CN115786151A - Recombinant strain of saccharomyces cerevisiae for producing retinal and construction method thereof - Google Patents

Abstract

The invention relates to a recombinant strain of saccharomyces cerevisiae for producing retinal and a construction method thereof, wherein the construction method comprises the steps of knocking out adh6 genes, adh7 genes, sfa genes, gre2 genes and hfd genes of saccharomyces cerevisiae to obtain an engineering strain, and enabling the engineering strain to heterologously express geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase/lycopene synthetase CrtYB, lycopene dehydrogenase CrtI and beta-carotene-15,15' -dioxygenase BCMO to obtain the recombinant strain of saccharomyces cerevisiae. The recombinant strain can improve the purity of the retinal, solves the problem that saccharomyces cerevisiae can not produce high-purity retinal, and has wide industrial application prospect.

Description

Recombinant strain of saccharomyces cerevisiae for producing retinal and construction method thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a saccharomyces cerevisiae recombinant strain for producing retinal and a construction method thereof.

Background

Retinaldehydes, which are the aldehyde form of vitamin a, have been used in food, cosmetics, pharmaceuticals, nutraceuticals, and animal feed additives primarily because of their antioxidant, anticancer, anti-infective, and anti-wrinkle properties. To meet the growing demand for health supplements, retinal has been commercially produced by 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 cumbersome purification procedures to remove the by-products produced. Therefore, the preparation of vitamin a by other mild substitution methods becomes a research hotspot. For example, β -carotene is converted to retinal by β -carotene-15,15' -dioxygenase derived from archaebacterium sp. However, the use of purified enzymes and beta-carotene also increases the cost of synthesis.

In recent years, the trend of producing retinal by fermentation of cheap raw materials by adopting a microbiological method is, and the method has the advantages of low cost, low energy consumption, environmental friendliness and the like. The microbial method generally uses glucose as an initial substrate, and uses microorganisms such as escherichia coli, ester-solubilizing yeast, saccharomyces cerevisiae and the like as basal disc cells, and the glucose passes through an endogenous methylerythritol phosphate (MEP) or Mevalonate (MVA) pathway, and then obtains retinal via geranyl diphosphate synthase (CrtE), bifunctional lycopene cyclase/lycopene synthase (crtbb), lycopene dehydrogenase (CrtI) and β -carotene-15,15' -dioxygenase (BCMO, encoded by BLH gene). In the current research on the synthesis of vitamin A by using Escherichia coli, the strain yield is 136 mg.L ^-1 Wherein the composition comprises a mixture of retinol, retinal and retinol acetate. In addition, vitamin A is synthesized from xylose by saccharomyces cerevisiae engineering bacteria, and the ratio of the final retinal yield to the retinol yield is 1.67. In a recent study, using an engineered strain of lipolytic yeast, the final engineered strain was fermented by fed-batch fermentation in a 5 liter fermentor producing 4.86 g.L by combining BHT treatment and extraction using Tween 80 ^-1 And 0.26 g.L of retinol ^-1 Retinaldehyde, as described heretoforeThe highest retinol production reported to date.

However, the above strains produce retinal with a by-product such as retinol and retinoic acid, or convert the produced retinal into retinol and retinoic acid. Limiting the yield and purity of retinal.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the present invention aims at providing a construction method of recombinant strain of Saccharomyces cerevisiae producing retinal. The recombinant strain can improve the purity of retinaldehyde.

To this end, in one aspect of the present invention, there is provided a method for constructing a recombinant strain of Saccharomyces cerevisiae producing retinal, comprising:

knocking out adh6 gene, adh7 gene, sfa gene, gre2 gene and hfd gene of saccharomyces cerevisiae to obtain an engineering strain, and allowing the engineering strain to heterologously express geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase/lycopene synthetase CrtYB, lycopene dehydrogenase CrtI and beta-carotene-15,15' -dioxygenase BCMO to obtain a saccharomyces cerevisiae recombinant strain.

According to the construction method of the saccharomyces cerevisiae recombinant strain for producing the retinal, disclosed by the embodiment of the invention, the strain capable of preventing the retinal from being converted into the retinol and the retinoic acid by endogenous oxidoreductase is obtained by knocking out saccharomyces cerevisiae oxidoreductase adh6, adh7, sfa, gre2 and hfd, geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase/lycopene synthetase CrtYB, lycopene dehydrogenase CrtI and beta-carotene-15,15' -dioxygenase BCMO are heterologously expressed by plasmids, and after the recombinant strain is cultured for 120 hours, the content of extracellular retinal (the purity is more than 99%) is 4.80 +/-0.20 mg/L. Solves the problem that the saccharomyces cerevisiae can not produce high-purity retinaldehyde, and has wide industrial application prospect.

In addition, the construction method of recombinant strain of saccharomyces cerevisiae for producing retinal according to the above embodiment of the present invention may further have the following additional technical features:

optionally, the method comprises the following steps:

knocking out Saccharomyces cerevisiae BY4741 endogenous oxidoreductase adh6 gene, adh7 gene, sfa gene, gre2 gene and hfd gene to construct Saccharomyces cerevisiae aldehyde accumulation underplate cell JS-M5;

taking the sequence shown in SEQ ID NO. 29 as a template, BLH-BamHI-F shown in SEQ ID NO. 27 and BLH-XhoI-R shown in SEQ ID NO. 28 as upstream and downstream primers, carrying out PCR amplification on the BLH gene, and connecting the BLH gene fragment with an expression vector pRS425TEF2 to obtain a recombinant plasmid pRS425TEF2-BLH;

and (3) transferring the pRS425TEF2-BLH plasmid and the YEplac195-YB/I/E plasmid into a saccharomyces cerevisiae JS-M5 competent cell to obtain a saccharomyces cerevisiae recombinant strain JS-M5-P.

Further, the method comprises the following steps:

PCR amplification to obtain gRNA expression fragment of gene editing technology: adh6, adh7, sfa1, gre2 and hfd, and connecting the gRNA expression fragments with an expression vector p426-SNR52-GGA respectively to obtain a recombinant plasmid p426-gRNA (adh 6), a recombinant plasmid p426-gRNA (adh 7), a recombinant plasmid p426-gRNA (sfa), a recombinant plasmid p426-gRNA (gre 2) and a recombinant plasmid p426-gRNA (hfd);

sequentially transferring the recombinant plasmid p426-gRNA (adh 6), the recombinant plasmid p426-gRNA (adh 7), the recombinant plasmid p426-gRNA (sfa), the recombinant plasmid p426-gRNA (gre 2), the recombinant plasmid p426-gRNA (hfd) and a corresponding gene editing integrated fragment thereof into a saccharomyces cerevisiae competent cell to obtain a recombinant strain;

and (3) introducing a recombinant plasmid pRS425TEF2-BLH plasmid and YEplac195-YB/I/E plasmid into the recombinant strain so as to obtain a saccharomyces cerevisiae recombinant strain JS-M5-P.

Further, the recombinant plasmid p426-gRNA (adh 6) is prepared by taking a p426-SNR52-gRNA vector as a template, taking F _ gRNA.adh6 with a nucleotide sequence shown as SEQ ID NO. 1 and R _ SUP4 with a nucleotide sequence shown as SEQ ID NO. 6 as primers, carrying out PCR amplification to obtain a gRNA (adh 6) fragment, and recycling a target band through a DNA purification kit to obtain a gel recycling product; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.

Further, the recombinant plasmid p426-gRNA (adh 7) is prepared by taking a p426-SNR52-gRNA vector as a template, taking F _ gRNA.adh7 with a nucleotide sequence shown as SEQ ID NO. 2 and R _ SUP4 with a nucleotide sequence shown as SEQ ID NO. 6 as primers, carrying out PCR amplification to obtain a gRNA (adh 7) fragment, and recycling a target band through a DNA purification kit to obtain a gel recycling product; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.

Further, the recombinant plasmid p426-gRNA (sfa 1) takes a p426-SNR52-gRNA vector as a template, F _ gRNA.sfa1 with a nucleotide sequence shown in SEQ ID NO:3 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:6 as primers, a gRNA (sfa) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.

Further, the recombinant plasmid p426-gRNA (gre 2) is prepared by taking a p426-SNR52-gRNA vector as a template, taking F _ gRNA.gre2 with a nucleotide sequence shown as SEQ ID NO. 4 and R _ SUP4 with a nucleotide sequence shown as SEQ ID NO. 6 as primers, carrying out PCR amplification to obtain a gRNA (gre 2) fragment, and recovering a target band through a DNA purification kit to obtain a gel recovery product; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.

Further, the recombinant plasmid p426-gRNA (hfd 1) takes a p426-SNR52-gRNA vector as a template, takes F _ gRNA.hfd1 with a nucleotide sequence shown in SEQ ID NO:5 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:6 as primers, obtains a gRNA (hfd) fragment through PCR amplification, and recovers a target band through a DNA purification kit to obtain a gel recovery product; the gel recovered product was ligated with the expression vector p426-SNR 52-GGA.

In a second aspect of the present invention, the present invention provides a recombinant strain of Saccharomyces cerevisiae producing retinal constructed by the above-described construction method.

According to the recombinant saccharomyces cerevisiae of the embodiment of the present invention, the recombinant strain can improve the purity of retinaldehyde.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 shows the pathways for the enzymatic synthesis of retinal by heterologous expression of four enzymes CrtE, crtYB, crtI, BCMO in Saccharomyces cerevisiae;

FIG. 2 is a diagram of the knock-out of endogenous oxidoreductases Adh6, adh7, sfa1, gre, hfd in BY4741 according to an embodiment of the present invention;

FIG. 3 is a liquid phase analysis diagram and a yield diagram of fermentation broth of engineering bacteria JS-WT-P and JS-M5-P of Saccharomyces cerevisiae according to the embodiment of the invention.

Detailed Description

The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The test materials adopted by the invention are all common commercial products and can be purchased in the market; the related experiments are all routine experimental methods unless otherwise specified.

Sources of materials used: saccharomyces cerevisiae BY4741 and DH 5. Alpha. Are commercially available, and DH 5. Alpha. Is used for vector construction. Saccharomyces cerevisiae expression vectors p426-SNR52-gRNA, p426-SNR52-GGA, YEplac195-YB/I/E, pRS TEF2 are commercially available. Phusion high fidelity DNA polymerase, T4 ligase, restriction enzyme were purchased from Xiamen Lulong Biotech development Inc. Plasmid extraction kit, DNA purification kit, gel recovery kit and yeast genome DNA extraction kit were purchased from Shanghai bioengineering, inc.

The LB medium consisted of: 10 g.L ^-1 Peptone, 5 g. L ^-1 Yeast powder, 5 g.L ^-1 NaCl and double distilled water to 1L, and sterilizing at 115 deg.C under 0.1Mpa for 30min.

YPD medium composition was: 10 g.L ^-1 Yeast powder, 20 g.L ^-1 Peptone, 20 g.L ^-1 Glucose and double distilled water are supplemented to 1L, and sterilized at 115 deg.C under 0.1Mpa for 30min.

The YNBD-LEU culture medium comprises the following components: 6.7 g.L ^-1 Yeast nitrogen source base, 1.4 g.L ^-1 Yeast auxotrophic medium supplement (leucine-free), 20 g.L ^-1 D-glucose, and double distilled water to 1L, sterilizing at 115 deg.C under 0.1Mpa for 20min.

The YNBD-LEU-URA culture medium comprises the following components: 6.7 g.L ^-1 Yeast nitrogen source base, 1.4 g.L ^-1 Yeast auxotrophic medium supplement (without leucine and uracil), 20 g.L ^-1 D-glucose, and double distilled water to 1L, sterilizing at 115 deg.C under 0.1Mpa for 20min.

100x 5-fluoroorotic acid: 100mg of 5-fluoroorotic acid was dissolved in 1mL using DMSO.

Detecting the content of retinene and retinol:

layering fermented liquid up and down, carefully sucking 600 mu L dodecane in the upper layer, centrifuging at 14000rpm for 5min by a centrifuge, and filtering by a filter membrane to enter a liquid phase bottle; detecting and analyzing by using a Shimadzu high performance liquid chromatograph, and adopting a photodiode array detector (with the working wavelength of 340 nm); the chromatographic conditions are as follows: the mobile phase was 95% methanol, 5% acetonitrile, and the content of retinal and retinol was measured by using Shimadzu C18 column (4.6X 250mm,5 μm), flow rate 1.5ml/min, column temperature 35 ℃ and sample size 10 μ L.

The construction of the plasmids involved in the following examples was performed in e.coli DH5 α, and after the plasmid construction was completed, the plasmids were used as knock-out or expression vectors and transformed into saccharomyces cerevisiae BY4741 for gene knock-out and heterologous expression.

TABLE 1 primers used for PCR amplification

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

Example 1 construction of a gRNA expression module for CRISPR

(1) Construction of p426-gRNA (adh 6) plasmid:

a p426-SNR52-gRNA vector (adddge # 43803) is used as a template, F _ gRNA. Adh6 with a nucleotide sequence shown in SEQ ID NO:1 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:6 are used as primers (Table 1), a gRNA (adh 6) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large and small fragments are recovered by gel, and then mixed with gRNA (adh 6) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO:1 and SEQ ID NO:6 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (adh 6), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(2) Construction of the p426-gRNA (adh 7) plasmid:

a p426-SNR52-gRNA vector (adddge # 43803) is used as a template, F _ gRNA (adh 7) with a nucleotide sequence shown as SEQ ID NO:2 and R _ SUP4 with a nucleotide sequence shown as SEQ ID NO:6 are used as primers (Table 1), a gRNA (adh 7) segment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large and small fragments are recovered by gel, and then mixed with gRNA (adh 7) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO:2 and SEQ ID NO:6 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (adh 7), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(3) Construction of p426-gRNA (sfa 1) plasmid:

a gRNA (sfa) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) as a template, F _ gRNA.sfa1 with a nucleotide sequence shown in SEQ ID NO:3 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:6 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large fragment and the small fragment are recovered by gel, and then mixed with a gRNA (sfa) fragment, T4 ligase and BsaI enzyme are used, and the ligation conditions are as follows: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by primers with nucleotide sequences shown as SEQ ID NO:3 and SEQ ID NO:6 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (sfa 1), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(4) Construction of the p426-gRNA (gre 2) plasmid:

a p426-SNR52-gRNA vector (adddge # 43803) is used as a template, F _ gRNA.gre2 with a nucleotide sequence shown in SEQ ID NO. 4 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO. 6 are used as primers (table 1), a gRNA (gre 2) segment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large fragment and the small fragment are recovered by gel, and then mixed with gRNA (gre 2) fragments, T4 ligase and BsaI enzyme are used, and the ligation conditions are as follows: 10min at 37 ℃; circulation is carried out for 4 times at 37 ℃ and 10min at 16 ℃; transferring the connected product into a competent cell of Escherichia coli DH5 alpha after 10min at 20 ℃, verifying by primers with nucleotide sequences such as SEQ ID NO. 4 and SEQ ID NO. 6 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (gre 2), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(5) Construction of p426-gRNA (hfd 1) plasmid:

a gRNA (hfd) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) as a template, F _ gRNA.hfd1 with a nucleotide sequence shown in SEQ ID NO:5 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:6 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large fragment and the small fragment are recovered by gel, and then mixed with gRNA (hfd) fragment, using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by primers with nucleotide sequences shown as SEQ ID NO:5 and SEQ ID NO:6 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (hfd 1), and completely conforming the sequencing result to the designed plasmid DNA sequence.

Example 2 Targeted knock-out fragment preparation

(1) F-adh6-Del with a nucleotide sequence shown as SEQ ID NO. 7 and R-adh6-Del with a nucleotide sequence shown as SEQ ID NO. 8 are used as primers (Table 1), adh6 knockout integration fragments are obtained through PCR amplification, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 15s, and the cycle is 30 times; 72 ℃ for 1min.

(2) F-adh7-Del with a nucleotide sequence shown as SEQ ID NO. 9 and R-adh7-Del with a nucleotide sequence shown as SEQ ID NO. 10 are used as primers (Table 1), adh7 knockout integration fragments are obtained through PCR amplification, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 15s, and the cycle is 30 times; 72 ℃ for 1min.

(3) F-sfa-Del with the nucleotide sequence shown as SEQ ID NO:11 and R-sfa1-Del with the nucleotide sequence shown as SEQ ID NO:12 are used as primers (table 1), sfa knockout integration fragments are obtained through PCR amplification, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s and 72 ℃ for 15s, and the cycle is 30 times; 72 ℃ for 1min.

(4) F-gre2-Del with a nucleotide sequence shown as SEQ ID NO. 13 and R-gre2-Del with a nucleotide sequence shown as SEQ ID NO. 14 are used as primers (table 1), and gre2 knockout integration fragments are obtained through PCR amplification under the conditions of 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 15s, and the cycle is 30 times; 72 ℃ for 1min.

(5) F-hfd-Del with the nucleotide sequence shown as SEQ ID NO:15 and R-hfd1-Del with the nucleotide sequence shown as SEQ ID NO:16 are used as primers (table 1), hfd knockout integration fragments are obtained through PCR amplification, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s and 72 ℃ for 15s, and the cycle is 30 times; 72 ℃ for 1min.

Example 3 construction of recombinant strains

(1) A competent cell is prepared from saccharomyces cerevisiae BY4741, transformed into a p415-GPD-Cas9 plasmid, cultured on an YNBD-LEU plate at 30 ℃ for 2-4 days, and an obtained single colony is named as saccharomyces cerevisiae Y1. Then preparing competent cells from Y1, transforming the plasmid p426-gRNA (adh 6) obtained in the step (1) of the example 1 and the adh6 knockout integration fragment obtained in the step (1) of the example 2 into the Y1 competent cells, culturing for 2-4 days at 30 ℃ on an YNBD-LEU-URA plate, respectively scribing the grown single colonies on the YNBD-LEU-URA solid plate, and carrying out PCR verification BY using primers SEQ ID NO:17 and SEQ ID NO:18 to obtain the correct saccharomyces cerevisiae strain BY4741 delta adh6 which is named as a recombinant strain JS-M1.

(2) The JS-M1 in the step (1) is streaked on an YNBD-LEU plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (adh 6) plasmid, then the plasmid is used for preparing competent cells, the plasmid is transformed into the p426-gRNA (adh 7) in the step (2) in the example 1 and an adh7 knockout integration fragment in the step (2) in the example 2, the integrated fragments are cultured on the YNBD-LEU-URA plate at 30 ℃ for 2-4 days, grown single colonies are streaked on the YNBD-LEU-URA solid plate respectively, PCR verification is carried out BY primers SEQ ID NO:19 and SEQ ID NO:20, and the correct Saccharomyces cerevisiae strain BY4741 delta adh6 delta adh7 is named as a recombinant JS adh-M2.

(3) The JS-M2 in the step (2) is streaked on an YNBD-LEU plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove the intracellular p426-gRNA (adh 7) plasmid, then used for preparing competent cells, the p426-gRNA (sfa 1) in the step (3) in the example 1 and sfa knockout integration fragment in the step (3) in the example 2 are transformed, cultured on the YNBD-LEU-URA plate for 2-4 days at 30 ℃, grown single colonies are streaked on the YNBD-LEU-URA solid plate respectively, and PCR verification is carried out BY using primers SEQ ID NO:21 and SEQ ID NO:22, and a correct saccharomyces cerevisiae strain BY4741 delta adh6 delta adh7 delta sfa is named as a recombinant strain-JS 3.

(4) The JS-M3 in the step (3) is streaked on an YNBD-LEU plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (sfa) plasmid, then the plasmid is used for preparing competent cells, the p426-gRNA (gre 2) in the step (4) of the example 1 and the gre2 knockout integration fragment in the step (4) of the example 2 are transformed, the gre2 knockout integration fragment is cultured on the YNBD-LEU-URA plate for 2 to 4 days at 30 ℃, the grown single colonies are streaked on the YNBD-LEU-URA solid plate respectively, and PCR verification is carried out BY primers SEQ ID NO:23 and SEQ ID NO:24, and the correct Saccharomyces cerevisiae strain BY4741 delta adh6 delta adh7 delta sfa Δ gre2 is named as a recombinant strain-M4.

(5) The JS-M4 in step (4) is streaked on YNBD-LEU plate containing 5-fluoroorotic acid with concentration of 1mg/mL to remove intracellular p426-gRNA (gre 2) plasmid, then used for preparing competent cells, transformed into p426-gRNA (hfd) in step (5) in example 1 and hfd knockout integrated fragment in step (5) in example 2, cultured on YNBD-LEU-URA plate at 30 ℃ for 2-4 days, grown single colony is streaked on YNBD-LEU-URA solid plate respectively, PCR is carried out BY primers SEQ ID NO:25 and SEQ ID NO:26, correct Saccharomyces cerevisiae BY4741 Δ adh6 Δ adh7 Δ sfa Δ gre2 Δ hfd is streaked on yeast strain containing 5-fluoroorotic acid with concentration of 1mg/mL, the recombinant strain is streaked on YPBD-LEU plate containing 5-fluoroorotic acid with concentration of 1mg/mL, the strain is subjected to remove intracellular p426-gRNA (g 2) plasmid, and cultured on YPBD 3732 strain through strain deletion culture medium, and finally passed through strain recombinant strain SAND 3732.

As a result, as shown in FIG. 2, the strain JS-M5 knocked out five genes of adh6, adh7, sfa, gre2 and hfd 1.

Example 4 construction of pRS425TEF2-BLH plasmid

The sequence shown in SEQ ID NO. 29 after artificial codon optimization is taken as a template, BLH-BamHI-F with the nucleotide sequence shown in SEQ ID NO. 27 and BLH-XhoI-R with the nucleotide sequence shown in SEQ ID NO. 28 are taken as an upstream primer and a downstream primer (table 1), the BLH gene is amplified, and the PCR amplification conditions are as follows: 2min at 94 ℃, 15s at 56 ℃, 2min at 72 ℃, and 30 times of circulation; 4min at 72 ℃. The BLH gene fragment was digested with BamHI and XhoI, followed by ligation into BamHI and XhoI double digested expression vector pRS425TEF2, resulting in vector pRS425TEF2-BLH.

Example 5 construction of synthetic retinal Strain JS-WT-P

BY4741 was used to prepare competent cells, which were transformed with pRS425TEF2-BLH plasmid and YEplac195-YB/I/E plasmid (obtained in Prof. Gerhard Sandmann, as described in Appl. Environ. Microbiol.73, 4342-4350) obtained in example 4, and cultured on YNBD-LEU-URA plates at 30 ℃ for 2-4 days to obtain JS-WT-P Saccharomyces cerevisiae recombinant strain as a positive clone.

EXAMPLE 6 construction of synthetic retinal Strain JS-M5-P

Competent cells were prepared from the engineered strain JS-M5 constructed in step (5) of example 3, and transformed into pRS425TEF2-BLH plasmid and YEplac195-YB/I/E plasmid obtained in example 4, which were cultured on YNBD-LEU-URA plate at 30 ℃ for 2 to 4 days to obtain a positive clone JS-M5-P Saccharomyces cerevisiae recombinant strain.

Example 7 production of retinal and retinol by recombinant strains under shake flask fermentation conditions

(1) The recombinant strain JS-WT-P and the recombinant strain JS-M5-P constructed in the examples 5 and 6 are respectively cultured for 12-16 h under the conditions of 30 ℃ and 250rpm to prepare seed liquid, the prepared seed liquid is inoculated into a 250mL triangular flask filled with 20mL YNBD-LEU-URA liquid culture medium and 4mL dodecane according to the inoculation amount of 1% (v/v), and the culture is carried out for 120h under the conditions of 30 ℃ and 250rpm to prepare fermentation liquid.

(2) Calculating the yield of extracellular retinal and retinol:

absorbing 600 mu L of dodecane on the upper layer of the fermentation liquid, centrifuging for 5min at 14000rpm of a centrifuge, filtering the dodecane on a liquid phase sample bottle through a filter membrane, detecting the dodecane by high performance liquid chromatography, and converting the dodecane with the peak areas of retinene and retinol standard products to obtain the fermentation yield of the engineering strain.

As shown in FIG. 3, the extracellular retinal content of JS-M5-P strain was 4.80. + -. 0.20mg L ^-1 While the control strain JS-WT-P synthesized 0.53. + -. 0.12mg L ^-1 Retinol and 1.33. + -. 0.12mg L ^-1 Retinal.

In conclusion, according to the recombinant saccharomyces cerevisiae strain disclosed by the embodiment of the invention, strains capable of preventing retinal from being converted into retinol and retinoic acid by endogenous oxidoreductases are obtained by knocking out saccharomyces cerevisiae oxidoreductases Adh6, adh7, sfa1, gre and Hfd, geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase/lycopene synthetase CrtYB, lycopene dehydrogenase CrtI and beta-carotene-15,15' -dioxygenase BCMO are expressed in a heterologous manner by plasmids, and after the recombinant strain is cultured for 120 hours, the content of extracellular retinal (the purity is more than 99%) is 4.80 +/-0.20 mg/L. Solves the problem that the saccharomyces cerevisiae can not produce high-purity retinal, and has wide industrial application prospect.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (9)

1. A method for constructing a recombinant strain of Saccharomyces cerevisiae producing retinal, comprising:

knocking out adh6 gene, adh7 gene, sfa gene, gre2 gene and hfd gene of saccharomyces cerevisiae to obtain an engineering strain, and allowing the engineering strain to heterologously express geranylgeranyl diphosphate synthase CrtE, bifunctional lycopene cyclase/lycopene synthetase CrtYB, lycopene dehydrogenase CrtI and beta-carotene-15,15' -dioxygenase BCMO to obtain a saccharomyces cerevisiae recombinant strain.

2. The method of construction of claim 1, comprising the steps of:

knocking out Saccharomyces cerevisiae BY4741 endogenous oxidoreductase adh6 gene, adh7 gene, sfa gene, gre2 gene and hfd gene to construct Saccharomyces cerevisiae aldehyde accumulation underplate cell JS-M5;

taking the sequence shown in SEQ ID NO. 29 as a template, BLH-BamHI-F shown in SEQ ID NO. 27 and BLH-XhoI-R shown in SEQ ID NO. 28 as upstream and downstream primers, carrying out PCR amplification on the BLH gene, and connecting the BLH gene fragment with an expression vector pRS425TEF2 to obtain a recombinant plasmid pRS425TEF2-BLH;

and (3) transferring the pRS425TEF2-BLH plasmid and the YEplac195-YB/I/E plasmid into a saccharomyces cerevisiae JS-M5 competent cell to obtain a saccharomyces cerevisiae recombinant strain JS-M5-P.

3. The method of construction of claim 2, comprising the steps of:

PCR amplification to obtain gRNA expression fragment of gene editing technology: adh6, adh7, sfa1, gre2 and hfd, and connecting the gRNA expression fragments with an expression vector p426-SNR52-GGA respectively to obtain a recombinant plasmid p426-gRNA (adh 6), a recombinant plasmid p426-gRNA (adh 7), a recombinant plasmid p426-gRNA (sfa), a recombinant plasmid p426-gRNA (gre 2) and a recombinant plasmid p426-gRNA (hfd);

sequentially transferring the recombinant plasmid p426-gRNA (adh 6), the recombinant plasmid p426-gRNA (adh 7), the recombinant plasmid p426-gRNA (sfa), the recombinant plasmid p426-gRNA (gre 2), the recombinant plasmid p426-gRNA (hfd) and a corresponding gene editing integrated fragment thereof into a saccharomyces cerevisiae competent cell to obtain a recombinant strain;

and (3) introducing a recombinant plasmid pRS425TEF2-BLH plasmid and a YEplac195-YB/I/E plasmid into the recombinant strain so as to obtain a saccharomyces cerevisiae recombinant strain JS-M5-P.

4. The construction method of claim 3, wherein the recombinant plasmid p426-gRNA (adh 6) is prepared by taking a p426-SNR52-gRNA vector as a template, taking F _ gRNA.adh6 with a nucleotide sequence shown in SEQ ID NO:1 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:6 as primers, performing PCR amplification to obtain a gRNA (adh 6) fragment, and recovering a target band through a DNA purification kit to obtain a gel recovery product; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.

5. The construction method of claim 3, wherein the recombinant plasmid p426-gRNA (adh 7) is a gRNA (adh 7) fragment obtained by PCR amplification using a p426-SNR52-gRNA vector as a template, F _ gRNA. Adh7 having a nucleotide sequence shown in SEQ ID NO:2 and R _ SUP4 having a nucleotide sequence shown in SEQ ID NO:6 as primers, and a target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovered product was ligated with the expression vector p426-SNR 52-GGA.

6. The construction method of claim 3, wherein the recombinant plasmid p426-gRNA (sfa 1) is a gel recovery product obtained by using a p426-SNR52-gRNA vector as a template, using F _ gRNA.sfa1 with a nucleotide sequence shown in SEQ ID NO:3 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:6 as primers, performing PCR amplification to obtain a gRNA (sfa) fragment, and recovering a target band through a DNA purification kit; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.

7. The construction method according to claim 3, wherein the recombinant plasmid p426-gRNA (gre 2) is prepared by taking a p426-SNR52-gRNA vector as a template, taking F _ gRNA.gre2 with a nucleotide sequence shown in SEQ ID NO. 4 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO. 6 as primers, performing PCR amplification to obtain a gRNA (gre 2) fragment, and recovering a target band through a DNA purification kit to obtain a gel recovery product; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.

8. The construction method of claim 3, wherein the recombinant plasmid p426-gRNA (hfd 1) is a gel recovered product obtained by using a p426-SNR52-gRNA vector as a template, using F _ gRNA.hfd1 with a nucleotide sequence shown in SEQ ID NO:5 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:6 as primers, performing PCR amplification to obtain a gRNA (hfd) fragment, and recovering a target band through a DNA purification kit; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.

9. A recombinant strain of saccharomyces cerevisiae producing retinal, constructed by the construction method according to any one of claims 1 to 8.

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