CN112391402B

CN112391402B - Method for improving expression level of target protein in yarrowia lipolytica

Info

Publication number: CN112391402B
Application number: CN202011283106.9A
Authority: CN
Inventors: 闫云君; 周清华; 焦梁成; 李文娟; 李云翀; 张后今; 阎金勇; 徐莉; 杨敏
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2020-11-17
Filing date: 2020-11-17
Publication date: 2023-03-28
Anticipated expiration: 2040-11-17
Also published as: CN112391402A

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a method for improving the expression level of a target protein in yarrowia lipolytica. Co-overexpression preferably comprises any of the following: (i) Co-overexpression of a single copy gene of a target protein gene and an accessory protein; (ii) Co-overexpression of a gene of a target protein and a multicopy gene of an accessory protein; (iii) Co-overexpression of a gene of a protein of interest with different copy number genes of various accessory proteins. The invention improves the whole action mechanism of the lifting method, and improves the expression level of the target protein in the yarrowia lipolytica by jointly over-expressing the protein to synthesize the auxiliary protein related to the secretory pathway, thereby obviously improving the yield of the target protein in the yarrowia lipolytica.

Description

Method for improving expression level of target protein in yarrowia lipolytica

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for improving the expression level of a target protein in yarrowia lipolytica, which is used for improving the expression level of the target protein in yarrowia lipolytica by co-overexpressing proteins to synthesize an accessory protein related to a secretory pathway.

Background

Yarrowia lipolytica is a widely used general purpose recombinant as safe (GRAS) non-conventional yeast, and many subtypes of the yeast have been sequenced, and currently over 180 proteins have been successfully expressed in this host. The expression level of the target protein in the genetic engineering bacteria is influenced by various factors, such as codon preference, promoters, signal peptides, gene dosage, protein synthesis and secretion, fermentation culture processes and the like. At present, the research for improving the expression level of target proteins in yarrowia lipolytica mainly focuses on codon preference, promoters, gene dosage, engineering bacteria culture process and the like, but a method for improving the expression level of the target proteins by optimizing protein synthesis and secretion pathways is lacked.

In yarrowia lipolytica, a number of cofactors are involved in protein synthesis and secretion. For example, the endoplasmic reticulum-resident protein Kar2 belonging to the Hsp70 protein family can bind to and protect the structure of polypeptides and provide an energy source for the polypeptides to cross the endoplasmic reticulum; the cofactor Sls1 of Kar2 can regulate the activity of Kar2 and is directly involved in the process of protein co-translation translocation. In addition, kar2 can also bind to the multifunctional type I transmembrane protein Ire1, thereby affecting Unfolded Protein Response (UPR) in cells; while Sls1 may promote the transition of Kar2 from ADP to ATP binding to facilitate the interaction between Kar2 and Ire 1.

Cofactors in the protein synthesis and secretion pathways can act as sensors and effectors at each quality control point to ensure homeostasis of the overall process and normal processing and maturation of proteins. However, in order to obtain higher protein yield, the target protein in the engineering bacteria usually needs to be over-expressed, but the content of the accessory factors involved in protein synthesis and secretion is limited, so that the steady state of protein synthesis and secretion is difficult to maintain, and the expression amount of the target protein in the engineering bacteria is limited. Therefore, overexpression of protein synthesis and secretion-related auxiliary proteins in the target genetically engineered bacterium to increase the content thereof may help to alleviate the above limitations and further increase the expression level of the target protein.

Disclosure of Invention

In view of the above-mentioned drawbacks or needs for improvement of the prior art, the present invention aims to provide a method for increasing the expression level of a protein of interest in yarrowia lipolytica, wherein the production of the protein of interest in yarrowia lipolytica can be significantly increased by improving the overall mechanism of action of the lifting method, by co-overexpressing protein synthesis accessory proteins associated with the secretory pathway to increase the expression level of the protein of interest in yarrowia lipolytica.

To achieve the above object, according to the present invention, there is provided a method for increasing the expression level of a target protein in yarrowia lipolytica, characterized in that the expression level of the target protein in yarrowia lipolytica is increased by coexpressing the gene for the target protein and the gene for an accessory protein involved in protein synthesis and secretion pathway.

As a further preference of the present invention, the co-overexpression specifically includes any of the following cases:

(i) Co-overexpression of a single copy gene of a target protein gene and an accessory protein;

(ii) Co-overexpression of a target protein gene and a multicopy gene of an accessory protein;

(iii) Co-overexpression of a gene of a protein of interest with different copy number genes of various accessory proteins.

As a further preferred aspect of the present invention, the co-overexpression is preferably co-overexpression of a gene of a protein of interest and different copy number genes of a plurality of helper proteins.

As a further preferred aspect of the present invention, the protein synthesis accessory protein associated with the secretory pathway can be directly or indirectly involved in the protein synthesis and secretory pathway of yarrowia lipolytica.

As a further preference of the invention, the accessory protein is preferably selected from the group consisting of Sls1, kar2, pdi, hac1, ire1, hrd1, bmh2.

As a further preferred aspect of the invention, the protein of interest is a yarrowia lipolytica homologous protein or a heterologous protein.

Through the technical scheme, compared with the prior art, the expression level of the target protein in the yarrowia lipolytica can be effectively improved through co-overexpression of the target protein gene and the single copy gene of at least one auxiliary protein. Based on the invention, the gene of the auxiliary protein related to the protein synthesis and secretion pathway in the yarrowia lipolytica can be integrated into the engineering bacteria containing the target gene, so that the over-expression of the target gene and the auxiliary protein gene is realized, and the expression level of the target protein in the yarrowia lipolytica is improved by over-expressing the auxiliary protein related to the protein synthesis and secretion pathway.

Compared with expression systems such as saccharomyces cerevisiae, protein expression examples and research on protein synthesis and secretion pathways in yarrowia lipolytica are fewer, and the problems of insufficient availability of screening markers, low efficiency of site-specific marker-free integration and the like further limit the progress of related research in yarrowia lipolytica. The invention selects the auxiliary protein directly or indirectly participating in the protein synthesis and secretion pathway in the yarrowia lipolytica, and respectively applies the integration method and the conventional integration method in the patent 'a system and method for integrating target genes in the yarrowia lipolytica without trace for multiple times' (Chinese patent application number: 201910747704.8) to jointly over-express the auxiliary protein and the target protein, thereby obviously improving the expression level of the target protein of the yarrowia lipolytica, making up the defects of relevant strategy methods in the host and laying a solid foundation for the development and the improvement of the protein expression system of the yarrowia lipolytica.

Drawings

FIG. 1 is a map of the plasmids Cre-Y2-sls1, cre-Y3-sls1, cre-Y2-kar2 and Cre-Y3-kar 2. Wherein Ura3 is a yarrowia lipolytica uracil-deficient selection marker; pPOX2-cre-lip2t is a cre gene expression cassette induced by oleic acid; lox71 is a left arm mutant lox site, lox66 is a right arm mutant lox site, and a triangular arrow indicates the sequence direction; hp12d-sls1-XPR2t is an expression cassette of an accessory protein gene sls1, and hp12d-kar2-XPR2t is an expression cassette of an accessory protein gene kar2; leu2 is taken as a homologous fragment of the Leu2 gene of the genome integration site; amp-ori is Escherichia coli ampicillin resistance gene expression box and plasmid replication origin.

FIG. 2 is a schematic diagram of the enzyme activity of strain FY5-4rml-sls 1.

FIG. 3 is a diagram showing the enzyme activities of the strains FY5-4rml-kar2 and FY5-4rml-2kar2.

FIG. 4 is a diagram showing the enzyme activity of strain FY5-4rml-kar2-sl 1.

FIG. 5 is a map of plasmid Ura-kar 2. Wherein Ura3 is a yarrowia lipolytica uracil auxotrophic screening marker and is used as a homologous fragment of a genome integration site; pTEF-kar2-lip2t is an auxiliary protein gene expression box; amp-ori is Escherichia coli ampicillin resistance gene expression box and plasmid replication origin.

FIG. 6 is a schematic diagram of the enzyme activity of the strain Po1f-3rol-kar2.

FIG. 7 is a graph showing comparison of enzyme activities of strains FY5-4rml, FY5-4rml-sls1, FY5-4rml-kar2, FY5-4rml-2kar2, and FY5-4rml-kar2-sls1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The invention takes Rhizomucor miehei lipase (RML, uniProtKB/Swiss-Prot: P19515) and Rhizopus miehei lipase (ROL, uniProtKB/Swiss-Prot: G8I0Z 0) as research objects to verify the influence of commonly over-expressed auxiliary protein Sls1 (UniProtKB/Swiss-Prot: Q99158) and Kar2 (UniProtKB/Swiss-Prot: Q99170) on the expression level of target protein in yarrowia lipolytica. In the invention, an RML initial strain FY5-4RML is constructed and stored by the laboratory, the strain is derived from yarrowia lipolytica Po1f and contains a gene expression cassette with 4 copies of RML, and the specific construction process is shown in the patent 'a system and a method for integrating a target gene in yarrowia lipolytica in a traceless way for multiple times'; the initial ROL strain Po1f-3ROL is constructed and stored in the laboratory, and the strain is obtained by transferring Po1f into a recombinant plasmid of a gene expression cassette containing 3 copies ROL and derived from a commercial vector pINA 1296. The plasmids used in the present invention were stored in the laboratory and their maps are shown in FIGS. 1 and 5. The plasmid used in the invention is transferred into yeast competent cells by a common lithium acetate conversion method in yarrowia lipolytica, and after screening and verification, the obtained positive transformant is fermented and cultured for 120h by BMSY culture medium (sorbitol 50g/L, yeast powder 10g/L, peptone 20g/L, phosphate buffer solution 100mM, amino-free yeast 13.4g/L and biotin 0.4 mg/L) and then supernatant is collected. RML and ROL lipase activities were determined by basic titration, i.e.lipase activity was determined by monitoring the yield of free fatty acids released by the lipase using olive oil as substrate. Three measurements per sample were averaged and one unit of enzyme activity (U) was defined as the amount of enzyme required for the lipase to hydrolyze olive oil to 1. Mu. Mol free fatty acids per minute.

The following are specific examples:

example 1

Co-expression of Sls1 increased the expression level of RML in yarrowia lipolytica

The plasmid Cre-Y2-sls1 (the plasmid map is shown in figure 1) is linearized by a restriction enzyme ApaI at Leu2 and then is transformed into a strain FY5-4rml, and the screening and inducing processes of the transformed strain adopt the method of the patent 'a system and a method for integrating a target gene in yarrowia lipolytica for multiple times without scars'. Positive transformants are selected as uracil-deficient transformants, and plasmid fragments of the non-target gene are recovered by the Cre/lox system. The target engineering strain obtained after screening verification is named as FY5-4rml-sls1, and the lipase activity of the supernatant after shaking flask fermentation is compared with FY5-4rml and is shown in figure 2. As can be seen from FIG. 2, the enzyme activity of RML is increased from 180U/mL to 210U/mL by coexpression of Sls1, which is improved by 17% compared with that of the initial strain FY5-4RML, and the result shows that the coexpression of the helper protein Sls1 can promote the expression of the target protein RML in yarrowia lipolytica.

Example 2

Co-expression of Kar2 increases the expression levels of RML in yarrowia lipolytica

The plasmid Cre-Y2-kar2 (a plasmid map is shown in figure 1) is linearized at Leu2 by a restriction enzyme ApaI and then is transformed into a strain FY5-4rml, the screening and inducing processes of the transformed strain both adopt the method of a system and a method for integrating a target gene in yarrowia lipolytica for multiple times without scars, and the target engineering strain obtained after screening verification is named as FY5-4rml-kar2. The plasmid Cre-Y3-kar2 (the plasmid map is shown in figure 1) is linearized at Leu2 by a restriction enzyme ApaI and then is transferred into the strain FY5-4rml-kar2 again, the screening and induction processes of the transformed strain both adopt the method of the patent 'a system and a method for multiple traceless integration of target genes in yarrowia lipolytica', and the target engineering strain obtained after screening verification is named FY5-4rml-2kar2. The results of the enzyme activities of the two engineering strains in the shake flask fermentation are shown in figure 3, and the enzyme activity of RML can be improved from 180U/mL to 258U/mL only by co-expressing 1 additional kar2 gene, and the improvement proportion reaches 43 percent; when 2 copies of kar2 genes are co-transformed, the RML enzyme activity expressed by the engineering bacteria FY5-4RML-2kar2 is further improved to 276U/mL, which is improved by 7 percent compared with the strain FY5-4RML-kar2 and is improved by 53 percent compared with the initial strain FY5-4 RML. It can be seen that co-expression of Kar2 can promote the production of RML in yarrowia lipolytica.

Example 3

Co-expression of Kar2 and Sls1 simultaneously increased the expression level of RML in yarrowia lipolytica

The plasmid Cre-Y3-sls1 (a plasmid map is shown in figure 1) is linearized at Leu2 by a restriction enzyme ApaI and then is transformed into a strain FY5-4rml-kar2, the screening and induction processes of the transformed strain both adopt the method of a system and a method for multiple traceless integration of a target gene in yarrowia lipolytica, and a target engineering strain obtained after screening verification is named as FY5-4rml-kar2-sls1. As shown in FIGS. 4 and 7, when Kar2 and Sls1 are co-expressed, the enzyme activity of the engineering bacteria reaches 327U/mL, is improved by 82% compared with the strain FY5-4rml, is improved by 27% compared with the strain FY5-4rml-Kar2, and is improved by 56% compared with the strain FY5-4rml-Sls 1. Thus, co-expression of Kar2 and Sls1 in combination can further increase the expression level of RML in yarrowia lipolytica.

Example 4

Co-expression of Kar2 increases the expression level of ROL in yarrowia lipolytica

The plasmid Ura-kar2 (see FIG. 5 for plasmid map) was digested with restriction enzyme BstXI and the fractions containing the kar2 expression cassette were recovered, and then transformed into strain Po1f-3rol competent cells and coated with uracil deficient plates using a general lithium acetate transformation method (see, for example, chen DC, beckerrich JM, gaillardin C.1997.One-step transformation of the dimorph yeast Yarrowia lipolytica. Appl Microbiol 48. The enzyme activity is shown in figure 6, the enzyme activity of ROL in the engineering bacteria Po1f-3ROL-Kar2 is 3680U/mL by co-expression Kar2, which is improved by 28% compared with the bacterial strain Po1f-3ROL (2870U/mL), and the co-expression auxiliary protein Kar2 can promote the expression of ROL in yarrowia lipolytica.

In the above embodiments, sls1 and Kar2 are taken as examples, and the accessory protein may also be a protein with important functions in other protein synthesis and secretion pathways, such as Pdi, hac1, ire1, hrd1, bmh2, and the like. Where the invention has not been described in detail reference is made to the relevant prior art.

It will be understood by those skilled in the art that the foregoing is only an exemplary embodiment of the present invention, and is not intended to limit the invention to the particular forms disclosed, since various modifications, substitutions and improvements within the spirit and scope of the invention are possible and within the scope of the appended claims.

Claims

1. A method for increasing the expression level of a protein of interest in yarrowia lipolytica by co-overexpressing a gene for the protein of interest and a gene for an accessory protein involved in the protein synthesis and secretion pathway;

wherein the co-overexpression is the co-overexpression of a target protein gene and different copy number genes of a plurality of auxiliary proteins; the multiple accessory proteins are Sls1 and Kar2; the target protein is Rhizomucor Miehei Lipase (RML).

2. The method of claim 1, wherein the protein synthesis and secretion pathway associated accessory proteins are involved directly or indirectly in the protein synthesis and secretion pathway of yarrowia lipolytica.

3. The method of claim 1 or 2, wherein the protein of interest is a yarrowia lipolytica homologous protein or a heterologous protein.