality of strains. And (3) carrying out shake flask fermentation on the obtained strains, and screening to obtain the strains which are most beneficial to high yield of the 7-dehydrocholesterol and the arrangement mode of the organelles of the enzyme.

The invention has the advantages that:

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

The invention is further illustrated by the following examples:

example 1 rearrangement of organelle distribution of downstream enzymes of zymosterol to improve 7-dehydrocholesterol production the preliminary construction method of the recombinant saccharomyces cerevisiae strain producing 7-dehydrocholesterol of the present invention is as follows:

1. the availability of high 7-dehydrocholesterol producing s.cerevisiae was provided by the original British subject group under strain number 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) metabolism engineering of Saccharomyces cerevisiae for 7-dehydrocholesterol production, Biotechnol Biofuels 11, 192).

2. Obtaining exogenous functional gene elements

The exogenous gene is a C24 reductase gene DHCR24 (shown as SEQ ID No. 1) for synthesizing 7-dehydrocholesterol, the source is a chicken source (gallolus), the genes are optimized by saccharomyces cerevisiae codons and appropriately avoid common restriction enzyme cutting sites, and 5' ends gcggccgcggtctcca (shown as SEQ ID No. 2) are additionally added at two ends of the genes; 3' taaaggagaccgcggccgc (shown in SEQ ID No. 3) was obtained by artificial synthesis.

3. Construction of a Modularly Integrated plasmid

Constructing a first module: firstly, the authors designed to relocate the genes of module one ERG2 (Access: KZV09055.1), ERG3 (Access: CAA97586.1) and module two gallinaceous DHCR24 simultaneously to liposomes, the liposome localization sequences used were in turn AAM-B (shown as SEQ ID No. 4), Oleosin (shown as SEQ ID No. 5) and AAM-B sequences, respectively. Specifically, ERG2 and DHCR24 were positioned with AAM-B; ERG3 was located with oleosin. To allow the module to exist stably, a method of integrating the module into the genome was selected. Since homologous recombination of repeated sequences is easily caused when adjacent sequences in Saccharomyces cerevisiae are repeated and is lost from the genome, it is necessary to avoid integration of two modules comprising AAM-B sequences at the same site, and therefore the experiment is designed to integrate three gene modules in two groups in steps: AAM-B-ERG2, Oleosin-ERG3 (or ERG2, ERG3) are integrated at HO site, AAM-B-DHCR24 (or DHCR24) are integrated at ERG6 site, and ERG6 is knocked out. The starting chassis of the two strains are both SyBE _ Sc0125X 001. The specific construction process is to carry out OE-PCR on the positioning sequence AAMB or oleosin and the gene sequence, and to connect the positioning sequence AAMB or oleosin and the gene sequence into an expression cassette TDH2t-gal1p-FBA1t or FBA1t-gal7p-PGK1t in a laboratory module library in an enzyme digestion connection mode. Among them, Oleosin-ERG3(ERG3) and (AAM-B-DHCR24) DHCR24 used the gal1p expression cassette, and AAM-B-ERG2(ERG2) used the gal7p expression cassette. Then, the Oleosin-ERG3(ERG3) and AAM-B-ERG2(ERG2) are connected into a large fragment TDH2t-gal1p-Oleosin-ERG3(ERG3) -FBA1t-gal 7p-AAM-B-ERG2(ERG2) -PGK1t in a seamless connection mode. Meanwhile, the left arm HO _ L-URA-TDH2t and the right arm PGK1t-HO _ R which are subjected to homologous recombination are constructed by an OE-PCR method and are connected into a Blunt-end vector pEAZY-Blunt. The constructed expression module and the homology arm module are respectively transformed into escherichia coli competence DH 5alpha, colony PCR screening is carried out, and single and double enzyme digestion verification and sequencing verification are carried out on the quality-improved grains so as to ensure that the connection of the target fragments is correct and the base sequence is not mutated.

4. Modular integration construction of recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol

Firstly, cutting an expression module and homologous recombination left and right arm module plasmids by a not1 enzyme cutting site to obtain an integrated fragment, firstly, converting the module TDH2t-gal1p-Oleosin-ERG3 (or ERG3) -FBA1t-gal 7p-AAM-B-ERG2 (or ERG2) -PGK1t and homologous recombination left and right arm HO _ L-URA-TDH2t and PGK1t-HO _ R into a high-yield 7-dehydrocholesterol yeast strain Sy _ Sc0125X001 by a lithium acetate method, and recombining the host left and right homologous sequences with a host site on a yeast genome to integrate the host genome. Strains with HO sites integrated into ERG2 and ERG3 and strains with HO sites integrated into AAMB-ERG2 and Oleosin-ERG3 were obtained, respectively. The right arm ERG6_ L-LEU-TDH2t and FBA1t-ERG6_ R of the homologous recombination group at the ERG6 site were obtained from a laboratory library of modules. It was cut with not1 and co-transformed with the fragment AAMB-DHCR24 (or DHCR24) into the two chassis strains obtained above. Four strains with high yield of 7-dehydrocholesterol were obtained.

After transformation, SD-TRP-LEU-HIS-URA solid plate (synthetic yeast nitrogen source YN)B6.7 g/L, glucose 20g/L, tryptophan, leucine, histidine and uracil lacking mixed amino acid powder 2g/L, 2% agar powder), carrying out streak purification culture on the obtained transformant, extracting yeast genome, carrying out PCR verification, and preserving glycerol from the recombinant strain with correct verification, wherein the recombinant strain is named as SyBE _ Sc01250034SyBE _ Sc01250035, SyBE _ Sc01250036 and SyBE _ Sc 01250037. Wherein, SyBE _ Sc 01250034: SyBE _ Sc0125X001,. DELTA.ho:: P_GAL1-ERG3-P_GAL7ERG2,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_GAL7AAMB-ERG2,ΔERG6::P_GAL1-DHCR24；SyBE_Sc01250037：SyBE_Sc0125X001，Δho::P_GAL1-ERG3-P_GAL7ERG2,

ΔERG6::P_GAL1-AAMB-DHCR24；

The strain SyBE _ Sc01250038 was obtained by further integrating the module TDH2t-gal1p-ERG3-FBA 1t-gal7p-ERG 2-PGK1t at the delta15 site in the strain SyBE _ Sc 01250035. SyBE _ Sc 01250038: SyBE _ Sc0125X001,. DELTA.ho:: P_GAL1-Oleosin-ERG3-P_GAL7-AAMB-ERG2,ΔERG6::P_GAL1-AAMB-DHCR24，Δdelta15::P_GAL1-ERG3-P_GAL7ERG2。

The specific construction method of the strain SyBE _ Sc01250038 is similar to that of the strain, and the difference is that an auxotrophic label is not selected as a screening mode, but a CRISPR mode is adopted for genome integration.

5. The shake flask yields of 7-dehydrocholesterol were compared for the newly constructed strain SyBE _ Sc01250034-SyBE _ SyBE _ Sc01250038 and the control strain SyBE _ Sc0125XJ 06. The genotype of SyBE _ Sc0125XJ06 is SyBE _ Sc0125XJ 06: SyBE _ Sc0125X001,. DELTA.ERG 6:: P_GAL1-DHCR24。

Test materials: strains SyBE _ Sc01250034-SyBE _ SyBE _ Sc01250038 and control strains SyBE _ Sc0125XJ06(Guo, X.J., Xiao, W.H., Wang, Y., Yao, M.D., Zeng, B.X., Liu, H.J., Zhao, G.R., and Yuan, Y.J. (2018). Metabolic engineering of Saccharomyces cerevisiae for 7-dehydrocholestenosed biofuels 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 powder and 10g/L D-galactose.

Inoculating the above strain into 5mL seed culture medium, culturing at 30 deg.C and 250rpm for 14-16h, and determining initial thallus concentration OD₆₀₀The cells were inoculated in 50mL of each fermentation medium at 0.2, galactose was added, the cells were cultured at 30 ℃ and 250rpm, and the cell density (OD600) and the yield of 7-dehydrocholesterol were monitored during the fermentation.

7-dehydrocholesterol quantification method: centrifuging fermentation liquor 4ml, 5000g for 2min to collect thallus, resuspending cells with 3N HCl, boiling in boiling water bath for 5min, centrifuging to collect thallus, washing with water once, resuspending with 3M NaOH/methanol solution, placing centrifuge tube in 60 deg.C water bath, and saponifying for 4 h. And adding 400mL of normal hexane after saponification, adding a certain amount of quartz sand, carrying out vortex oscillation for 20min, and centrifuging to collect the upper-layer solution. The extraction process was repeated three times to ensure that the product was adequately extracted. Vacuum centrifuging for 30min to remove the n-hexane phase, and drying to obtain solid powder containing 7-dehydrocholesterol. The powder was derivatized with MSTFA and diluted prior to gas loading.

The test results are shown in fig. 8 and table 1.

Table 1 figure 8 data

The results of the test in FIG. 8 show that:

the five strains were classified according to the localization of ERG2/ERG 3: the first are SyBE _ Sc01250034 and SyBE _ Sc 01250037; the second is SyBE _ Sc01250036 and SyBE _ Sc 01250035; the third class is SyBE _ Sc 01250038. In the first strain, ERG2-3 is over-expressed in endoplasmic reticulum; in the second type of strain, ERG2-3 is overexpressed in liposomes; a third group of strains comprises ERG2/ERG3 overexpressed by endoplasmic reticulum and liposomes.

The total sterols of the first strain did not change significantly compared to the control strain, with only one copy of liposomal DHCR 24-expressing strain SyBE _ Sc01250037 having improved 7-dehydrocholesterol production or occupancy (46.1% to 274.2mg/L, 40.7% increase in production). The increase in 7-dehydrocholesterol was due to liposome-expressed DHCR24, a phenomenon that was also demonstrated when comparing the second species of strains SyBE _ Sc01250036 and SyBE _ Sc 01250035. The second strain shares one copy of liposome-expressed ERG 2-3. The total sterol amounts of the second species strains SyBE _ Sc01250036 and SyBE _ Sc01250035 were increased by 20.5% and 16.8%, respectively, compared to the control strains; the yield of 7-dehydrocholesterol is respectively improved by 51.0 percent (to 283.4mg/L) and 64.2 percent (to 308.2 mg/L); the proportion of 7-dehydrocholesterol is increased by 25.1 percent and 40.7 percent respectively. These results indicate that the localization of ERG2/3 to liposomes not only pulls the entire post-squalene pathway metabolic flux, but also further improves the production of 7-dehydrocholesterol. A third group of strains possess both endoplasmic reticulum and liposome-expressed ERG2/ERG 3. Compared with the control strain, the total solid content, the 7-dehydrocholesterol ratio and the 7-dehydrocholesterol yield are respectively improved by 21.0 percent, 18.0 percent and 42.7 percent (to 267.8mg/L), but the 7-dehydrocholesterol yield is reduced compared with the strain SyBE _ Sc 01250035.

Among the 5 newly constructed strains, the strain SyBE _ Sc01250035, in which ERG2, ERG3 and DHCR24 were all relocated to liposomes, reached the highest production of 7-dehydrocholesterol. It is demonstrated that the distribution of ERG2, ERG3, DHCR24 in liposomes with one copy of DHCR24 in the bottom disk is most favorable for 7-dehydrocholesterol production. It is noteworthy that the proportion of 7-dehydrocholesterol in the downstream products of zymosterol was highest in the strain SyBE _ Sc01250034, indicating that over-expression of ERG2-ERG3-DHCR24 at its original position can reduce the content of other intermediates downstream of zymosterol.

Example 2 rearrangement of organelle distribution of zymosterol upstream enzymes to improve 7-dehydrocholesterol production

1. Construction of a Modularly Integrated plasmid

First, the applicant designed to separate the enzymes present in the endoplasmic reticulum upstream of zymosterol in the post-squalene pathway into two modules: ERG1-ERG11-ERG24 are module one, and ERG25-ERG26-ERG27 are module two. To allow the module to exist stably, a method of integrating the module into the genome was selected. Since homologous recombination of the adjacent sequences in Saccharomyces cerevisiae is likely to occur when the adjacent sequences are repeated, and loss from the genome must be avoided by integrating two modules comprising AAM-B sequences at the same site, the experiment is designed to fuse the first two of the three genes in each module and place an Oleosin sequence between the two genes for localization; the third gene is located by AAM-B sequence, and the two modules are integrated into delta22 site and delta15 site in two steps. The starting chassis of the two strains are both SyBE _ Sc 01250035.

The specific construction process is to carry out OE-PCR on the positioning sequence AAMB or oleosin and the gene sequence, and to connect the positioning sequence AAMB or oleosin and the gene sequence into an expression cassette TDH2t-gal1p-FBA1t or FBA1t-gal7p-PGK1t in a laboratory module library in a seamless connection mode. Among them, ERG1-Oleosin-ERG11(ERG1-ERG11) and ERG25-Oleosin-ERG26(ERG25-ERG26) used gal1p expression cassettes, and AAM-B-ERG24(ERG24) and AAM-B-ERG27(ERG27) used gal7p expression cassettes. Then, homologous recombination of left arm delta22 (or 15) _ L-TDH2t and right arm FBA1t-GAL7p-AAM-B-ERG24(ERG24) -PGK1t-delta22 (or 15) _ R, or FBA1t-GAL7p-AAM-B-ERG27(ERG27) -PGK1t-delta22 (or 15) _ R is constructed by using an OE-PCR method and is connected into a Blunt-end vector pEAZY-Blunt or 425 PRS425K plasmid. In addition, CRISPR plasmids targeting delta22 and delta15 need to be constructed to cleave the corresponding sites instead of screening for nutritional tags. The constructed expression module and the homology arm module are respectively transformed into escherichia coli competence DH 5alpha, colony PCR screening is carried out, and single and double enzyme digestion verification and sequencing verification are carried out on the quality-improved grains so as to ensure that the connection of the target fragments is correct and the base sequence is not mutated.

2. Modular integration construction of recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol

Firstly, the expression module and homologous recombination left and right arm module plasmid are cut by a not1 enzyme cutting site to obtain an integrated fragment, the module ERG1-Oleosin-ERG11(ERG1-ERG11) or ERG25-Oleosin-ERG26(ERG25-ERG26) and homologous recombination left and right arms HO _ L-TDH2t and FBA1t-GAL7p-AAM-B-ERG24(ERG24) -PGK1t-delta22_ R or FBA1t-GAL7p-AAM-B-ERG27(ERG27) -PGK1 t-22 are independently transformed into a high-yield 7-dehydrocholesterol yeast strain SyBE _ Sc01250035, and the high-yield 7-dehydrocholesterol yeast strain is integrated with the delta22 on a yeast genome through delta22 and left and right homologous sequences to recombine on the genome 22. Respectively obtaining a strain with delta22 site integrated into GAL1p-ERG25-ERG26-FBA1t-GAL7p-ERG27-PGK1t/GAL1p-ERG25-Oleosin-ERG26-FBA1t-GAL7p-AAMBERG27-PGK1t and a strain with delta22 site integrated into GAL1p-ERG1-ERG11-FBA1t-GAL7p-ERG24-PGK1t/GAL1p-ERG1-Oleosin-ERG11-FBA1 t-7 p-AAMBERG24-PGK1 t. The plasmid in the yeast cells is discarded by successive passages. Strains SyBE _ Sc01250039, SyBE _ Sc01250040, SyBE _ Sc01250041 and SyBE _ Sc01250042 are obtained respectively. Then, in a strain SyBE _ Sc01250040 in which ERG25-Oleosin-ERG26-AAMB-ERG27 is introduced into a delta22 site, GAL1p-ERG1-ERG11-FBA1t-GAL7p-ERG24-PGK1t and GAL1p-ERG1-Oleosin-ERG11-FBA1t-GAL7p-AAMBERG24-PGK1t are respectively integrated into the delta15 site, and strains SyBE _ Sc01250043 and SyBE _ Sc01250044 are respectively obtained. Thus, six recombinant strains with different arrangements are obtained in the experiment of rearranging the organelle location of the zymosterol upstream enzyme.

After transformation, SD-TRP-LEU-HIS-URA solid plates (6.7 g/L of synthesized yeast nitrogen source YNB, 20g/L of glucose, 2g/L of mixed amino acid powder lacking tryptophan, leucine, histidine and uracil, 2% agar powder) are adopted for screening, the obtained transformants are subjected to streak-line purification culture, yeast genomes are extracted for PCR verification, glycerol bacteria are preserved for the recombinant strains which are verified to be correct and are respectively named as SyBE _ Sc01250039, SyBE _ Sc01250035 and Delta22, PGAL1-ERG 25-GGGGGS-ERG 26-TFBA1 6-PGAL 7-ERG27-TPGK1 SyBE _ Sc 50040, SyBE _ Sc01250035, Delta22, PGAL 4642-ERG 25-Oleosin-ERG 26-PGBA t-PGBA 5-HIS-URA solid plates, PSE-5-PSE-50035, PSE-SAG-50035, PSE-G-50035, PSE-80, PSE-G-50035, PSE-50035, PSE-80-PSE-50035, PSE-80, delta22, PGAL1-ERG1-Oleosin-ERG11-TFBA1t-PGAL7-AAMB-ERG24-TPGK1t SyBE _ Sc01250043, SyBE _ Sc01250040, Delta15, PGAL1-ERG 1-GGGGGGS-ERG 11-TFBA1t-PGAL7-ERG24-TPGK1t SyBE _ Sc01250044, SyBE _ Sc01250040, Delta15, PGAL1-ERG1-Oleosin-ERG11-TFBA1t-PGAL7-AAMB-ERG24-TPGK1t

3. The shake flask yields of 7-dehydrocholesterol were compared for the newly constructed strain SyBE _ Sc01250039-SyBE _ SyBE _ Sc01250044 and the control strain SyBE _ Sc 01250035.

Test materials: strains SyBE _ Sc01250039-SyBE _ SyBE _ Sc01250044 and control strain SyBE _ Sc 01250035.

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 results of the test in FIG. 9 show that:

the strains constructed in this section were classified into three groups according to the location of ERG 1-11-24. Class I: SyBE _ Sc01250039, 40; and II: SyBE _ Sc01250042, 44; class III: SyBE _ Sc01250041, 43. The first strains all contained modules ERG25-26-27 with either endoplasmic reticulum overexpression or liposome relocation, and no ERG1-11-24 overexpression; ERG1-11-24 in the second strain is located in liposome; the third type of strain, ERG1-11-24, localizes to the endoplasmic reticulum. Compared with the control strain SyBE _ Sc01250035, the total cholesterol content and the 7-dehydrocholesterol content in the first strain are reduced, and the 7-dehydrocholesterol content is not increased. In the second type of strain, liposome-localized ERG1-11-24 was introduced into the control strain SyBE _ Sc01250035 and the first type of strain SyBE _ Sc 01250040. In the two strains, 7-dehydrocholesterol is not obviously changed, the total sterol content is reduced, and the 7-dehydrocholesterol percentage is increased by 22.9 percent. Under the pulling of endoplasmic reticulum ERG1-11-24, although the total sterol content in the third type of strain is lower than that of the control strain, the 7-dehydrocholesterol content of the SyBE _ Sc01250041 strain and the SyBE _ Sc01250043 strain are respectively increased by 10.5% (to 342.2mg/L) and 16.4% (to 360.6mg/L), and the 7-dehydrocholesterol ratio is also increased. Furthermore, in the third type of strain, the 7-dehydrocholesterol production and ratio of SyBE _ Sc01250043 was higher than that of SyBE _ Sc01250041 due to the introduction of liposome ERG 25-26-27. The applicant therefore speculated that the introduction of liposomal ERG25-26-27 in certain strains could pull the metabolic flux down and increase the production of 7-dehydrocholesterol, while the strain SyBE _ Sc01250043 is currently the highest producing strain at shake flask level.

Example 3 organelle distribution binding upstream and downstream enzymes of zymosterol further improves 7-dehydrocholesterol production

1. Construction of a Modularly Integrated plasmid

To further reduce the by-products in the strains, the authors introduced the downstream genes ERG2, ERG3 in the post-squalene pathway into the tau3 site in the high 7-dehydrocholesterol producing strains. The specific construction process is to amplify 700bp fragments of tau3 upstream and downstream, terminator TDH2t and PGK1t by using genome as a template. Tau3_ up700 and TDH2t were connected by OE-PCR to obtain upstream homology arm tau3_ L, and PGK1t and tau3_ down700 were connected to obtain downstream homology arm tau3_ R.

Firstly, the expression module TDH2t-gal1p-ERG3-FBA 1t-gal7p-ERG 2-PGK1t constructed in the previous paragraph is cut by a not1 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 jointly transformed into the bacterial strain SyBE _ Sc01250043 with the highest 7-dehydrocholesterol yield in the previous paragraph by a lithium acetate method to obtain the bacterial strain SyBE _ Sc01250045.

SyBE_Sc01250045:SyBE_Sc01250040,Delta15::PGAL1-ERG1-GGGGS-ERG11-TFBA1t-PGAL7-ERG24-TPGK1tΔtau3::P_GAL1-ERG3-P_GAL7ERG2。

3. The shake flask yields of 7-dehydrocholesterol were compared for the newly constructed strain SyBE _ Sc01250045 and the control strain SyBE _ Sc 01250043.

Test materials: strain SyBE _ Sc01250045 and control strain SyBE _ Sc 01250043.

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

Table 3 figure 10 data

	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

	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 results of the test in FIG. 10 show that: although the yield of 7-dehydrocholesterol was not significantly changed in the strain SyBE _ Sc01250045, the content of the by-product 5alpha-Cholest-8-en-3beta-ol in the strain SyBE _ Sc01250045 was reduced by 49.9%, the content of lathosterol was reduced by 44.7%, and the content of total cholesterol was reduced by 17.0%, thus the yield of 7-dehydrocholesterol was improved by 20.9%. It is known that the combined rearrangement of the upstream and downstream genes of the yeast sterol can reduce the content of byproducts simultaneously on the basis of obtaining a strain with high 7-dehydrocholesterol yield, thereby increasing the ratio of 7-dehydrocholesterol in sterol.

Compared with the reported yield of the highest-producing strain SyBE _ Sc01250043, the yield of the highest-producing strain SyBE _ Sc0125XJ08 is remarkably improved (P is less than 0.05).

TABLE 5

SyBE_Sc0125XJ08	SyBE_Sc01250043
		251.8±5.66	360.61±3.98

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Tianjin university

<120> recombinant saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and construction method thereof

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ggtttgtttc aacatacctg tatggcatac gaattggttt tagccgatgg ttcattagtc 600

agatgctccc caacagaaaa cagtgacttg ttttatgccg ttccttggtc ttgtggtacc 660

ttaggtttct tggtcgctgc tgaaattaaa atgatcccag ctaaaaagta catcagattg 720

cattacgaac ctgttagagg tttgagatca atctgcgaaa agtttactga agaatctaaa 780

aataaggaaa actcattcgt cgaaggttta gtatactcct tggaagaagc tgtaattatg 840

actggtgttt taacagatga agcagaacct agtaagatta atagaatcgg taactactac 900

aagccttggt ttttcaagca cgttgaaaag tatttgaagg ccaataagac tggtatcgaa 960

tacattccat ccagacatta ctaccataga cacacaagaa gtattttctg ggaattacaa 1020

gatatcatcc cattcggtaa caaccctgtc tttagatatt tgttcggttg gatggtacca 1080

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ttttgggata tgttcgacgg tagtttatac cactctttga gagaacaaat gaactgtaag 1500

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agaagaatgg ccgaggctgc tgctcatgct gggcataaaa ctgcccaagc tggtcaagct 480

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Claims

Use of overexpression of a 7-dehydrocholesterol pathway enzyme and/or rearrangement thereof in organelles for the synthesis of 7-dehydrocholesterol, increasing the yield of 7-dehydrocholesterol and/or reducing intermediates or by-products of 7-dehydrocholesterol.
2. The use of claim 1, wherein the 7-dehydrocholesterol pathway enzyme comprises one or more of DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, ERG 27.
3. The use according to claim 1 or 2, wherein the organelles comprise endoplasmic reticulum and/or liposomes.
4. A recombinant strain of saccharomyces cerevisiae characterised in that its 7-dehydrocholesterol pathway enzyme is overexpressed and/or said 7-dehydrocholesterol pathway enzyme is rearranged in an organelle.
5. The recombinant strain of saccharomyces cerevisiae according to claim 4, wherein said 7-dehydrocholesterol pathway enzymes comprise one or more of DHCR24, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG26, ERG 27.
6. The recombinant strain of saccharomyces cerevisiae according to claim 4 or 5, wherein said recombinant strain of saccharomyces cerevisiae comprises the following modules:

a first module: ERG2-ERG 3;

and a second module: DHCR 24;

wherein ERG2 and DHCR24 are located with AAM-B; ERG3 was located with oleosin;

AAM-B-ERG2, Oleosin-ERG3 (or ERG2, ERG3) are integrated at HO site, AAM-B-DHCR24 or DHCR24 are integrated at ERG6 site, and ERG6 is knocked out; or

And a third module: ERG1-ERG11-ERG 24;

and a module IV: ERG25-ERG26-ERG 27;

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

or introduce module one at the tau3 site.
7. The recombinant strain of Saccharomyces cerevisiae according to any one of claims 4 to 6, wherein DHCR24, ERG2 and/or ERG3 are localized in liposomes; or

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

ERG2, ERG3 localized to liposomes and DHCR24 was overexpressed in the endoplasmic reticulum; or

ERG2, ERG3 were overexpressed in the endoplasmic reticulum and DHCR24 localized to the liposomes; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG2 and/or ERG3 are overexpressed in the endoplasmic reticulum; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG25, ERG26 and/or ERG27 are overexpressed in the endoplasmic reticulum; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG25, ERG26 and/or ERG27 are localized to the liposomes; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG1, ERG11 and/or ERG24 are overexpressed in the endoplasmic reticulum; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes and ERG1, ERG11 and/or ERG24 are localized to the liposomes; or

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

DHCR24, ERG2 and/or ERG3 are localized to liposomes, ERG25, ERG26 and/or ERG27 are localized to liposomes and ERG1, ERG11 and/or ERG24 are localized to liposomes;

DHCR24, ERG2 and/or ERG3 are localized to the liposomes, ERG25, ERG26 and/or ERG27 are localized to the liposomes and ERG1, ERG11 and/or ERG24 are overexpressed in the endoplasmic reticulum, and ERG2 and/or ERG3 are overexpressed in the endoplasmic reticulum.
8. The method for constructing a recombinant strain of Saccharomyces cerevisiae according to any one of claims 4 to 7, wherein a module is introduced into the starting strain;

the module comprises:

a first module: ERG2-ERG 3;

and a second module: DHCR 24;

wherein ERG2 and DHCR24 are located with AAM-B; ERG3 was located with oleosin;

AAM-B-ERG2, Oleosin-ERG3 (or ERG2, ERG3) are integrated at HO site, AAM-B-DHCR24 or DHCR24 are integrated at ERG6 site, and ERG6 is knocked out; or

And a third module: ERG1-ERG11-ERG 24;

and a module IV: ERG25-ERG26-ERG 27;

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

or introducing module one into the tau3 site;

specifically, the construction method comprises the following steps:

DHCR24, ERG2, and/or ERG3 are localized to the liposomes; or

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

ERG2, ERG3 localized to liposomes and DHCR24 localized to the endoplasmic reticulum; or

ERG2, ERG3 were overexpressed in the endoplasmic reticulum and DHCR24 localized to the liposomes;

DHCR24, ERG2 and/or ERG3 are localized to the liposomes, and ERG2 and/or ERG3 are overexpressed in the endoplasmic reticulum;

DHCR24, ERG2 and/or ERG3 are localized to the liposomes, ERG25, ERG26 and/or ERG27 are overexpressed in the endoplasmic reticulum; or

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

DHCR24, ERG2 and/or ERG3 are localized to the liposomes, ERG1, ERG11 and/or ERG24 are localized to the liposomes; or

DHCR24, ERG2 and/or ERG3 are localized to the liposomes, ERG25, ERG26 and/or ERG27 are localized to the liposomes and ERG1, ERG11 and/or ERG24 are localized to the endoplasmic reticulum;

DHCR24, ERG2 and/or ERG3 are localized to liposomes, ERG25, ERG26 and/or ERG27 are localized to liposomes and ERG1, ERG11 and/or ERG24 are localized to liposomes;

DHCR24, ERG2 and/or ERG3 are localized to the liposomes, ERG25, ERG26 and/or ERG27 are localized to the liposomes and ERG1, ERG11 and/or ERG24 are localized to the endoplasmic reticulum, and ERG2 and/or ERG3 are overexpressed to the endoplasmic reticulum.
9. Use of a recombinant strain of saccharomyces cerevisiae according to any of claims 4 to 7 for the synthesis of 7-dehydrocholesterol, for increasing the production of 7-dehydrocholesterol and/or for reducing intermediates or by-products of 7-dehydrocholesterol.
10. The method for synthesizing 7-dehydrocholesterol by using saccharomyces cerevisiae recombinant strain is characterized by taking the saccharomyces cerevisiae recombinant strain as claimed in any one of claims 4 to 7, inoculating, culturing, collecting culture solution, separating and purifying.