A recombinant strain of Saccharomyces cerevisiae for producing retinal and a method for constructing the same

Abstract

The present invention relates to a recombinant strain of Saccharomyces cerevisiae for producing retinal and a method for constructing the same. The method comprises knocking out the adh6, adh7, sfa1, gre2, and hfd1 genes of Saccharomyces cerevisiae to obtain an engineered strain, wherein the engineered strain heterologously expresses geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase / lycopene synthase CrtYB, lycopene dehydrogenase CrtI, and β-carotene-15,15'-dioxygenase BCMO to obtain the recombinant strain. The recombinant strain can improve the purity of retinal, solving the problem that Saccharomyces cerevisiae cannot produce high-purity retinal, and has broad prospects for industrial application.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a recombinant strain of Saccharomyces cerevisiae for producing retinal and a construction method thereof. Background Art

[0002] Retinal, the aldehyde form of vitamin A, has been used in food, cosmetics, pharmaceuticals, health supplements, and animal feed additives primarily for its antioxidant, anticancer, anti-infective, and anti-wrinkle properties. To meet the growing demand for health supplements, retinal has been commercially produced through chemical synthesis. However, chemical synthesis requires the addition of petroleum-based chemicals, such as acetone and acetylene, for acidification and hydrolysis. Post-processing also requires tedious purification to remove the resulting byproducts. Therefore, the use of milder alternative methods for the preparation of vitamin A has become a hot topic. For example, β-carotene-15,15'-dioxygenase from the archaeon Halobacterium sp. NRC-1 is used to convert β-carotene to retinal. However, the use of purified enzymes and β-carotene also increases the cost of synthesis.

[0003] In recent years, the use of microbial methods to produce retinal through fermentation of cheap raw materials has become a trend, with advantages such as low cost, low energy consumption, and environmental protection. Microbial methods usually use glucose as the initial substrate and use microorganisms such as Escherichia coli, Saccharomyces cerevisiae or Saccharomyces cerevisiae as chassis cells to convert glucose through the endogenous methylerythritol phosphate (MEP) or mevalonate (MVA) pathway, and then through geranyl diphosphate synthase (CrtE), bifunctional lycopene cyclase / lycopene synthase (CrtYB), lycopene dehydrogenase (CrtI) and β-carotene-15,15'-dioxygenase (BCMO, encoded by the BLH gene) to obtain retinal. In the current research on the synthesis of vitamin A using Escherichia coli, the strain yield is 136 mg·L -1 , which contains a mixture of retinol, retinal and retinyl acetate. In addition, the production ratio of retinal to retinol was 1.67 when vitamin A was synthesized from xylose by engineering yeast Saccharomyces cerevisiae. In a recent study, an engineered strain of esterolytic yeast was used to produce 4.86 g·L by fed-batch fermentation in a 5-liter fermenter by combining BHT treatment and Tween 80 extraction. -1 of retinol and 0.26g·L -1 of retinal, which is the highest retinol production reported to date.

[0004] However, when these strains produce retinal, they also produce byproducts such as retinol and retinoic acid, or convert the produced retinal into retinol and retinoic acid, which limits the yield and purity of retinal. Summary of the Invention

[0005] The present invention aims to at least partially address one of the technical problems in the related art. To this end, the present invention provides a method for constructing a recombinant strain of Saccharomyces cerevisiae that produces retinal. This recombinant strain can improve the purity of retinal.

[0006] To this end, in one aspect of the present invention, a method for constructing a recombinant strain of Saccharomyces cerevisiae that produces retinal is provided, comprising:

[0007] The adh6 gene, adh7 gene, sfa1 gene, gre2 gene and hfd1 gene of Saccharomyces cerevisiae are knocked out to obtain an engineered strain, and the engineered strain is made to heterologously express geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase / lycopene synthase CrtYB, lycopene dehydrogenase CrtI and β-carotene-15,15'-dioxygenase BCMO to obtain a recombinant Saccharomyces cerevisiae strain.

[0008] According to an embodiment of the present invention, a method for constructing a recombinant Saccharomyces cerevisiae strain for producing retinal is described. This method eliminates the Saccharomyces cerevisiae oxidoreductases adh6, adh7, sfa1, gre2, and hfd1, thereby preventing retinal from being converted to retinol and retinoic acid by endogenous oxidoreductases. The strain then heterologously expresses geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase / lycopene synthase CrtYB, lycopene dehydrogenase CrtI, and β-carotene-15,15'-dioxygenase BCMO using plasmids. After 120 hours of culture, the recombinant strain exhibits an extracellular retinal content (purity >99%) of 4.80±0.20 mg / L. This method addresses the problem of Saccharomyces cerevisiae's inability to produce high-purity retinal and has broad prospects for industrial application.

[0009] In addition, the method for constructing a recombinant strain of Saccharomyces cerevisiae for producing retinal according to the above embodiment of the present invention may also have the following additional technical features:

[0010] Optionally, the following steps are included:

[0011] The endogenous oxidoreductase genes adh6, adh7, sfa1, gre2, and hfd1 of Saccharomyces cerevisiae BY4741 were knocked out to construct the Saccharomyces cerevisiae aldehyde-accumulating chassis cell JS-M5.

[0012] Using the BLH genome as a template, BLH-BamHI-F shown in SEQ ID NO:27 and BLH-XhoI-R shown in SEQ ID NO:28 as the upstream and downstream primers, PCR amplified the BLH gene, and ligated the BLH gene fragment with the expression vector pRS425TEF2 to obtain the recombinant plasmid pRS425TEF2-BLH.

[0013] The pRS425TEF2-BLH plasmid and the YEplac195-YB / I / E plasmid were transformed into Saccharomyces cerevisiae JS-M5 competent cells to obtain the recombinant Saccharomyces cerevisiae strain JS-M5-P.

[0014] Further, the following steps are included:

[0015] The gRNA expression fragments for gene editing technology were obtained by PCR amplification: adh6, adh7, sfa1, gre2, and hfd1. The gRNA expression fragments were ligated with the expression vector p426-SNR52-GGA to obtain recombinant plasmids p426-gRNA (adh6), recombinant plasmid p426-gRNA (adh7), recombinant plasmid p426-gRNA (sfa1), recombinant plasmid p426-gRNA (gre2), and recombinant plasmid p426-gRNA (hfd1).

[0016] The recombinant plasmid p426-gRNA (adh6), recombinant plasmid p426-gRNA (adh7), recombinant plasmid p426-gRNA (sfa1), recombinant plasmid p426-gRNA (gre2) and recombinant plasmid p426-gRNA (hfd1) and their corresponding gene editing integration fragments are sequentially transformed into Saccharomyces cerevisiae competent cells to obtain recombinant strains;

[0017] The recombinant plasmids pRS425TEF2-BLH plasmid and YEplac195-YB / I / E plasmid were introduced into the recombinant strain to obtain the recombinant Saccharomyces cerevisiae strain JS-M5-P.

[0018] Furthermore, the recombinant plasmid p426-gRNA (adh6) is based on the p426-SNR52-gRNA vector as a template, and the nucleotide sequence F_gRNA.adh6 shown in SEQ ID NO: 1 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 are used as primers to obtain the gRNA (adh6) fragment by PCR amplification, and the target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product is connected to the expression vector p426-SNR52-GGA.

[0019] Furthermore, the recombinant plasmid p426-gRNA (adh7) is based on the p426-SNR52-gRNA vector as a template, and the nucleotide sequence F_gRNA.adh7 shown in SEQ ID NO: 2 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 are used as primers to obtain the gRNA (adh7) fragment by PCR amplification, and the target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product is connected to the expression vector p426-SNR52-GGA.

[0020] Furthermore, the recombinant plasmid p426-gRNA (sfa1) is based on the p426-SNR52-gRNA vector as a template, and the nucleotide sequence F_gRNA.sfa1 shown in SEQ ID NO: 3 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 are used as primers to obtain the gRNA (sfa1) fragment by PCR amplification, and the target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product is connected to the expression vector p426-SNR52-GGA.

[0021] Furthermore, the recombinant plasmid p426-gRNA (gre2) is based on the p426-SNR52-gRNA vector as a template, with the nucleotide sequence F_gRNA.gre2 shown in SEQ ID NO: 4 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 as primers, and the gRNA (gre2) fragment is obtained by PCR amplification, and the target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product is connected to the expression vector p426-SNR52-GGA.

[0022] Furthermore, the recombinant plasmid p426-gRNA (hfd1) is based on the p426-SNR52-gRNA vector as a template, with the nucleotide sequence F_gRNA.hfd1 shown in SEQ ID NO: 5 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 as primers, and the gRNA (hfd1) fragment is obtained by PCR amplification. The target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product is connected to the expression vector p426-SNR52-GGA.

[0023] In a second aspect of the present invention, the present invention provides a recombinant strain of Saccharomyces cerevisiae for producing retinal constructed by the above-mentioned construction method.

[0024] According to the recombinant Saccharomyces cerevisiae strain of the embodiment of the present invention, the recombinant strain can improve the purity of retinal.

[0025] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The pathway for retinal synthesis catalyzed by four enzymes, CrtE, CrtYB, CrtI, and BCMO, expressed heterologously in Saccharomyces cerevisiae;

[0027] Figure 2 According to an embodiment of the present invention, endogenous oxidoreductases Adh6, Adh7, Sfa1, Gre2, and Hfd1 are knocked out in BY4741;

[0028] Figure 3 1 is a liquid phase analysis diagram and yield diagram of the fermentation broth of the engineered yeast Saccharomyces cerevisiae strains JS-WT-P and JS-M5-P according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships, without substantially changing the technical content, should also be regarded as the scope of the present invention.

[0030] In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0031] The test materials used in the present invention are all common commercial products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0032] Sources of Materials: Saccharomyces cerevisiae BY4741 and DH5α were commercially available; DH5α was used for vector construction. The Saccharomyces cerevisiae expression vectors p426-SNR52-gRNA, p426-SNR52-GGA, YEplac195-YB / I / E, and pRS425TEF2 were commercially available. Phusion high-fidelity DNA polymerase, T4 ligase, and restriction endonucleases were purchased from Xiamen Lulong Biotechnology Development Co., Ltd. Plasmid extraction kits, DNA purification kits, gel recovery kits, and yeast genomic DNA extraction kits were purchased from Shanghai Bioengineering Co., Ltd.

[0033] LB medium composition: 10 g·L -1 Peptone, 5g·L -1 Yeast powder, 5g·L -1 NaCl, make up to 1 L with double-distilled water, and sterilize at 0.1 MPa pressure and 115 °C for 30 min.

[0034] The composition of YPD medium is: 10 g L -1 Yeast powder, 20g·L -1 Peptone, 20 g L -1 Glucose was added and made up to 1 L with double distilled water, and sterilized at 0.1 MPa pressure and 115°C for 30 min.

[0035] The composition of YNBD-LEU culture medium is: 6.7 g·L -1 Yeast nitrogen base, 1.4 g·L -1 Yeast nutrient deficiency medium supplement (without leucine), 20g·L -1 D-glucose was added to 1 L with double-distilled water and sterilized at 0.1 MPa pressure and 115°C for 20 min.

[0036] The composition of YNBD-LEU-URA culture medium is: 6.7 g L -1 Yeast nitrogen base, 1.4 g·L -1 Yeast nutrient deficiency medium supplement (without leucine and uracil), 20g·L -1 D-glucose was added to 1 L with double-distilled water and sterilized at 0.1 MPa pressure and 115°C for 20 min.

[0037] 100x 5-Fluoroorotic Acid: Dissolve 100 mg of 5-Fluoroorotic Acid in 1 mL of DMSO.

[0038] Detection of retinal and retinol content:

[0039] After fermentation, the fermentation broth was separated into upper and lower layers. 600 μL of the upper layer of dodecane was carefully aspirated, the mixture was centrifuged at 14,000 rpm for 5 minutes, and the mixture was filtered through a filter membrane into a liquid phase bottle. The chromatographic conditions were as follows: a mobile phase of 95% methanol and 5% acetonitrile, a Shimadzu C18 column (4.6×250 mm, 5 μm), a flow rate of 1.5 ml / min, a column temperature of 35°C, and an injection volume of 10 μL. The retinal and retinol contents were determined.

[0040] The plasmids involved in the following examples were constructed in E. coli DH5α. After the plasmid construction was completed, it was used as a knockout or expression vector and transformed into Saccharomyces cerevisiae BY4741 for gene knockout and heterologous expression.

[0041] Table 1 Primers used for PCR amplification

[0042]

[0043]

[0044] The present invention is described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way.

[0045] Example 1 Construction of CRISPR gRNA expression module

[0046] (1) Construction of p426-gRNA (adh6) plasmid:

[0047] Using the p426-SNR52-gRNA vector (Addgene #43803) as a template, primers F_gRNA.adh6 (SEQ ID NO: 1) and R_SUP4 (SEQ ID NO: 6) as primers (Table 1), PCR amplification was performed to obtain the gRNA (adh6) fragment. The target band was recovered using a DNA purification kit to obtain a gel-recovered product. PCR amplification conditions were: 98°C for 2 minutes, 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 1 minute, 30 cycles, and 72°C for 2 minutes. After the p426-SNR52-GGA plasmid was digested with BsaI for 4 hours, the large and small fragments were recovered by gel recovery and then mixed with the gRNA (adh6) fragment. T4 ligase and BsaI enzyme were used, and the ligation conditions were: 37°C for 10 minutes; 37°C for 10 minutes, 16°C for 10 minutes, and four cycles; 20°C for 10 minutes. The ligated product was transformed into Escherichia coli DH5α competent cells, and positive clones were obtained by verification using nucleotide sequences such as SEQ ID NO: 1 and SEQ ID NO: 6 primers. The gRNA expression plasmid p426-gRNA (adh6) was extracted, and the sequencing results were completely consistent with the designed plasmid DNA sequence.

[0048] (2) Construction of p426-gRNA (adh7) plasmid:

[0049] Using the p426-SNR52-gRNA vector (Addgene #43803) as a template, F_gRNA.adh7 (SEQ ID NO: 2) and R_SUP4 (SEQ ID NO: 6) as primers (Table 1), PCR amplification yielded the gRNA (adh7) fragment. The target band was recovered using a DNA purification kit to obtain a gel-recovered product. PCR amplification conditions were: 98°C for 2 minutes, 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 1 minute, and 72°C for 2 minutes for 30 cycles. After the p426-SNR52-GGA plasmid was digested with BsaI for 4 hours, the large and small fragments were recovered by gel recovery and then mixed with the gRNA (adh7) fragment. T4 ligase and BsaI enzyme were used, and the ligation conditions were: 37°C for 10 minutes; 37°C for 10 minutes, 16°C for 10 minutes, and four cycles; 20°C for 10 minutes. The ligated product was transformed into Escherichia coli DH5α competent cells, and positive clones were obtained by verification using nucleotide sequences such as SEQ ID NO: 2 and SEQ ID NO: 6 primers. The gRNA expression plasmid p426-gRNA (adh7) was extracted, and the sequencing results were completely consistent with the designed plasmid DNA sequence.

[0050] (3) Construction of p426-gRNA (sfa1) plasmid:

[0051] Using the p426-SNR52-gRNA vector (Addgene #43803) as a template, primers F_gRNA.sfa1 (SEQ ID NO: 3) and R_SUP4 (SEQ ID NO: 6) as primers (Table 1), PCR amplification was performed to obtain the gRNA (sfa1) fragment. The target band was recovered using a DNA purification kit to obtain a gel-recovered product. PCR amplification conditions were: 98°C for 2 minutes, 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 1 minute, 30 cycles, and 72°C for 2 minutes. After the p426-SNR52-GGA plasmid was digested with BsaI for 4 hours, the large and small fragments were recovered by gel recovery and then mixed with the gRNA (sfa1) fragment. T4 ligase and BsaI enzyme were used, and the ligation conditions were: 37°C for 10 minutes; 37°C for 10 minutes, 16°C for 10 minutes, and four cycles; 20°C for 10 minutes. The ligated product was transformed into Escherichia coli DH5α competent cells, and positive clones were obtained by verification using nucleotide sequences such as SEQ ID NO: 3 and SEQ ID NO: 6 primers. The gRNA expression plasmid p426-gRNA (sfa1) was extracted, and the sequencing results were completely consistent with the designed plasmid DNA sequence.

[0052] (4) Construction of p426-gRNA (gre2) plasmid:

[0053] Using the p426-SNR52-gRNA vector (Addgene #43803) as a template, primers F_gRNA.gre2 (SEQ ID NO: 4) and R_SUP4 (SEQ ID NO: 6) were used as primers (Table 1) to amplify the gRNA (gre2) fragment. The target band was recovered using a DNA purification kit to obtain a gel-recovered product. PCR amplification conditions were: 98°C for 2 minutes, 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 1 minute, 30 cycles, and 72°C for 2 minutes. After the p426-SNR52-GGA plasmid was digested with BsaI for 4 hours, the large and small fragments were recovered by gel recovery and then mixed with the gRNA (gre2) fragment. T4 ligase and BsaI enzyme were used, and the ligation conditions were as follows: 37°C for 10 minutes; 37°C for 10 minutes, 16°C for 10 minutes, and four cycles; 20°C for 10 minutes. The ligated product was transformed into Escherichia coli DH5α competent cells, and positive clones were obtained by verification using nucleotide sequences such as SEQ ID NO: 4 and SEQ ID NO: 6 primers. The gRNA expression plasmid p426-gRNA (gre2) was extracted, and the sequencing results were completely consistent with the designed plasmid DNA sequence.

[0054] (5) Construction of p426-gRNA (hfd1) plasmid:

[0055] Using the p426-SNR52-gRNA vector (Addgene #43803) as a template, primers F_gRNA.hfd1 (SEQ ID NO: 5) and R_SUP4 (SEQ ID NO: 6) as primers (Table 1), PCR amplification was performed to obtain the gRNA (hfd1) fragment. The target band was recovered using a DNA purification kit to obtain a gel-recovered product. PCR amplification conditions were: 98°C for 2 minutes, 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 1 minute, 30 cycles, and 72°C for 2 minutes. After the p426-SNR52-GGA plasmid was digested with BsaI for 4 hours, the large and small fragments were recovered by gel recovery and then mixed with the gRNA (hfd1) fragment. T4 ligase and BsaI enzyme were used, and the ligation conditions were: 37°C for 10 minutes; 37°C for 10 minutes, 16°C for 10 minutes, and four cycles; 20°C for 10 minutes. The ligated product was transformed into Escherichia coli DH5α competent cells, and positive clones were obtained by verification using primers with nucleotide sequences such as SEQ ID NO: 5 and SEQ ID NO: 6. The gRNA expression plasmid p426-gRNA (hfd1) was extracted, and the sequencing results were completely consistent with the designed plasmid DNA sequence.

[0056] Example 2 Preparation of targeted knockout fragments

[0057] (1) Using the nucleotide sequence F-adh6-Del shown in SEQ ID NO: 7 and the nucleotide sequence R-adh6-Del shown in SEQ ID NO: 8 as primers (Table 1), the adh6 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were 94°C for 2 min, 94°C for 15 s, 50°C for 15 s, and 72°C for 15 s, for 30 cycles; and 72°C for 1 min.

[0058] (2) Using the nucleotide sequence F-adh7-Del shown in SEQ ID NO: 9 and the nucleotide sequence R-adh7-Del shown in SEQ ID NO: 10 as primers (Table 1), the adh7 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were 94°C for 2 min, 94°C for 15 s, 50°C for 15 s, and 72°C for 15 s, for 30 cycles; 72°C for 1 min.

[0059] (3) Using the nucleotide sequence F-sfa1-Del shown in SEQ ID NO: 11 and the nucleotide sequence R-sfa1-Del shown in SEQ ID NO: 12 as primers (Table 1), the sfa1 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were 94°C for 2 min, 94°C for 15 s, 50°C for 15 s, and 72°C for 15 s, for 30 cycles; 72°C for 1 min.

[0060] (4) Using the nucleotide sequence F-gre2-Del shown in SEQ ID NO: 13 and the nucleotide sequence R-gre2-Del shown in SEQ ID NO: 14 as primers (Table 1), the gre2 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were 94°C for 2 min, 94°C for 15 s, 50°C for 15 s, and 72°C for 15 s, for 30 cycles; 72°C for 1 min.

[0061] (5) Using the nucleotide sequence F-hfd1-Del shown in SEQ ID NO: 15 and the nucleotide sequence R-hfd1-Del shown in SEQ ID NO: 16 as primers (Table 1), the hfd1 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were 94°C for 2 min, 94°C for 15 s, 50°C for 15 s, and 72°C for 15 s, for 30 cycles; 72°C for 1 min.

[0062] Example 3 Construction of recombinant strains

[0063] (1) Competent cells of Saccharomyces cerevisiae BY4741 were prepared, and the p415-GPD-Cas9 plasmid was transformed into the cells. The cells were cultured on YNBD-LEU plates at 30°C for 2 to 4 days, and the obtained single colonies were named Saccharomyces cerevisiae Y1. Competent cells were then prepared from Y1, and the plasmid p426-gRNA (adh6) obtained in step (1) of Example 1 and the adh6 knockout integration fragment obtained in step (1) of Example 2 were transformed into Y1 competent cells. The cells were cultured on YNBD-LEU-URA plates at 30°C for 2 to 4 days. The grown single colonies were streaked on YNBD-LEU-URA solid plates, and PCR verification was performed using primers SEQ ID NO: 17 and SEQ ID NO: 18. The correct Saccharomyces cerevisiae strain BY4741Δadh6 was named the recombinant strain JS-M1.

[0064] (2) JS-M1 in step (1) was streaked onto a YNBD-LEU plate containing 5-fluoroorotic acid at a concentration of 1 mg / mL to remove the intracellular p426-gRNA (adh6) plasmid, and then used to prepare competent cells, transformed into the p426-gRNA (adh7) in step (2) of Example 1 and the adh7 knockout integration fragment in step (2) of Example 2, and cultured on a YNBD-LEU-URA plate at 30°C for 2 to 4 days. The grown single colonies were streaked onto YNBD-LEU-URA solid plates, and PCR verification was performed using primers SEQ ID NO: 19 and SEQ ID NO: 20. The correct Saccharomyces cerevisiae strain BY4741Δadh6Δadh7 was named recombinant strain JS-M2.

[0065] (3) JS-M2 in step (2) was streaked onto a YNBD-LEU plate containing 5-fluoroorotic acid at a concentration of 1 mg / mL to remove the intracellular p426-gRNA (adh7) plasmid, and then used to prepare competent cells, transformed into p426-gRNA (sfa1) in step (3) of Example 1 and the sfa1 knockout integration fragment in step (3) of Example 2, and cultured on a YNBD-LEU-URA plate at 30°C for 2 to 4 days. The grown single colonies were streaked onto YNBD-LEU-URA solid plates, and PCR verification was performed using primers SEQ ID NO: 21 and SEQ ID NO: 22. The correct Saccharomyces cerevisiae strain BY4741Δadh6Δadh7Δsfa1 was named recombinant strain JS-M3.

[0066] (4) JS-M3 in step (3) was streaked onto a YNBD-LEU plate containing 5-fluoroorotic acid at a concentration of 1 mg / mL to remove the intracellular p426-gRNA (sfa1) plasmid, and then used to prepare competent cells, transformed into p426-gRNA (gre2) in step (4) of Example 1 and the gre2 knockout integration fragment in step (4) of Example 2, and cultured on a YNBD-LEU-URA plate at 30°C for 2 to 4 days. The grown single colonies were streaked onto YNBD-LEU-URA solid plates, and PCR verification was performed using primers SEQ ID NO: 23 and SEQ ID NO: 24. The correct Saccharomyces cerevisiae strain BY4741Δadh6Δadh7Δsfa1Δgre2 was named the recombinant strain JS-M4.

[0067] (5) The JS-M4 in step (4) was streaked onto a YNBD-LEU plate containing 5-fluoroorotic acid at a concentration of 1 mg / mL to remove the intracellular p426-gRNA (gre2) plasmid, and then used to prepare competent cells, transformed into the p426-gRNA (hfd1) in step (5) of Example 1 and the hfd1 knockout integration fragment in step (5) of Example 2, and cultured on a YNBD-LEU-URA plate at 30°C for 2 to 4 days. The grown single colonies were streaked onto a YNBD-LEU-URA solid plate, and primers SEQ ID NO: 25, SEQ ID NO: 30, and SEQ ID NO: 31 were used to generate the competent cells. NO:26 was verified by PCR, and the correct Saccharomyces cerevisiae strain BY4741Δadh6Δadh7Δsfa1Δgre2Δhfd1 was obtained. The positive clones were streaked onto YNBD-LEU plates containing 1 mg / mL 5-fluoroorotic acid to remove the intracellular p426-gRNA (hfd1) plasmid, and the Cas9 expression vector was removed by subculture on YPD solid medium to obtain the recombinant strain JS-M5.

[0068] The results are as follows Figure 2 As shown, the JS-M5 strain knocked out five genes: adh6, adh7, sfa1, gre2, and hfd1.

[0069] Example 4 Construction of pRS425TEF2-BLH plasmid

[0070] The BLH genome was used as a template, and the BLH gene was amplified using the nucleotide sequence of SEQ ID NO:27 (BLH-BamHI-F) and the nucleotide sequence of SEQ ID NO:28 (BLH-XhoI-R) as the upstream and downstream primers (Table 1). PCR amplification conditions were: 94°C for 2 minutes, 94°C for 15 seconds, 56°C for 15 seconds, 72°C for 2 minutes, 30 cycles; and 72°C for 4 minutes. The BLH gene fragment was digested with BamHI and XhoI and then ligated into the expression vector pRS425TEF2, which had been double-digested with BamHI and XhoI, to generate the vector pRS425TEF2-BLH.

[0071] Example 5 Construction of retinal synthesis strain JS-WT-P

[0072] BY4741 was used to prepare competent cells, which were transformed into the pRS425TEF2-BLH plasmid and YEplac195-YB / I / E plasmid obtained in Example 4 (this vector was from Prof. Gerhard Sandmann, for specific information, please refer to the literature: Appl. Environ. Microbiol. 73, 4342–4350), and cultured on YNBD-LEU-URA plates at 30°C for 2-4 days to obtain the positive clone JS-WT-P Saccharomyces cerevisiae recombinant strain.

[0073] Example 6 Construction of Retinaldehyde Synthesis Strain JS-M5-P

[0074] The engineered strain JS-M5 constructed in step (5) of Example 3 was used to prepare competent cells, which were transformed into the pRS425TEF2-BLH plasmid and YEplac195-YB / I / E plasmid obtained in Example 4. The cells were cultured on YNBD-LEU-URA plates at 30°C for 2 to 4 days to obtain the positive clone JS-M5-P Saccharomyces cerevisiae recombinant strain.

[0075] Example 7 Retinaldehyde and retinol production of recombinant strains under shake flask fermentation conditions

[0076] (1) The recombinant strain JS-WT-P and the recombinant strain JS-M5-P constructed in Example 5 and Example 6 were cultured at 30°C and 250 rpm for 12 to 16 h to prepare a seed solution. The prepared seed solution was inoculated at an inoculum rate of 1% (v / v) into a 250 mL Erlenmeyer flask containing 20 mL of YNBD-LEU-URA liquid culture medium and 4 mL of dodecane, and cultured at 30°C and 250 rpm for 120 h to prepare a fermentation broth.

[0077] (2) Calculate the production of extracellular retinal and retinol:

[0078] 600 μL of dodecane from the upper layer of the fermentation broth was aspirated, centrifuged at 14,000 rpm for 5 min, filtered through a membrane into a liquid injection bottle, and subjected to high-performance liquid chromatography detection. The fermentation yield of the engineered strain was calculated by converting it with the peak area of retinal and retinol standards.

[0079] The results are as follows Figure 3 As shown, the extracellular retinal content of JS-M5-P strain was 4.80±0.20 mg L -1 , while the control strain JS-WT-P synthesized 0.53±0.12 mg L -1 Retinol and 1.33±0.12mg L -1 Retinaldehyde.

[0080] In summary, the recombinant Saccharomyces cerevisiae strain according to the embodiments of the present invention, by knocking out the Saccharomyces cerevisiae oxidoreductases Adh6, Adh7, Sfa1, Gre2, and Hfd1, obtained a strain that prevents retinal from being converted to retinol and retinoic acid by endogenous oxidoreductases. The recombinant strain heterologously expresses geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase / lycopene synthase CrtYB, lycopene dehydrogenase CrtI, and β-carotene-15,15'-dioxygenase BCMO using plasmids. After 120 hours of culture, the recombinant strain has an extracellular retinal content (purity >99%) of 4.80±0.20 mg / L. This solves the problem of Saccharomyces cerevisiae's inability to produce high-purity retinal and has broad prospects for industrial application.

[0081] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.

[0082] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a recombinant strain of Saccharomyces cerevisiae for producing retinal, characterized in that: include: The adh6 gene, adh7 gene, sfa1 gene, gre2 gene and hfd1 gene of Saccharomyces cerevisiae were knocked out to obtain an engineered strain; Using the BLH genome as a template, BLH-BamHI-F shown in SEQ ID NO:27 and BLH-XhoI-R shown in SEQ ID NO:28 as the upstream and downstream primers, PCR amplified the BLH gene, and ligated the BLH gene fragment with the expression vector pRS425TEF2 to obtain the recombinant plasmid pRS425TEF2-BLH. The pRS425TEF2-BLH plasmid and the YEplac195-YB / I / E plasmid were transformed into the engineered strain, so that the engineered strain heterologously expressed geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase / lycopene synthase CrtYB, lycopene dehydrogenase CrtI and β-carotene-15,15'-dioxygenase BCMO, thereby obtaining a recombinant Saccharomyces cerevisiae strain.

2. The construction method according to claim 1, wherein The following steps are involved: The endogenous oxidoreductase genes adh6, adh7, sfa1, gre2, and hfd1 of Saccharomyces cerevisiae BY4741 were knocked out to construct the Saccharomyces cerevisiae aldehyde-accumulating chassis cell JS-M5. The pRS425TEF2-BLH plasmid and the YEplac195-YB / I / E plasmid were transformed into Saccharomyces cerevisiae JS-M5 competent cells to obtain the Saccharomyces cerevisiae recombinant strain JS-M5-P.

3. The construction method according to claim 2, wherein: The following steps are involved: The gRNA expression fragments for gene editing technology were obtained by PCR amplification: adh6, adh7, sfa1, gre2, and hfd1. The gRNA expression fragments were ligated with the expression vector p426-SNR52-GGA to obtain recombinant plasmids p426-gRNA (adh6), recombinant plasmid p426-gRNA (adh7), recombinant plasmid p426-gRNA (sfa1), recombinant plasmid p426-gRNA (gre2), and recombinant plasmid p426-gRNA (hfd1). The recombinant plasmid p426-gRNA (adh6), recombinant plasmid p426-gRNA (adh7), recombinant plasmid p426-gRNA (sfa1), recombinant plasmid p426-gRNA (gre2) and recombinant plasmid p426-gRNA (hfd1) and their corresponding gene editing integration fragments are sequentially transformed into Saccharomyces cerevisiae competent cells to obtain recombinant strains; The recombinant plasmids pRS425TEF2-BLH plasmid and YEplac195-YB / I / E plasmid were introduced into the recombinant strain to obtain the recombinant Saccharomyces cerevisiae strain JS-M5-P.

4. The construction method according to claim 3, wherein: The recombinant plasmid p426-gRNA (adh6) uses the p426-SNR52-gRNA vector as a template, uses the nucleotide sequence F_gRNA.adh6 shown in SEQ ID NO: 1 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 as primers, and obtains the gRNA (adh6) fragment by PCR amplification. The target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product is connected to the expression vector p426-SNR52-GGA.

5. The construction method according to claim 3, wherein: The recombinant plasmid p426-gRNA (adh7) uses the p426-SNR52-gRNA vector as a template, uses the nucleotide sequence F_gRNA.adh7 shown in SEQ ID NO: 2 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 as primers, and obtains the gRNA (adh7) fragment by PCR amplification. The target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product is connected to the expression vector p426-SNR52-GGA.

6. The construction method according to claim 3, wherein: The recombinant plasmid p426-gRNA (sfa1) uses the p426-SNR52-gRNA vector as a template, uses the nucleotide sequence F_gRNA.sfa1 shown in SEQ ID NO: 3 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 as primers, and obtains the gRNA (sfa1) fragment by PCR amplification. The target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product is connected to the expression vector p426-SNR52-GGA.

7. The construction method according to claim 3, wherein: The recombinant plasmid p426-gRNA (gre2) uses the p426-SNR52-gRNA vector as a template, uses the nucleotide sequence F_gRNA.gre2 shown in SEQ ID NO: 4 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 as primers, and obtains the gRNA (gre2) fragment by PCR amplification. The target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product is connected to the expression vector p426-SNR52-GGA.

8. The construction method according to claim 3, wherein: The recombinant plasmid p426-gRNA (hfd1) uses the p426-SNR52-gRNA vector as a template, uses the nucleotide sequence F_gRNA.hfd1 shown in SEQ ID NO: 5 and the nucleotide sequence R_SUP4 shown in SEQ ID NO: 6 as primers, and obtains the gRNA (hfd1) fragment by PCR amplification. The target band is recovered by a DNA purification kit to obtain a gel recovery product; and the gel recovery product is connected to the expression vector p426-SNR52-GGA.

9. A recombinant strain of Saccharomyces cerevisiae for producing retinal, characterized in that: The method is constructed according to any one of claims 1 to 8.

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