Yeast source cholesterol feed additive

Abstract

The invention belongs to the technical field of biological feed additives, and particularly relates to a yeast source cholesterol feed additive. The feed additive is prepared by preparing saccharomyces cerevisiae engineering bacteria into freeze-dried powder, and the saccharomyces cerevisiae engineering bacteria are prepared by replacing a promoter of an ERG6 gene of wild saccharomyces cerevisiae with a promoter ERG7p, inserting genes GgDHCR24 and StDWF5 into an X-3 site, inserting genes tHMG1 and IDI1 into an X-4 site, inserting genes ERG20 and ERG9 into an XII-4 site and inserting a gene INO2 into an XII-5 site. According to the method, the culture cost can be reduced, the culture benefits can be improved, the production of cholesterol is not influenced by factors such as seasons and environments, and sustainable and healthy development of the prawn feed industry is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological feed additives, and particularly relates to a yeast-derived cholesterol feed additive. Background Art

[0002] Cholesterol, also known as cholesterin, is a derivative of cyclopentane polyhydrophenanthrene and is an important component of animal cell membranes. Research shows that shrimp cannot synthesize cholesterol in their bodies, and cholesterol is the basis for shrimp to form steroid hormones, molt, and grow. Lack of cholesterol will make it difficult for shrimp to molt or easily die during the molting process. Therefore, adding cholesterol to shrimp feed is one of the necessary measures to ensure the healthy growth of shrimp. With the rapid development of the world's shrimp aquaculture industry, the relative shortage of fish meal resources has led many shrimp feed production enterprises to try to use plant protein sources to replace fish meal. However, this method cannot meet the demand for cholesterol during the growth process of shrimp, affecting the growth and health of shrimp, and thus affecting the yield and quality of aquaculture. Therefore, it is necessary to add cholesterol to shrimp feed as an additional supplement.

[0003] Currently, the main way to obtain cholesterol is to extract it from animal tissues and lanolin. However, this extraction method has disadvantages such as the risk of disease transmission (such as mad cow disease and swine streptococcus), limited raw material sources, complex extraction processes, and serious process pollution. It has high technical requirements and cannot meet the needs of green and sustainable development. Moreover, the cholesterol from this source has a relatively high price, increasing the production cost of shrimp feed enterprises. In addition, the chemical synthesis method has complex process flows, large pollution, and expensive catalysts, and is not suitable for industrial production.

[0004] Yeast is a eukaryote with a relatively wide range of applications, rich in various nutrients such as proteins, fatty acids, polysaccharides, and B vitamins. It is commonly used in the production of fermented feed and yeast-derived biological feed, not only improving the utilization rate of feed but also promoting the growth of aquatic animals. Therefore, biosynthesizing cholesterol in yeast and applying it to shrimp feed not only provides an efficient and environmentally friendly solution for the shrimp farming industry but also provides a new direction for the development of biosynthesis technology. Summary of the Invention

[0005] The present invention improves the yield of cholesterol by strategies such as knocking out or weakening yeast endogenous genes, optimizing the key enzymes for cholesterol synthesis, sterol C-24 reductase and sterol C-7 reductase, increasing precursor supply, and expanding the organelles anchored by key genes, laying a solid research foundation for the green biological manufacturing and industrial application of cholesterol. Then, the engineered Saccharomyces cerevisiae strain constructed is fermented to obtain cells, which are freeze-dried to obtain freeze-dried yeast powder, and the freeze-dried yeast powder is added to the shrimp basal feed in a certain proportion.

[0006] The present invention provides a yeast-derived cholesterol feed additive, which is obtained by preparing freeze-dried powder from an engineered Saccharomyces cerevisiae strain. The engineered Saccharomyces cerevisiae strain is obtained by replacing the promoter of the ERG6 gene of wild-type Saccharomyces cerevisiae with the promoter ERG7p, inserting the genes GgDHCR24 and StDWF5 into the X-3 site, inserting the genes tHMG1 and IDI1 into the X-4 site, inserting the genes ERG20 and ERG9 into the XII-4 site, and inserting the gene INO2 into the XII-5 site. The sequence of the promoter of the ERG6 gene is shown in SEQ ID NO:1, the sequence of the promoter ERG7p is shown in SEQ ID NO:2, the sequence of the gene GgDHCR24 is shown in SEQ ID NO:3, the sequence of the gene StDWF5 is shown in SEQ ID NO:4, the sequence of the gene tHMG1 is shown in SEQ ID NO:5, the sequence of the gene IDI1 is shown in SEQ ID NO:6, the sequence of the gene ERG20 is shown in SEQ ID NO:7, the sequence of the gene ERG9 is shown in SEQ ID NO:8, and the sequence of the gene INO2 is shown in SEQ ID NO:9.

[0007] In a specific embodiment, the starting strain of the engineered Saccharomyces cerevisiae strain is the wild-type Saccharomyces cerevisiae CEN.PK2-1C strain.

[0008] In a specific embodiment, the method for preparing the freeze-dried powder is as follows: After fermenting and culturing the engineered yeast strain, collect the cells and freeze-dry them to obtain the freeze-dried powder. The fermentation and culture method is as follows: Enrich the colonies of the engineered yeast strain, inoculate them into YPD liquid medium, and culture them overnight in a shaker at 30 °C. The next day, transfer them to YPD liquid medium for fermentation and culture for 96 h according to an initial OD 600 of 0.2.

[0009] The present invention also provides a method for constructing the engineered Saccharomyces cerevisiae strain, which includes the following steps:

[0010] (1) Using the CRISPR-Cas9 technology, replace the promoter of the ERG6 gene of wild-type Saccharomyces cerevisiae with the promoter ERG7p to obtain recombinant strain 1.

[0011] (2) Using the CRISPR-Cas9 technology, integrate the cholesterol synthesis genes GgDHCR24 and StDWF5 at the X-3 site of recombinant strain 1 to obtain recombinant strain 2.

[0012] (3) Using the CRISPR-Cas9 technology, integrate the genes tHMG1 and IDI1 at the X-4 site of recombinant strain 2 to obtain recombinant strain 3;

[0013] (4) Using the CRISPR-Cas9 technology, the genes ERG20 and ERG9 were integrated at the XII-4 locus of recombinant strain 3 to obtain recombinant strain 4;

[0014] (5) Using the CRISPR-Cas9 technology, the gene INO2 was integrated at the XII-5 locus of recombinant strain 4 to obtain recombinant strain 5, which is the engineered Saccharomyces cerevisiae for producing cholesterol.

[0015] The present invention constructs a synthesis pathway for cholesterol by expressing sterol C-24 reductase and sterol C-7 reductase with high catalytic activity; the present invention replaces the promoter of the ERG6 gene with the promoter of the ERG7 gene to reduce the synthesis of ergosterol in the competing pathway; the present invention overexpresses the INO2 gene to expand the area of the endoplasmic reticulum, improve the synthesis and folding ability of endoplasmic reticulum proteins, and thereby increase the yield of cholesterol.

[0016] As a specific embodiment, step (1) includes:

[0017] The promoter ERG7p was used as a DNA donor and transferred into wild-type Saccharomyces cerevisiae together with a plasmid containing the Cas9 protein gene and gRNA (pCas-Promoter-ERG6, which is used to cut the promoter region of ERG6 in wild-type Saccharomyces cerevisiae and allow the DNA donor to be inserted) to obtain recombinant strain 1.

[0018] The promoter ERG7p can be obtained by the following method: Using the wild-type Saccharomyces cerevisiae genome as a template, PCR amplification was performed to obtain the gene fragment of the promoter ERG7p (using Perg7-ERG6-donor-F and Perg7-ERG6-donor-R as primers). The preparation method of plasmid pCas-Promoter-ERG6 is as follows: Using plasmid pST.URA as a template, PCR amplification was performed to obtain a fragment of the gRNA gene containing the ERG7p promoter (using Perg6-URA-1F and Perg6-URA-2R as primers). Then, the fragment of the gRNA gene containing the ERG7p promoter was inserted into plasmid pCas9 by Golden-Gate reaction to obtain plasmid pCas-Promoter-ERG6.

[0019] As a specific embodiment, step (2) includes:

[0020] The GgDHCR24-StDWF5 gene fragment was used as a DNA donor and transferred into recombinant strain 1 together with a plasmid containing the Cas9 protein gene and gRNA (pCas-X-3, which is used to cut the X-3 locus in recombinant strain 1 and then allow the DNA donor to be inserted) to obtain recombinant strain 2.

[0021] The GgDHCR24-StDWF5 gene fragment can be obtained by the following method: Using plasmid pSU-GgDHCR24-StDWF5 as a template, the GgDHCR24-StDWF5 gene fragment is obtained by PCR amplification (using X-3-donor-F and X-3-donor-R as primers). The preparation method of the plasmid pSU-GgDHCR24-StDWF5 is as follows: The gene StDWF5 is inserted between PGK1p and ADH1t of the plasmid pSU, and the gene GgDHCR24 is inserted between TEF1p and CYC1t of the plasmid pSU.

[0022] As a specific embodiment, step (3) includes:

[0023] The tHMG1-IDI1 gene fragment is used as a DNA donor and a plasmid containing the Cas9 protein gene and gRNA (pCas-X-4, used to cut the X-4 site in recombinant bacterium 2 and then allow the DNA donor to insert) are transferred into recombinant bacterium 2 to obtain recombinant bacterium 3.

[0024] The tHMG1-IDI1 gene fragment can be obtained by the following method: Using plasmid pSU-tHMG1-IDI1 as a template, the tHMG1-IDI1 gene fragment is obtained by PCR amplification (using X-4-donor-F and X-4-donor-R as primers). The preparation method of the plasmid pSU-tHMG1-IDI1 is as follows: Using the Saccharomyces cerevisiae genome as a template, the fragment of the tHMG1 gene (using tHMG1-F and tHMG1-R as primers) and the fragment of the IDI1 gene (using IDI1-F and IDI1-R as primers) are obtained by PCR amplification. The tHMG1 gene fragment is inserted between the EcoRI and SacI restriction enzyme cleavage sites of the pSU vector, and the IDI1 gene fragment is inserted between the BamHI and NheI restriction enzyme cleavage sites of the pSU vector to obtain the plasmid pSU-tHMG1-IDI1.

[0025] As a specific embodiment, step (4) includes:

[0026] The ERG20-ERG9 gene fragment is used as a DNA donor and a plasmid containing the Cas9 protein gene and gRNA (pCas-XII-4, used to cut the XII-4 site in recombinant bacterium 3 and then allow the DNA donor to insert) are transferred into recombinant bacterium 3 to obtain recombinant bacterium 4.

[0027] The ERG20-ERG9 gene fragment can be obtained by the following method: Using plasmid pSU-ERG20-ERG9 as a template, the ERG20-ERG9 gene fragment is obtained by PCR amplification (using XII-4-donor-F and XII-4-donor-R as primers). The preparation method of the plasmid pSU-ERG20-ERG9 is as follows: Using the Saccharomyces cerevisiae genome as a template, the fragment of the ERG20 gene is obtained by PCR amplification (using ERG20-F and ERG20-R as primers) and the fragment of the ERG9 gene is obtained by PCR amplification (using ERG9-F and ERG9-R as primers). The ERG20 gene fragment is inserted between the EcoRI and SacI restriction enzyme cleavage sites of the pSU vector, and the ERG9 gene fragment is inserted between the BamHI and NheI restriction enzyme cleavage sites of the pSU vector to obtain the plasmid pSU-ERG20-ERG9.

[0028] As a specific embodiment, step (5) includes:

[0029] Using the INO2 gene fragment as a DNA donor and transferring it into recombinant bacterium 4 together with a plasmid containing the Cas9 protein gene and gRNA (pCas-XII-5, which is used to cut the XII-5 site of recombinant bacterium 4 and then allow the DNA donor to insert), to obtain recombinant bacterium 5.

[0030] The INO2 gene fragment can be obtained by the following method: Using plasmid pSU-INO2 as a template, the INO2 gene fragment is obtained by PCR amplification (using XII-5-donor-F and XII-5-donor-R as primers). The preparation method of the plasmid pSU-INO2 is as follows: Using the Saccharomyces cerevisiae genome as a template, the fragment of the INO2 gene is obtained by PCR amplification (using INO2-F and INO2-R as primers), and the INO2 gene fragment is inserted between the EcoRI and SacI restriction enzyme cleavage sites of the pSU vector to obtain the plasmid pSU-INO2.

[0031] The present invention also provides a feed, which contains the feed additive, and this feed replaces cholesterol from chemical extraction sources therein with the feed additive. As a specific embodiment, the feed is a prawn feed, and 4.0% by mass of the feed additive is added to the prawn feed; the feed also contains a basic feed, and the content of each component of the basic feed is as follows: crude protein ≥ 43%, crude fiber ≤ 11%, crude fat ≥ 5%, crude ash ≤ 17%, lysine ≥ 2.1%, sodium chloride ≤ 3.8%, moisture ≤ 12%, calcium ≤ 5%, total phosphorus ≥ 0.5%.

[0032] The beneficial effects of the present invention are:

[0033] By preparing the engineered Saccharomyces cerevisiae for synthesizing cholesterol into yeast powder and replacing the cholesterol from chemical extraction sources in the shrimp feed, the source of cholesterol is changed in a green and environmentally friendly production method, which can not only reduce the breeding cost and improve the breeding efficiency, but also the production of cholesterol is not affected by factors such as seasons and environments, being conducive to the sustainable and healthy development of the shrimp feed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the plasmid pCas-Promoter-ERG5 map.

[0035] Figure 2 It is a schematic diagram of the plasmid pCas-Promoter-ERG6 map.

[0036] Figure 3 It is a schematic diagram of the plasmid pCas-ERG5 map.

[0037] Figure 4 It is a schematic diagram of the plasmid pCas-ERG6 map. Figure 1 、 2 The plasmid size compositions shown in 2, 3, and 4 have the same framework, only the gRNA of 20bp is different.

[0038] Figure 5 It is a schematic diagram of the plasmid pSU-tHMG1-IDI1 map.

[0039] Figure 6 It is a schematic diagram of the plasmid pSU-ERG20-ERG9 map.

[0040] Figure 7 It is a schematic diagram of the plasmid pSU-INO2 map.

[0041] Figure 8 It is a schematic diagram of the cholesterol synthesis pathway in Saccharomyces cerevisiae.

[0042] Figure 9 It is the cholesterol yield in Saccharomyces cerevisiae under different conditions.

[0043] Figure 10 It is the influence of providing supply before strengthening and expanding the endoplasmic reticulum area on the cholesterol yield.

[0044] Figure 11 It is the analysis result of the shrimp survival rate.

[0045] Figure 12 It is the analysis result of the shrimp weight gain rate. DETAILED DESCRIPTION OF THE INVENTION

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0047] Example 1

[0048] 1. Experimental Materials and Methods

[0049] 1.1 Strains, Plasmids, and Primers

[0050] The starting strain used in this example is Saccharomyces cerevisiae CEN.PK2-1C, for the construction of Saccharomyces cerevisiae engineering bacteria for cholesterol production. The strains involved in this example are shown in Table 1, the plasmids involved are shown in Table 2, and the primers used in the plasmid construction process are shown in Table 3. The genes used were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primers used were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0051] Table 1. Strains Involved in This Example

[0052]

[0053]

[0054] Table 2. Plasmids Involved in This Example

[0055]

[0056] Table 3. Primers Used in This Example

[0057]

[0058]

[0059]

[0060]

[0061] 1.2 Culture Media and Culture Conditions

[0062] The activation and culture of Saccharomyces cerevisiae strains were carried out using YPD medium (1% yeast extract, 2% peptone, 2% anhydrous glucose), and the culture of Escherichia coli was carried out using LBA medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 100 μg / mL ampicillin). Autoclave at 121 °C for 20 min and store at room temperature.

[0063] During the construction of yeast strains, SC-URA auxotrophic medium with glucose as the carbon source is used for transformation, strain screening, and streak plating. The culture temperature of Saccharomyces cerevisiae is 30 °C, and the culture temperature of Escherichia coli is 37 °C.

[0064] 1.3 Construction of plasmids

[0065] (1) Construction of plasmids pCas-Promoter-ERG5 and pCas-Promoter-ERG6: Using plasmid pST.URA as a template, PCR amplifications were carried out with Perg5-URA-1F and Perg5-URA-2R, Perg6-URA-1F and Perg6-URA-2R as primers respectively. After gel extraction, two fragments containing the gRNA gene with the ERG7p promoter were obtained respectively. Then, by using the Golden-Gate reaction, the two fragments containing the gRNA gene with the ERG7p promoter were inserted into the BsaI digestion site of plasmid pCas9 respectively. Then, E. coli transformation and plasmid extraction were carried out to obtain plasmids pCas-Promoter-ERG5 (schematic diagram of plasmid map as shown in Figure 1 ) and pCas-Promoter-ERG6 (schematic diagram of plasmid map as shown in Figure 2 ), and they were verified by sequencing. The plasmids with correct verification were used for the construction of subsequent integration strains.

[0066] (2) Construction of plasmids pCas-ERG5 and pCas-ERG6: Using plasmid pST.URA as a template, PCR amplifications were carried out with ERG5-URA-1F and ERG5-URA-2R, ERG6-URA-1F and ERG6-URA-2R as primers respectively. After gel extraction, two fragments containing the gRNA genes of ERG5 and ERG6 were obtained respectively. Then, by using the Golden-Gate reaction, the two fragments containing the gRNA genes of ERG5 and ERG6 were inserted into the BsaI digestion site of plasmid pCas9 respectively. Then, E. coli transformation and plasmid extraction were carried out to obtain plasmids pCas-ERG5 (schematic diagram of plasmid map as shown in Figure 3 ) and pCas-ERG6 (schematic diagram of plasmid map as shown in Figure 4 ), and they were verified by sequencing. The plasmids with correct verification were used for the construction of subsequent integration strains.

[0067] (3) Construction of plasmid pSU-tHMG1-IDI1: Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was carried out with tHMG1-F and tHMG1-R as primers. After gel recovery, the fragment of the tHMG1 gene was obtained. Then, plasmid pSU was double-digested with EcoRI and SacI, and after gel recovery, the digested fragment of the pSU vector was obtained. Finally, the tHMG1 gene fragment and the digested fragment of the pSU vector were ligated homologously, transformed into E. coli, the plasmid was extracted, and after sequencing verification, plasmid pSU-tHMG1 was obtained. Next, using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was carried out with IDI1-F and IDI1-R as primers. After gel recovery, the fragment of the IDI1 gene was obtained. Then, plasmid pSU-tHMG1 was double-digested with BamHI and NheI, and after gel recovery, the digested fragment of the pSU-tHMG1 vector was obtained. Finally, the IDI1 gene fragment and the digested fragment of the pSU-tHMG1 vector were ligated homologously, transformed into E. coli, the plasmid was extracted, and plasmid pSU-tHMG1-IDI1 was obtained (the schematic diagram of the plasmid map is as shown in Figure 5 ), and after sequencing verification, the plasmid with correct verification was used for the construction of subsequent integrated strains.

[0068] (4) Construction of plasmid pSU-ERG20-ERG9: Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was carried out with ERG20-F and ERG20-R as primers. After gel recovery, the fragment of the ERG20 gene was obtained. Then, plasmid pSU was double-digested with EcoRI and SacI, and after gel recovery, the digested fragment of the pSU vector was obtained. Finally, the ERG20 gene fragment and the digested fragment of the pSU vector were ligated homologously, transformed into E. coli, the plasmid was extracted, and after sequencing verification, plasmid pSU-ERG20 was obtained. Next, using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was carried out with ERG9-F and ERG9-R as primers. After gel recovery, the fragment of the ERG9 gene was obtained. Then, plasmid pSU-ERG20 was double-digested with BamHI and NheI, and after gel recovery, the digested fragment of the pSU-ERG20 vector was obtained. Finally, the ERG9 gene fragment and the digested fragment of the pSU-ERG20 vector were ligated homologously, transformed into E. coli, the plasmid was extracted, and plasmid pSU-ERG20-ERG9 was obtained (the schematic diagram of the plasmid map is as shown in Figure 6 ), and after sequencing verification, the plasmid with correct verification was used for the construction of subsequent integrated strains.

[0069] (5) Construction of plasmid pSU-INO2: Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was performed with INO2-F and INO2-R as primers. After gel recovery, the fragment of the INO2 gene was obtained. Then, plasmid pSU was double digested with EcoRI and SacI, and after gel recovery, the digested fragment of the pSU vector was obtained. Finally, the INO2 gene fragment and the digested fragment of the pSU vector were ligated homologously, transformed into E. coli, and the plasmid was extracted to obtain plasmid pSU-INO2 (the schematic diagram of the plasmid map is as shown in Figure 7 ), and it was verified by sequencing. The plasmid with correct verification was used for the construction of subsequent integrated strains.

[0070] 1.4 Construction of Saccharomyces cerevisiae strains

[0071] (1) Construction of strain CEN.PK2-1C-Werg5: First, using the genome of CEN.PK2-1C as a template, PCR amplification was performed with Perg7-ERG5-donor-F and Perg7-ERG5-donor-R as primers. After gel recovery, the gene fragment of promoter ERG7p was obtained as the DNA donor. Then, the plasmid pCas-Promoter-ERG5 containing the Cas9 protein gene and gRNA and the DNA donor were transformed into Saccharomyces cerevisiae CEN.PK2-1C by chemical transformation. The primers Perg7-ERG5-CK-F and Perg7-ERG5-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CEN.PK2-1C-Werg5 was obtained.

[0072] (2) Construction of strain CEN.PK2-1C-Werg6: First, using the genome of CEN.PK2-1C as a template, PCR amplification was performed with Perg7-ERG6-donor-F and Perg7-ERG6-donor-R as primers. After gel recovery, the gene fragment of promoter ERG7p was obtained as the DNA donor. Then, the plasmid pCas-Promoter-ERG6 containing the Cas9 protein gene and gRNA and the DNA donor were transformed into Saccharomyces cerevisiae CEN.PK2-1C by chemical transformation. The primers Perg7-ERG6-CK-F and Perg7-ERG6-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CEN.PK2-1C-Werg6 was obtained.

[0073] (3) Construction of Strain CH-1: First, using plasmid pSU-GgDHCR24-StDWF5 as a template (GeneBank of gene GgDHCR24: NP_001026459.1; GeneBank of StDWF5: XP_006362628.1), PCR amplification was carried out with primers EGR5-donor-F and EGR5-donor-R. After gel extraction, the GgDHCR24-StDWF5 gene fragment (containing PGK1p-StDWF5-ADH1t-TEF1p-GgDHCR24-CYC1t) was obtained as the DNA donor. Then, the plasmid pCas-ERG5 containing the Cas9 protein gene and gRNA and the DNA donor were introduced into Saccharomyces cerevisiae CEN.PK2-1C by chemical transformation. Primers ERG5-CK-F and ERG5-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CH-1 was obtained.

[0074] (4) Construction of Strain CH-2: First, using plasmid pSU-GgDHCR24-StDWF5 as a template, PCR amplification was carried out with primers EGR6-donor-F and EGR6-donor-R. After gel extraction, the GgDHCR24-StDWF5 gene fragment was obtained as the DNA donor. Then, the plasmid pCas-ERG6 containing the Cas9 protein gene and gRNA and the DNA donor were introduced into Saccharomyces cerevisiae CEN.PK2-1C by chemical transformation. Primers ERG6-CK-F and ERG6-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CH-2 was obtained.

[0075] (5) Construction of Strain CH-3: First, using plasmid pSU-GgDHCR24-StDWF5 as a template, PCR amplification was carried out with primers X-3-donor-F and X-3-donor-R. After gel extraction, the GgDHCR24-StDWF5 gene fragment was obtained as the DNA donor. Then, the plasmid pCas-X-3 containing the Cas9 protein gene and gRNA and the DNA donor were introduced into Saccharomyces cerevisiae CEN.PK2-1C by chemical transformation. Primers X-3-CK-F and X-3-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CH-3 was obtained.

[0076] Construction of strain CH-4: First, using plasmid pSU-GgDHCR24-StDWF5 as a template, and X-3-donor-F and X-3-donor-R as primers for PCR amplification. After gel extraction, the GgDHCR24-StDWF5 gene fragment was obtained as the DNA donor. Then, the plasmid pCas-X-3 containing the Cas9 protein gene and gRNA and the DNA donor were transferred into Saccharomyces cerevisiae CEN.PK2-1C-Werg5 by chemical transformation. Primers X-3-CK-F and X-3-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CH-4 was obtained.

[0077] Construction of strain CH-5: First, using plasmid pSU-GgDHCR24-StDWF5 as a template, and X-3-donor-F and X-3-donor-R as primers for PCR amplification. After gel extraction, the GgDHCR24-StDWF5 gene fragment was obtained as the DNA donor. Then, the plasmid pCas-X-3 containing the Cas9 protein gene and gRNA and the DNA donor were transferred into Saccharomyces cerevisiae CEN.PK2-1C-Werg6 by chemical transformation. Primers X-3-CK-F and X-3-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CH-5 was obtained.

[0078] Construction of strain CH-6: First, using plasmid pSU-tHMG1-IDI1 as a template, and X-4-donor-F and X-4-donor-R as primers for PCR amplification. After gel extraction, the tHMG1-IDI1 gene fragment was obtained as the DNA donor. Then, the plasmid pCas-X-4 containing the Cas9 protein gene and gRNA and the DNA donor were transferred into Saccharomyces cerevisiae CH-5 by chemical transformation. Primers X-4-CK-F and X-4-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CH-6 was obtained.

[0079] Construction of strain CH-7: First, using plasmid pSU-ERG20-ERG9 as a template, and XII-4-donor-F and XII-4-donor-R as primers for PCR amplification. After gel extraction, the ERG20-ERG9 gene fragment was obtained as the DNA donor. Then, the plasmid pCas-XII-4 containing the Cas9 protein gene and gRNA and the DNA donor were transferred into Saccharomyces cerevisiae CH-6 by chemical transformation. Primers XII-4-CK-F and XII-4-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CH-7 was obtained.

[0080] Construction of strain CH-8: First, using plasmid pSU-INO2 as a template, and XII-5-donor-F and XII-5-donor-R as primers for PCR amplification. After gel recovery, the INO2 gene fragment was obtained as the DNA donor. Then, the plasmid pCas-XII-5 containing the Cas9 protein gene and gRNA and the DNA donor were transferred into Saccharomyces cerevisiae CH-7 by chemical transformation. The primers XII-5-CK-F and XII-5-CK-R were used to verify whether the transformants were correct. After losing the plasmid, strain CH-8 was obtained.

[0081] 1.5 Chemical transformation of Saccharomyces cerevisiae

[0082] In this example, the method of transferring the plasmid into Saccharomyces cerevisiae was the lithium acetate / salmon sperm DNA / polyethylene glycol method (LiOAc / ssDNA / PEG) reported in the literature (Dong G, Zhao Y, Ding W, et al. Metabolic engineering of Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids[J]. Metabolic Engineering. 2024, 82: 100 - 109.). The transformation system is shown in Table 4.

[0083] Table 4. Transformation system of Saccharomyces cerevisiae

[0084] Reagent Dosage 1M Lithium Acetate (LiOAc) 36 μL 2 mg / mL Single-stranded DNA (ssDNA) 50 μL 50% Polyethylene Glycol (PEG3350) 240 μL Plasmid 300 - 500 ng <![CDATA[Make up to ddH2O]]> 360 μL

[0085] 1.6 Fermentation culture of Saccharomyces cerevisiae

[0086] Pick the correctly transformed single colonies on the plate and streak them on the corresponding solid plate, and place them in a constant temperature incubator at 30 °C for enrichment culture; then inoculate them into 3 mL of YPD medium and culture them overnight in a shaker at 220 rpm and 30 °C; the next day, transfer them to a 100 mL shake flask of YPD medium at an initial OD 600 ratio of 0.2, and ferment and culture them in a shaker at 220 rpm and 30 °C for 72 h. Set three parallels for each sample. After 72 h, collect the fermentation broth and measure the cholesterol content in the fermentation broth.

[0087] 1.7 Cholesterol detection method

[0088] Take 2 mL of the fermentation broth, centrifuge it at 12,000 g for 3 min, discard the supernatant, and wash it twice with deionized water; then add an appropriate amount of acidic glass beads and shake it with a vortex oscillator for 30 min; then add 3M NaOH - methanol solution and saponify it in a water bath at 80°C for 1 h; after saponification, add 2 mL of n - hexane, shake it with a vortex oscillator for 10 min, and then centrifuge it at 12,000 g for 1 min; aspirate the upper n - hexane phase and transfer it to a new EP tube, and evaporate it to dryness in a vacuum concentrator; add 200 μL of silanization derivatization reagent, mix well, and place it in an oven at 60°C for 1 h for reaction; after the reaction is completed, dilute the derivatized sample with n - hexane by 10 times, take an appropriate amount and place it in a gas chromatography injection vial, and analyze and detect it by using GC - MS (GC - MS QP2020, Shimadzu, Japan).

[0089] The detection conditions are as follows: DB - 5MS chromatographic column (30 m×0.25 mm×0.25 μm, Agilent), the EI source bombardment energy is 70 eV, the carrier gas is helium, the flow rate is 3.0 mL / min, the inlet temperature is 280°C, the ion source temperature is 250°C, the sample injection volume is 1 μL, the split ratio is 10:1, and the temperature programming is that the column temperature is maintained at 70°C for 2 min, then increased to 250°C at a rate of 20°C / min, maintained at 250°C for 2 min, then increased to 280°C at a rate of 10°C / min, and maintained at 280°C for 15 min.

[0090] 2. Results and Discussion

[0091] 2.1 Construction of the de novo cholesterol synthesis pathway

[0092] In order to achieve de novo synthesis of cholesterol in Saccharomyces cerevisiae, it is necessary to express the key enzymes sterol C - 24 reductase and sterol C - 7 reductase, and the synthesis pathway is as Figure 8As shown in the figure. Therefore, in this example, chicken-derived C-24 reductase GgDHCR24 and potato-derived C-7 reductase StDWF5 were selected for expression in the Saccharomyces cerevisiae CEN.PK2-1C strain. Since the yeast endogenous ergosterol synthesis pathway is a competing pathway for cholesterol synthesis, in order to reduce the influence of this competing pathway, it is necessary to knockout or weaken the key genes ERG5 or ERG6 for ergosterol synthesis. By the CRISPR-Cas9 method (refer to Zhang Y, Wang J, Wang Z, et al. AgRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae [J]. Nature Communications, 2019, 10(1): 1053.), the cholesterol synthesis genes GgDHCR24 and StDWF5 were integrated at the ERG5 or ERG6 locus of the CEN.PK2-1C strain to obtain strains CH-1 and CH-2. At the same time, in order to investigate the effects of knocking out or weakening the ergosterol synthesis pathway on the growth and cholesterol production of yeast strains, the promoters of the ERG5 and ERG6 genes were replaced with the weak promoter ERG7p to obtain strains CEN.PK2-1C-Werg5 and CEN.PK2-1C-Werg6, and then the cholesterol synthesis genes GgDHCR24 and StDWF5 were integrated at the X-3 locus of the strains CEN.PK2-1C, CEN.PK2-1C-Werg5 or CEN.PK2-1C-Werg6 to obtain strains CH-3, CH-4 and CH-5.

[0093] The five constructed strains (CH-1, CH-2, CH-3, CH-4, CH-5) were fermented and cultured in YPD liquid medium. The 72-hour fermentation broth was taken for derivatization pretreatment, and finally the cholesterol content in the sample was analyzed and detected by GC-MS. The detection results are as Figure 9 shown. From Figure 9 it can be seen that cholesterol was detected in all strains. Compared with strain CH-3, the cholesterol content in strains CH-1 and CH-2 was increased to a certain extent, indicating that knocking out the key genes ERG5 or ERG6 for ergosterol synthesis is beneficial to cholesterol synthesis. The cholesterol content in strains CH-4 and CH-5 was further increased, and the cholesterol content in strain CH-5 reached 12.83 mg / L, indicating that weakening the expression of the key genes ERG5 or ERG6 for ergosterol synthesis is more beneficial to cholesterol synthesis and does not affect the growth of the strain. Therefore, strain CH-5 was selected for further optimization and improvement in the follow-up.

[0094] 2.2 Effect of strengthening precursor supply on cholesterol production

[0095] In the mevalonate pathway, tHmg1p and Idi1p are two key rate-limiting enzymes. Therefore, to increase cholesterol production, it is necessary to adjust the expression of key enzymes and genes in the mevalonate pathway to promote the maximum metabolic flux towards the target product. We integrated the genes tHMG1 and IDI1 at the X-4 locus of strain CH-5 to obtain strain CH-6. Additionally, ERG20 and ERG9 are key genes for synthesizing the cholesterol precursor squalene. We integrated the above two genes at the XII-4 locus of strain CH-6 to obtain strain CH-7. Then, strains CH-6 and CH-7 were fermented and cultured in YPD liquid medium. The 72-hour fermentation broth was taken for derivatization pretreatment, and finally, the cholesterol content in the sample was analyzed and detected by GC-MS. The detection results are as Figure 10 shown. As can be seen from Figure 10 it, the cholesterol content in strains CH-6 and CH-7 was significantly increased, reaching 38.41 mg / L and 51.86 mg / L respectively. Compared with strain CH-5, they were increased by 1.99 times and 3.04 times respectively. The above results indicate that overexpressing key genes in the mevalonate pathway can effectively increase cholesterol production in Saccharomyces cerevisiae.

[0096] 2.3 Effect of expanding endoplasmic reticulum area on cholesterol production

[0097] In the metabolic pathway of cholesterol synthesis, the enzymes after the mevalonate pathway are all localized in the endoplasmic reticulum. Therefore, the size of the endoplasmic reticulum space area has an important impact on the correct folding and expression of related proteases. INO2 is a regulatory gene for yeast phospholipid biosynthesis, which can effectively expand the surface area of the endoplasmic reticulum, thereby enhancing the synthesis and folding ability of endoplasmic reticulum proteins. To further increase cholesterol production, we integrated the INO2 gene at the XII-5 locus of strain CH-7 to obtain strain CH-8. Then, strain CH-8 was fermented and cultured in YPD liquid medium. The 72-hour fermentation broth was taken for derivatization pretreatment, and finally, the cholesterol content in the sample was analyzed and detected by GC-MS. The detection results are as Figure 10 shown. As can be seen from Figure 10 it, the cholesterol content in strain CH-8 was further increased, reaching 60.63 mg / L. Compared with strain CH-5, it was increased by 3.72 times. Therefore, enhancing the area of the endoplasmic reticulum by overexpressing the INO2 gene is an effective strategy to increase cholesterol production in Saccharomyces cerevisiae.

[0098] References:

[0099] [1] Xu S, Qiao W, Wang Z, et al. Exploiting a heterologous construction of the 3-hydroxypropionic acid carbon fixation pathway with mesaconate as an indicator in Saccharomyces cerevisiae[J]. Bioresources and Bioprocessing, 2023, 10:33.

[0100] [2] Zhang Y, Wang J, Wang Z, et al. AgRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae[J]. Nature Communications, 2019, 10(1):1053.

[0101] [3] Meng J, Qiu Y, Zhang Y, et al. CMI: CRISPR / Cas9 based efficient multiplexed integration in Saccharomyces cerevisiae[J]. ACS synthetic biology, 2023, 12(5):1408-1414.

[0102] Example 2 Prawn Culture Experiment

[0103] 1. Preparation of Yeast Powder

[0104] Pick the correctly transformed strain CH-8 colonies on the plate for streak enrichment culture, then inoculate them into 4 mL of YPD liquid medium and culture overnight at 220 rpm in a shaker at 30 °C. The next day, transfer according to the initial OD 600 of 0.2 to a 100 mL shake flask containing YPD liquid medium for fermentation culture for 96 h. Collect the cells and freeze-dry for 24 h to obtain yeast powder containing cholesterol. In the later stage, according to the production volume, gradually expand the culture of the cells to prepare a large amount of yeast powder as a prawn feed additive.

[0105] 2. Preparation of Prawn Feed

[0106] The purchased basic feed (produced by New Hope Group Co., Ltd., content: crude protein ≥ 43%, crude fiber ≤ 11%, crude fat ≥ 5%, crude ash ≤ 17%, lysine ≥ 2.1%, sodium chloride ≤ 3.8%, moisture ≤ 12%, calcium ≤ 5%, total phosphorus ≥ 0.5%) was pulverized into powder with a pulverizer and passed through an 80-mesh sieve. Then, different amounts of yeast powder containing cholesterol were added to the basic feed powder according to the mass ratio:

[0107] Group A: Add 0.0% yeast powder (control);

[0108] Group B: Add 1.0% yeast powder;

[0109] Group C: Add 2.0% yeast powder;

[0110] Group D: Add 4.0% yeast powder;

[0111] Group E: Add 6.0% yeast powder;

[0112] Group F: Add 8.0% yeast powder.

[0113] The above 6 groups of feeds were respectively mixed evenly, and then prepared into feed pellets with a particle size of 1.5 mm by an automatic granulator to obtain shrimp feed.

[0114] 3. Analysis of the breeding experiment

[0115] 3.1 Selection of shrimp and experimental design

[0116] Select 900 healthy juvenile shrimp with uniform size (purchased from the aquatic product market), with an initial weight of about 2.8 ± 0.1 g per tail. Three parallels were randomly set for each type of shrimp feed, and 50 tails were raised in each water bucket (100 cm × 100 cm × 80 cm), totaling 18 buckets. Feed once at 06:00 and 18:00 every day, and the feeding amount was about 5% of the shrimp weight. The breeding period was 60 days. After 60 days of breeding, samples were collected to count the survival rate and weight change of the shrimp.

[0117] 3.2 Data analysis

[0118] (1) Effect of different contents of yeast powder on the survival rate of shrimp

[0119] After 60 days, the survival rates of the 6 groups fed with different shrimp feeds are as Figure 11 shown, and the calculation formula for the survival rate is: Survival rate = (Number remaining after 60 days ÷ Initial number released) × 100%.

[0120] From Figure 11It can be seen that the survival rates of all groups of shrimp fed with yeast powder (1.0%: 78.6%; 2.0%: 80.2%; 4.0%: 84.7%; 6.0%: 81.6%; 8.0%: 76.3%) were higher than those of the control group (0.0%: 68.2%). When the addition amount of yeast powder was 4.0%, the survival rate reached the maximum value (84.7%), which was significantly higher than that of the control group (68.2%). The above results indicate that when 4.0% cholesterol-containing yeast powder is added to the basic feed for shrimp, the survival rate of shrimp can be significantly improved.

[0121] (2) Effects of yeast powder with different contents on the growth performance of shrimp

[0122] After 60 days, the weight gain rates of the 6 groups fed with different shrimp feeds are as Figure 12 shown, and the calculation formula for the weight gain rate is: weight gain rate = (average weight of shrimp after 60 days - average initial weight of shrimp) ÷ average initial weight of shrimp × 100%.

[0123] It can be Figure 12 seen that the weight gain rates of all groups of shrimp fed with yeast powder (1.0%: 297.5%; 2.0%: 328.1%; 4.0%: 344.8%; 6.0%: 331.7%; 8.0%: 318.9%) were higher than those of the control group (0.0%: 286.4%). When the addition amount of yeast powder was 4.0%, the weight gain rate reached the maximum value (344.8%), which was significantly higher than that of the control group (286.4%). The above results indicate that when 4.0% cholesterol-containing yeast powder is added to the basic feed for shrimp, the weight gain rate of shrimp can be significantly improved.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A yeast-derived cholesterol feed additive, characterized in that, The feed additive is obtained by preparing the engineered Saccharomyces cerevisiae into freeze-dried powder. The engineered Saccharomyces cerevisiae has replaced the promoter of the ERG6 gene with the promoter ERG7p, inserted the gene GgDHCR24 and StDWF5 into the X-3 site, inserted the genes tHMG1 and IDI1 into the X-4 site, inserted the genes ERG20 and ERG9 into the XII-4 site, and inserted the gene INO2 into the XII-5 site; The ERG6 sequence of the promoter of the gene is shown in SEQ ID NO:1, the sequence of the promoter ERG7p is shown in SEQ ID NO:2, the sequence of the gene GgDHCR24 is shown in SEQ ID NO:3, the sequence of the gene StDWF5 is shown in SEQ ID NO:4, the sequence of the gene tHMG1 is shown in SEQ ID NO:5, the sequence of the gene IDI1 is shown in SEQ ID NO:6, the sequence of the gene ERG20 is shown in SEQ ID NO:7, the sequence of the gene ERG9 is shown in SEQ ID NO:8, the sequence of the gene INO2 is shown in SEQ ID NO:

9.

2. The yeast-derived cholesterol feed additive according to claim 1, wherein, The starting strain of the engineered bacterium is the wild-type Saccharomyces cerevisiae CEN.PK2-1C strain.

3. A yeast-derived cholesterol feed additive according to claim 1, characterized in that, The preparation method of the freeze-dried powder is as follows: the yeast engineered bacterium is fermented and cultured, and then the cells are collected and freeze-dried to obtain the product.

4. The yeast-derived cholesterol feed additive according to claim 3, wherein The fermentation culture method is as follows: enrich the yeast engineering bacteria colonies, inoculate them into YPD liquid medium, and culture them overnight in a shaker at 30 °C. The next day, transfer them to YPD liquid medium for fermentation culture for 96 h according to an initial OD 600 of 0.

2.

5. A yeast-derived cholesterol feed additive according to claim 1, characterized in that, The construction method of the Saccharomyces cerevisiae engineered bacterium comprises the following steps: (1) Using the CRISPR-Cas9 technology, replace the promoter of the ERG6 gene in wild-type Saccharomyces cerevisiae with the promoter ERG7p to obtain recombinant strain 1; (2)Using the CRISPR-Cas9 technology, integrate the cholesterol synthesis gene GgDHCR24 and StDWF5 at the X-3 locus of recombinant bacterium 1 to obtain recombinant bacterium 2; (3) Using the CRISPR-Cas9 technology, integrate the gene tHMG1 and IDI1 at the X-4 locus of recombinant strain 2 to obtain recombinant strain 3; (4)Using the CRISPR-Cas9 technology, integrate the genes ERG20 and ERG9 at the XII-4 locus of recombinant strain 3 to obtain recombinant strain 4; (5) Using the CRISPR-Cas9 technology, the gene INO2 was integrated at the XII-5 locus of recombinant strain 4 to obtain recombinant strain 5, which is the engineered Saccharomyces cerevisiae for cholesterol production.

6. A feed, characterized in that, The feed contains the feed additive described in any one of claims 1 to 4.

7. The feed according to claim 6, characterized in that, The feed is prawn feed, and 4.0% by mass of the feed additive is added to the prawn feed.

8. The feed according to claim 6, characterized in that, The feed further contains a basal feed, and the component contents of the basal feed are as follows: crude protein ≥ 43%, crude fiber ≤ 11%, crude fat ≥ 5%, crude ash ≤ 17%, lysine ≥ 2.1%, sodium chloride ≤ 3.8%, moisture ≤ 12%, calcium ≤ 5%, total phosphorus ≥ 0.5%.

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