CN113249239A

CN113249239A - Saccharomyces cerevisiae for high yield of crocetin and construction method and application thereof

Info

Publication number: CN113249239A
Application number: CN202110516673.2A
Authority: CN
Inventors: 元英进; 梁楠; 肖文海; 姚明东; 王颖
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2021-08-13

Abstract

The invention relates to the technical field of microorganisms, and discloses saccharomyces cerevisiae for high yield of crocetin and a construction method and application thereof. The saccharomyces cerevisiae is integrated with coding genes of CCD2 protease and ALD protease, or is transformed with a vector for expressing CCD2 protease and ALD protease; meanwhile, the HIS label is fused to the N segment of the CCD2 protease coding gene. According to the invention, the 6 xHis tag is fused at the N-terminal of the CsCCD2 gene to improve the soluble expression of the CsCCD2 in yeast, so that the crocetin yield is improved, and compared with a control strain not fused with the 6 xHis tag, the yield is improved by 36%.

Description

Saccharomyces cerevisiae for high yield of crocetin and construction method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to saccharomyces cerevisiae for high yield of crocetin and a construction method and application thereof.

Background

Crocetin (Crocetin) is an important medicinal active ingredient in saffron. In the previous research of the invention, the de novo synthesis of crocetin in saccharomyces cerevisiae is successfully realized, and the catalytic efficiency of the crocetin and beta-carotene is greatly improved by the hybrid design of a substrate channel of a key enzyme CCD2 (patent: 201910243448.9). The need for saccharomyces cerevisiae strains with high production of crocetin has been a generally sought goal, and there is still room for further improvement of crocetin production based on the aforementioned studies.

Disclosure of Invention

In view of this, the invention aims to provide a saccharomyces cerevisiae with high yield of crocetin and a construction method thereof, so that the constructed saccharomyces cerevisiae can obviously improve the yield of crocetin;

another object of the present invention is to provide the use of the above constructed Saccharomyces cerevisiae in the synthesis of crocetin.

In order to achieve the above purpose, the invention provides the following technical scheme:

a high-yield crocetin Saccharomyces cerevisiae, wherein the chromosome of the Saccharomyces cerevisiae is integrated with coding genes of CCD2 and ALD two proteases, or the Saccharomyces cerevisiae is transformed with a vector for expressing CCD2 and ALD two proteases; meanwhile, the HIS label is fused to the N segment of the CCD2 protease coding gene.

Preferably, the CCD2 protease is a CCD2 derived from saffron, namely CsCCD 2.

The research of the invention finds that the N end of the full-length gene CsCCD2L comprises a signal peptide positioned in plastid, and although CsCCD2 is the N-end truncated version of CsCCD2L, the alpha helical structure formed by hydrophobic amino acid residues at the N end can interact with cell membranes and possibly influence the soluble expression of CsCCD 2. Therefore, the invention improves the soluble expression of CsCCD2 in yeast by fusing a 6 xHis label at the N end of the CsCCD2 gene, thereby improving the yield of crocetin.

Preferably, the saccharomyces cerevisiae is capable of self-synthesizing zeaxanthin and/or beta-carotene.

Preferably, the CCD2 protease is wild type CCD2 protease derived from saffron or mutant CCD2 protease having one or more than two site mutations based on the wild type protease:

V120F、Y190K/A、R192V/F、E211A、E212A、T290V、K320A、S323F/A/T。

more preferably, the mutant CCD2 protease has an S323F/a/T site mutation and/or has one or more of the following site mutations:

V120F、Y190K/A、R192V/F、E211A、E212A、T290V、K320A。

in a specific implementation process, the saffron-derived CCD2 mutant has site mutation in any one of the following situations:

(1) S323F/A site mutation + R192V/F site mutation;

(2) (ii) S323F/a site mutation + T290V site mutation;

(3) S323F/A site mutation + R192V/F site mutation + T290V site mutation.

The mutant CsCCD2 protease is disclosed to have better catalytic efficiency on zeaxanthin or beta-carotene in the prior application 201910243448.9, and the invention selects the mutant CCD2_ mut29(R192F & T290V & S323A) in the prior application to carry out relevant experimental verification in the specific embodiment of the invention; the ALD protease and the coding sequence thereof are Syn _ ALD derived from Synechocystis sp.PCC6803 and the coding sequence thereof. Any information on CsCCD2 protease and SynALD protease can be found in earlier application 201910243448.9, which is equivalent to the present invention and can be included as part of the present invention.

The strain stability can be increased relative to the use of a vector plasmid format in which the genes encoding the two proteases CCD2 and ALD are integrated at the HO integration site of the Saccharomyces cerevisiae chromosome in a specific embodiment of the present invention.

According to the invention, 6 His amino acids (HIS labels) are fused at the N-terminal of the CsCCD2, so that the yield of the crocetin is improved by 36%, an unexpected technical effect is achieved, and the application of the saccharomyces cerevisiae in the synthesis of the crocetin is provided based on the invention.

Simultaneously, the invention also provides a construction method of the saccharomyces cerevisiae, which comprises the steps of fusing an HIS label sequence on the N section of the CCD2 protease coding gene, designing a homology arm to be integrated on an initial saccharomyces cerevisiae chromosome, and integrating the ALD protease coding gene on the initial saccharomyces cerevisiae chromosome in the same way to obtain the saccharomyces cerevisiae; or

The HIS tag sequence is fused with the N segment of the CCD2 protease coding gene, then is connected to a carrier plasmid and transformed into the initial Saccharomyces cerevisiae, and the ALD protease coding gene is connected to the carrier plasmid (the two protease coding genes can be on different plasmids or the same plasmid) and transformed into the initial Saccharomyces cerevisiae in the same way, so as to obtain the Saccharomyces cerevisiae.

In addition, the invention also provides a method for synthesizing crocetin, which is characterized in that the saccharomyces cerevisiae is adopted for fermentation synthesis of crocetin, and if the saccharomyces cerevisiae can synthesize zeaxanthin and/or beta-carotene, zeaxanthin and/or beta-carotene can not be added or can be added in a fermentation medium in a small amount; if the Saccharomyces cerevisiae itself is not capable of synthesizing zeaxanthin and/or beta-carotene, then zeaxanthin and/or beta-carotene needs to be added to the fermentation medium.

Preferably, the fermentation medium is a YPD medium containing the induction substrates determined according to the inducible expression system in Saccharomyces cerevisiae. For example, the present invention in the embodiment uses the previous application of SyBE _ Sc0123Cz14, the inducible expression system is galactose inducible expression system, so the induction substrate in the fermentation medium is selected to be galactose, preferably at a concentration of 10 g/L.

In a specific embodiment of the invention, reference is made to the following constituents of the YPD medium:

40g/L glucose, 20g/L peptone, 10g/L yeast extract;

in the field of microbial fermentation, seed liquid of a strain is generally prepared before fermentation, and the method can also comprise a process for preparing the seed liquid, wherein the process can be used for determining primary seed liquid or secondary seed liquid according to conditions, and a seed culture medium of the seed liquid is a fermentation culture medium without an induction substrate.

In a specific embodiment of the invention, the seed culture medium is a YPD medium; the seed liquid preparation process can be referred to as follows:

first-stage seed liquid: inoculating the strain to 3mL YPD seed culture medium, and culturing at 220rpm30 ℃ for 17h as primary seed;

secondary seed liquid: the first seed is expressed as the initial OD₆₀₀Transferring to 10mL of fresh YPD seed culture medium at 0.2, and culturing at 220rpm30 ℃ for 7h as secondary seeds;

during inoculation and fermentation, the secondary seed liquid is treated according to the initial OD₆₀₀Fermenting 0.1 and 50mL YPDG (YPD fermentation medium containing galactose) at 250rpm at 20 deg.C for 144 h.

According to the technical scheme, the 6 xHis tag is fused at the N end of the CsCCD2 gene, so that the soluble expression of the CsCCD2 in yeast is improved, the yield of crocetin is further improved, and compared with a control strain which is not fused with the 6 xHis tag, the yield is improved by 36%.

Drawings

FIG. 1 shows the fermentation yields of crocetin of different Saccharomyces cerevisiae unfused with Histag fused at the N-terminal, Histag fused at the C-terminal and Histag fused at the N-terminal, and Histag fused at the C-terminal and C-terminal.

Detailed Description

The embodiment of the invention discloses saccharomyces cerevisiae for high yield of crocetin and a construction method and application thereof, and a person skilled in the art can realize the high yield of crocetin by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. The Saccharomyces cerevisiae and its construction method and application have been described in the preferred embodiments, and it is obvious to those skilled in the art that the technology of the present invention can be implemented and applied by modifying or appropriately changing and combining the Saccharomyces cerevisiae and its construction method and application described herein without departing from the content, spirit and scope of the present invention.

The invention further provides the saccharomyces cerevisiae for high yield of crocetin and a construction method and application thereof.

Example 1: construction of chromosome-integrated Strain

In the prior application 201910243448.9, the invention finds that the CCD2 mutant CCD2_ mut29(R192F & T290V & S323A) has better effect on catalyzing beta-carotene substrate and zeaxanthin substrate, and the characterization is carried out by cloning genes on an episomal plasmid pRS 416. In the present invention, the CCD2_ mut29 expression cassette is integrated on the chromosome of zeaxanthin producing strain SyBE _ Sc0123Cz14 to improve the stability of the strain. Meanwhile, His labels are respectively fused at the N end, the C end, the N end and the C end of the CCD2_ mut29 and are integrated at a chromosome HO site of a zeaxanthin producing strain Cz14, so that the influence of the hydrotropic label on the catalytic efficiency of the CCD2 enzyme is researched. The specific construction process is as follows:

step1, obtaining upstream and downstream homologous arms of HO integration sites, GAL10p-CCD2_ mut29-TEF2t expression cassettes, GAL10p-CCD2_ mut29-TEF2t expression cassettes (Histag fused with the N end, the C end, the N end and the C end) and URA3 screening labels through PCR, and obtaining TEF2t-GAL7p-SynALD-PGI1t expression cassettes through restriction enzyme digestion of plasmid pRS425 k-A-04; the above expression cassette can also be prepared in the manner described in the prior application;

step2. the DNA fragment obtained in Step1 was integrated into the HO site of Cz14 strain by yeast homologous recombination, and SC-URA plates were coated to select correct transformants.

The constructed strains were as follows:

SyBE_Sc02070334：Cz14,HO::GAL10p-CCD2_mut29-TEF2t-GAL7p-Syn ALD-PGI1t-URA3；

SyBE_Sc02070335：Cz14,HO::GAL10p-Histag-CCD2_mut29-TEF2t-GAL7p-SynALD-PGI1t-URA3；

SyBE_Sc02070336：Cz14,HO::GAL10p-CCD2_mut29-Histag-TEF2t-GA L7p-SynALD-PGI1t-URA3；

SyBE_Sc02070337：Cz14,HO::GAL10p-Histag-CCD2_mut29-Histag-TEF2t-GAL7p-SynALD-PGI1t-URA3。

example 2: fermentation characterization of chromosomally integrated strains

Seed medium YPD: 40g/L glucose, 20g/L peptone, 10g/L yeast extract;

fermentation medium YPDG: 40g/L glucose, 20g/L peptone, 10g/L yeast extract, 10g/L galactose;

fermenting the strain:

step1. inoculating 4 strains of yeast strain glycerol strain constructed in example 1 into 3mL YPD seed culture medium, and culturing at 220rpm30 ℃ for 17h as primary seeds;

step2. first seed is adjusted to initial OD₆₀₀Transferring to 5mL of fresh YPD seed culture medium at 0.2, and culturing at 220rpm as secondary seed at 30 ℃ for 7 h;

step3. Secondary seeds were pressed to initial OD₆₀₀Fermenting 0.1 and 50mL YPDG at 250rpm at 20 deg.C for 144 h.

After the fermentation is finished, the crocetin product is extracted according to the method of patent 201910243448.9 and detected by HPLC, and the result is shown in figure 1;

after chromosome integration, the crocetin yield of the mutant strain containing the CCD2_ mut29 reaches 13.22 mg/L. The yield of crocetin in the CCD2_ mut29 strain with Histag fused at the N end reaches 18.05mg/L, and the yield is improved by nearly 36 percent; the yield of the crocetin is not improved by the CCD2_ mut29 strain fused with Histag only at the C end, and the yield of the crocetin is also obviously improved by the CCD2_ mut29 strain fused with Histag at the N + C end. This shows that crocetin production can be greatly improved while strain stability is increased by chromosomal integration. Meanwhile, the N-terminal Histag fusion of the CCD2 protein also contributes to improving the yield of crocetin.

The foregoing is only for the purpose of understanding the method of the present invention and the core concept thereof, and it will be understood by those skilled in the art that various changes and modifications may be made without departing from the principle of the invention, and the invention also falls within the scope of the appended claims.

Claims

1. The saccharomyces cerevisiae for high yield of crocetin is characterized in that coding genes of CCD2 and ALD protease are integrated into a chromosome of the saccharomyces cerevisiae, or a vector for expressing CCD2 and ALD protease is transformed into the saccharomyces cerevisiae; meanwhile, the HIS label is fused to the N segment of the CCD2 protease coding gene.

2. The Saccharomyces cerevisiae of claim 1, wherein the Saccharomyces cerevisiae is capable of self-synthesizing zeaxanthin and/or β -carotene.

3. The Saccharomyces cerevisiae according to claim 1, wherein the CCD2 protease is wild-type CCD2 protease derived from saffron or mutant CCD2 protease with one or more site mutations based on the wild-type protease as follows:

V120F、Y190K/A、R192V/F、E211A、E212A、T290V、K320A、S323F/A/T。

4. the Saccharomyces cerevisiae according to claim 3, wherein the mutant CCD2 protease has S323F/A/T site mutation and/or has one or more of the following site mutations:

V120F、Y190K/A、R192V/F、E211A、E212A、T290V、K320A。

5. the Saccharomyces cerevisiae according to claim 1, wherein the ALD protease is derived from Synechocystis.

6. The Saccharomyces cerevisiae of claim 1, wherein the genes encoding CCD2 and ALD protease are integrated at the HO integration site of Saccharomyces cerevisiae chromosome.

7. Use of the saccharomyces cerevisiae according to any of the claims 1-6 for the synthesis of crocetin.

8. The method for constructing Saccharomyces cerevisiae as claimed in claim 1, wherein HIS tag sequence is fused to N segment of CCD2 protease coding gene, then homology arm is designed to integrate to initial Saccharomyces cerevisiae chromosome, ALD protease coding gene is integrated to initial Saccharomyces cerevisiae chromosome in the same way to obtain Saccharomyces cerevisiae; or

The HIS tag sequence was fused to the N-fragment of the CCD2 protease-encoding gene, then ligated to a vector plasmid and transformed into the original s.cerevisiae, and the gene encoding ALD protease was ligated to the vector plasmid and transformed into the original s.cerevisiae in the same manner to obtain the s.cerevisiae.

9. A method for synthesizing crocetin, characterized in that the saccharomyces cerevisiae of any one of claims 1-6 is adopted for fermentation synthesis of crocetin, if the saccharomyces cerevisiae can synthesize zeaxanthin and/or beta-carotene by itself, zeaxanthin and/or beta-carotene can be not added or is slightly added in a fermentation medium; if the Saccharomyces cerevisiae itself is not capable of synthesizing zeaxanthin and/or beta-carotene, then zeaxanthin and/or beta-carotene needs to be added to the fermentation medium.