Genetically engineered bacterium of high-yield lipase as well as preparation method and application of genetically engineered bacterium
By selecting specific integration sites in Pichia cerevisiae, and combining signal peptide optimization, multi-copy integration and molecular chaperone co-expression, a genetically engineered strain with high lipase yield was constructed, solving the problem of insufficient ROL expression level, and achieving efficient secretion expression and good enzyme activity level.
Patent Information
- Application Number
- CN202411810949.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-10
- Publication Date
- 2025-03-21
AI Technical Summary
In the prior art, the expression level of ROL in Pichia cerevisia cannot meet industrial needs, and there are few studies on the expression of recombinant proteins in the optimization of integration site.
Genetically engineered strains of high-yield lipase yield were constructed by selecting specific integration sites on chromosomes 3 or 4 of Pichia yeast, and combining signal peptide optimization, multi-copy integration and chaperone co-expression.
The efficient secretion and expression of lipase in Pichia yeast was achieved, with a yield of 8.42 mg/mL and a good enzyme activity level, overcoming the shortcoming of low expression in the prior art.
Smart Images
Figure CN119662433A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of genetic engineering, and specifically relates to a genetic engineering bacterium with high lipase production, and a preparation method and application thereof. Background Art
[0002] Lipase (EC 3.1.1.3, lipase) is a general term for hydrolases that use triglycerides as substrates. It can catalyze the hydrolysis of triglycerides to produce corresponding fatty acids, diglycerides, monoglycerides and glycerol. Lipases from Rhizopus microorganisms are one of the important sources of microbial lipase resources. At present, more than 30 Rhizopus lipases have been successfully isolated, including Rhizopus oryzae lipase (ROL), Rhizopus arrhizus lipase and Rhizopus chinensis lipase. Among them, ROL has high 1,3-position specificity, excellent stereoselectivity and stability, and has broad application prospects in industrial fields such as oil processing, chiral compound separation and biodiesel preparation. However, the natural yield of ROL is extremely low, which is difficult to meet industrial needs. Efficient heterologous secretory expression through the Pichia pastoris expression system is an important means to solve this problem.
[0003] Pichia pastoris is one of the most commonly used microbial cell factories. It is a eukaryotic expression system for foreign proteins jointly developed by Salk Institute Biotechnology / Industrial Associates and Phillips Petroleum in the early 1980s. The Pichia pastoris expression system has many advantages: (1) It can use methanol as the only carbon source and energy source; (2) It has a variety of expression hosts and vectors, and genetic manipulation is relatively simple; (3) It has an alcohol oxidase promoter that can be strictly induced and regulated by methanol; (4) Foreign genes are integrated into the genome through homologous recombination single exchange or double exchange, which can effectively avoid foreign gene loss; (5) The culture conditions are relatively simple and it is easy to culture to a high cell density; (6) It can perform complex protein post-translational modifications, which is conducive to the folding and secretion of foreign genes; (7) The secretion of impurities is relatively small, which is conducive to subsequent separation and purification. At present, Pichia pastoris has achieved the active expression of more than 5,000 proteins from fungi, bacteria, plants, animals and viruses. As a cell factory for producing foreign proteins, it has played a huge role in the production of enzymes, vaccines and protein polymers.
[0004] In previous studies, we found that the heterologous secretory expression level of ROL after glycosylation modification in Pichia pastoris could not meet industrial needs. Generally speaking, when the Pichia pastoris expression system is secreted, the total amount of exogenous protein produced usually does not exceed 5% of the total intracellular protein, so there is still a lot of room for improvement in the expression level of ROL in Pichia pastoris. At present, the strategies to improve the exogenous secretory expression level of Pichia pastoris mainly include: optimization of signal peptides, promoters and terminators; multi-copy integration of target genes; co-expression of molecular chaperones, etc. In addition, the study also found that the choice of integration site has an important influence on the expression efficiency of Pichia pastoris. However, the current research on integration site optimization is mostly concentrated in the field of metabolic engineering, while there are relatively few related studies on recombinant protein expression. Summary of the invention
[0005] In view of the need for improving the expression level of lipase ROL in Pichia pastoris in the prior art, the present invention provides a genetically engineered bacterium with high lipase production and a preparation method and application thereof. The specific technical scheme is as follows:
[0006] In a first aspect, the present invention provides a genetically engineered bacterium, wherein the starting strain is Pichia pastoris, a lipase gene is inserted into the genome of the Pichia pastoris, and the insertion site of the lipase gene is between PAS_chr3_1156 and PAS_chr3_0154 genes on chromosome 3 of Pichia pastoris or between PAS_chr4_0465 and PAS_chr4_0467 genes on chromosome 4;
[0007] The nucleotide sequence of the lipase gene is shown in SEQ ID NO.1.
[0008] Furthermore, 1 to 5 copies of lipase gene are inserted into the genetically engineered bacteria.
[0009] Furthermore, four copies of lipase gene are inserted into the genetically engineered bacteria.
[0010] Furthermore, the upstream of the lipase gene is connected to the α-MF signal peptide gene of Pichia pastoris; the nucleotide sequence of the α-MF signal peptide gene is shown in SEQ ID NO.3; and the expression product of the α-MF signal peptide gene is connected to the N-terminus of the expression product of the lipase gene.
[0011] Furthermore, the genetically engineered bacteria have inserted therein a gene fragment encoding a molecular chaperone Pdi and a gene fragment encoding a molecular chaperone Sbh1.
[0012] Furthermore, the gene encoding the molecular chaperone Pdi and the gene encoding the molecular chaperone Sbh1 are inserted into the genetically engineered bacteria; the copy number of the gene encoding the molecular chaperone Pdi is 2, and the copy number of the gene encoding the molecular chaperone Sbh1 is 1.
[0013] In a second aspect, the present invention provides a method for preparing the above-mentioned genetically engineered bacteria, comprising the following steps:
[0014] (1) Four copies of the lipase gene with an α-MF signal peptide were constructed between the PAS_chr3_1156 and PAS_chr3_0154 genes using the CRISPR-Cas9 method;
[0015] (2) Using the pAO815 plasmid as the original expression vector, two copies of the coding gene fragment of the molecular chaperone Pdi with a GAP promoter and terminator and one copy of the coding gene fragment of the molecular chaperone Sbh1 with a GAP promoter and terminator were inserted to construct a molecular chaperone combination gene expression vector, which was then inserted into the His4 integration site of the strain in (1) by homologous recombination to obtain a genetically engineered bacterium.
[0016] In a third aspect, the present invention provides a method for producing lipase, wherein the genetically engineered bacteria as described in any one of claims 1 to 5 or the genetically engineered bacteria prepared by the preparation method as described in claim 6 are used as fermentation bacteria to produce lipase.
[0017] Furthermore, during the fermentation process, OD 600 The temperature is 450~500℃ and the fermentation time is 100~150h.
[0018] Furthermore, during the fermentation process, glycerol is added after the first dissolved oxygen rebound; methanol is added after the dissolved oxygen rebounds again. The dissolved oxygen rebound means that during the fermentation process, microorganisms multiply rapidly at the beginning of the fermentation, the oxygen demand increases, resulting in a rapid decrease in dissolved oxygen concentration. As the fermentation proceeds, the dissolved oxygen concentration begins to rise. After feeding, the fermentation process continues, the oxygen demand increases, and the dissolved oxygen concentration decreases. As the fermentation proceeds, the dissolved oxygen concentration begins to rise again. When the dissolved oxygen concentration rises, it is called dissolved oxygen rebound.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The present invention utilizes the Pichia pastoris eukaryotic expression system to produce lipase ROL, and improves the secretion expression level of lipase in Pichia pastoris by adjusting the integration site of the lipase ROL gene in the Pichia pastoris genome, thereby achieving a better effect. The yield of the lipase high-yield strain prepared by the present invention reaches 8.42 mg / mL, and has a good enzyme activity level, thereby overcoming the deficiency of low expression of related lipase expressed by existing Pichia pastoris genetically engineered bacteria. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a schematic diagram of SDS-PAGE analysis of protein expression in the fermentation supernatant of strains with different integration sites; among them, Int1~Int7 represent different integration sites.
[0022] Figure 2 Schematic diagram of SDS-PAGE analysis of protein expression in the fermentation supernatant of strains with different copy numbers of ROL genes.
[0023] Figure 3 Schematic diagram of SDS-PAGE analysis of protein expression in the supernatant of co-expressing molecular chaperone strains; wherein, Sbh1~Sso2 represent Pichia pastoris with 4 copies of ROL gene inserted using different molecular chaperones, and I3-4ROL represents Pichia pastoris with 4 copies of ROL gene inserted.
[0024] Figure 4 Schematic diagram of SDS-PAGE analysis of protein expression in supernatant of molecular chaperones Pdi and Sbh1 combination and multi-copy strains; wherein, I3-4ROL represents Pichia pastoris with 4 copies of ROL gene inserted, 2Pdi-Sbh1 represents Pichia pastoris with 4 copies of ROL gene, 2 copies of Pdi gene and 1 copy of Sbh1 gene inserted, Pdi-Sbh1 represents Pichia pastoris with 4 copies of ROL gene, 1 copy of Pdi gene and 1 copy of Sbh1 gene inserted, and 2Pdi represents Pichia pastoris with 4 copies of ROL gene and 2 copies of Pdi gene inserted. DETAILED DESCRIPTION
[0025] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.
[0026] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.
[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.
[0028] In the present application, a lipase derived from Rhizopus oryzae that has been glycosylated is used. The nucleotide sequence of the lipase gene is shown in SEQ ID NO.1, and the amino acid sequence of the lipase is shown in SEQ ID NO.2.
[0029] SEQ ID NO.1:
[0030]
[0031] SEQ ID NO.2:
[0032] VSGKSGSSNTAVSASDNAALPPLISSRCAPPSNKGSKSDLQAEPYNMQKNTEWYESHGGNTSIGKRDDNLVGGMTLDLPSDAPPISLSSSTNSASDGGKVVAATTAQIQEFTKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSFR SAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVVQEQLTAHPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVGGPRVGNPTFAYYVCSTGIPFQRTVHKRDIVPHLPPQSFGFLHPGVESWIKNGTSNVQICTSCIETKDCSNSIVPFTSILDHLSYFGINEGSCL.
[0033] In the present application, the enzyme activity is defined as the amount of enzyme required to catalyze the substrate to generate 1 μmol pNP per minute.
[0034] Example 1 Selection and Optimization of Signal Peptides
[0035] In this application, the lipase derived from Rhizopus oryzae after glycosylation modification was selected for use.
[0036] As shown in Table 1, 13 Pichia pastoris homologous secretory signal peptides were selected: α-MF, PIR1, PRY2, GAS1, MSB2, FRE2, DAN4, FLO10, DSE4, PHO5, FLO9, BGL2, EXG1, of which α-MF is carried by the pPICZα plasmid, and the remaining signal peptides were amplified by designing corresponding signal peptide primers with Pichia pastoris as a template, and were connected to the pPICZ plasmid after being digested with BstB I and EcoR I. Subsequently, the glycosylated ROL (SEQ ID NO.1) was constructed on all plasmids, and after linearization, it was integrated into the genome of Pichia pastoris GS115 by traditional homologous recombination.
[0037] After the corresponding strains were induced by methanol, the enzyme activity of the supernatant was determined by the pNPP method to characterize the secretion expression level of lipases with different signal peptides in Pichia pastoris, and the results are shown in Table 1. Among them, the α-MF signal peptide had the best enzyme activity, reaching 61.26 U / mL, so the α-MF signal peptide was selected as the preferred signal peptide for subsequent optimization experiments.
[0038] Table 1 Selection of lipase signal peptides and their enzyme activity levels
[0039]
[0040] Example 2 Integration site selection
[0041] The lipase with α-MF signal peptide was inserted into seven different integration sites (as shown in Table 2) and integrated by CRISPR-Cas9 method. The Cas9 gene was first transformed into Pichia pastoris GS115 in the form of a free plasmid, and then the gRNA plasmid and donor DNA were constructed respectively. The gRNA plasmid and donor DNA were then transformed into Pichia pastoris with CAS9 gene. Under the guidance of gRNA, Cas9 destroyed the specific site on the genome, and the donor DNA contained the site homology arm, which underwent homologous recombination repair with the genome, thereby integrating the target gene ROL into the specific site of the genome. Finally, Pichia pastoris strains with 7 different integration sites were successfully constructed.
[0042] In order to characterize the secretory expression level of the lipase gene in Pichia pastoris, the lipase activity of different integration sites was first determined by the pNPP method, and the results are shown in Table 2. The lipase activity of the Int3 and Int5 insertion sites was improved compared with the traditional AOX1 insertion site, especially Int3, where the enzyme activity increased from 61.26 U / mL to 97.89 U / mL, an increase of 59.8%. Figure 1 The protein gel characterization shown in the figure shows that the secretory expression level of Int3 is significantly improved, indicating that the selection of a suitable integration site can significantly improve the secretory expression level of Pichia pastoris.
[0043] Table 2 Lipase integration site selection and enzyme activity level in Pichia pastoris
[0044]
[0045] Example 3 Construction of multiple copies of lipase
[0046] The CRISPR-Cas9 method was used to construct 1-5 copies of the ROL gene at the Int3 integration site. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) in the Pichia pastoris GS115 genome was used as a control gene, and the ROL copy number was verified by fluorescent quantitative PCR. It was verified that the Pichia pastoris expression strain containing 1-5 copies was successfully constructed.
[0047] The supernatant enzyme activity was determined by the pNPP method after methanol induction culture, and the results are shown in Table 3. As the copy number increased, the enzyme activity of the supernatant gradually increased, reaching the highest at 4 copies, which was 148.79 U / mL, 1.52 times that of a single copy. After 5 copies, the enzyme activity began to decrease. Figure 2 The protein gel image shown shows that the 4-copy strain has the highest secretory expression level. Therefore, 4 copies were selected as the optimal copy number and the strain was named I3-4ROL.
[0048] Table 3 Construction of multi-copy lipase in Pichia pastoris and its enzyme activity level
[0049]
[0050] Example 4 Co-expression of Molecular Chaperones
[0051] The present invention selects 14 molecular chaperones for co-expression: Pdi, Bip, Ero1, Cne1, Sec53, Hrd1, Ubc1, Ssa4, Sso2, Bmh2, Sly1, Sbh1, Kar2p and Ydj1p. The 14 molecular chaperones are amplified by designing corresponding sequences using the genome as a template, and are connected to the pGAPZ plasmid after enzyme digestion. After the plasmid is linearized, it is integrated into the strain I3-4ROL obtained in Example 3 by traditional homologous recombination.
[0052] The supernatant enzyme activity was determined by methanol induction culture using the pNPP method. The results are shown in Table 4. The enzyme activity of the strain co-expressing the molecular chaperones Pdi, Cne1, Hrd1, Sso2, and Sbh1 was higher than that of I3-4ROL, with the highest being Pdi and Sbh1, reaching 193.91 U / mL and 195.75 U / mL, respectively. The supernatant of the co-expressing molecular chaperone strain with improved enzyme activity was analyzed by SDS-PAGE. Figure 3 As shown, it was found that co-expression of Pdi and Sbh1 had a significant effect on improving the secretory expression of lipase in Pichia pastoris.
[0053] Table 4 Enzyme activity levels in the supernatant of co-expressing molecular chaperone strains
[0054]
[0055] Example 5 Combination Expression of Molecular Chaperones
[0056] Pdi and Sbh1 were expressed in combination and in multiple copies. pGAPZ-Pdi and pGAPZ-Sbh1 constructed in Example 4 were digested with BglII and BamHI to obtain molecular chaperone fragments GAP-Pdi and GAP-Sbh1 with GAP promoter and terminator. After the plasmid pAO815 was digested with BamHI, the fragments GAP-Pdi and GAP-Sbh1 were connected respectively to obtain plasmids pAO815-Pdi and pAO815-Sbh1, and then the above two plasmids were digested with BamHI again, and the fragments GAP-Pdi and GAP-Sbh1 were continued to be connected. In this way, 2 copies of Pdi plasmid pAO815-2Pdi, 2 copies of Sbh1 plasmid pAO815-2Sbh1, Pdi and Sbh1 combination plasmid pAO815-Pdi-Sbh1, 2 copies of Pdi and Sbh1 combination plasmid pAO815-2Pdi-Sbh1 and Pdi and 2 copies of Sbh1 combination plasmid pAO815-Pdi-2Sbh1 were finally constructed. Finally, the plasmid was linearized with SalI and integrated into the strain I3-4ROL genome prepared in Example 3 by traditional homologous recombination.
[0057] The supernatant enzyme activity was determined by methanol-induced culture using the pNPP method, and the results are shown in Table 5. The molecular chaperones Pdi and Sbh1 were expressed in two copies, and the enzyme activity of the two-copy Pdi strain was higher than that of the single copy, while the enzyme activity of the two-copy Sbh1 was reduced. After the combined expression of Pdi and Sbh1, the enzyme activity was further improved compared to the expression of a single molecular chaperone. Finally, after trying to combine Pdi and Sbh1 and express multiple copies, it was found that the highest enzyme activity was obtained after the combination of two copies of Pdi and Sbh1, and the enzyme activity reached 233.91U / mL, which was 57% higher than that of the I3-4ROL strain. The strain was named I3-4ROL-2Pdi-Sbh1. The supernatant of the strain with improved enzyme activity was subjected to SDS-PAGE analysis, as shown Figure 4 As shown, it was found that the lipase expression level in the supernatant of the I3-4ROL-2Pdi-Sbh1 strain was significantly higher than that of the I3-4ROL strain.
[0058] Table 5 Enzyme activity levels in supernatants of molecular chaperones Pdi and Sbh1 combinations and multi-copy strains
[0059]
[0060] Example 6 High-density fermentation in fermenter
[0061] The strain I3-4ROL-2Pdi-Sbh1 and the strain ROL before transformation were fermented at high density in a 15L fermenter to characterize the secretory expression level. The fermentation medium was 6L FM22 medium, pH 5.0, and the fermentation temperature was 30°C. After the inoculation and fermentation until the first dissolved oxygen rebound, glycerol was fed. After the feeding was completed, the dissolved oxygen rebounded again and starvation treatment was performed. Then, the methanol feeding induction stage was started. The fermentation was stopped until the enzyme activity began to decrease.
[0062] During the 15L high-density fermentation, the total fermentation time of strain I3-4ROL-2Pdi-Sbh1 and strain ROL before transformation was about 120 h, and the OD 600 Both are about 480. The fermentation supernatant of the strain was determined by the pNPP method. The ROL of the strain before the transformation was 979.31U / mL, while the supernatant enzyme activity of the strain I3-4ROL-2Pdi-Sbh1 reached 4524.41U / mL, which was 4.62 times that of the starting strain. The protein concentrations of the supernatants of the two strains were determined. The protein concentration of strain ROL was 1.72mg / mL, and the supernatant protein concentration of strain I3-4ROL-2Pdi-Sbh1 reached 8.42mg / mL, which was 4.89 times that of the starting strain. The results of 15L high-density fermentation showed that the combination of strategies such as signal peptide optimization, integration site optimization, multiple copies of the target gene, and molecular chaperone co-expression can effectively improve the secretory expression level of the lipase ROL after glycosylation modification in Pichia pastoris.
Claims
1. A genetically engineered bacterium, characterized in that: The starting strain is Pichia pastoris, and a lipase gene is inserted into the genome of the Pichia pastoris. The insertion site of the lipase gene is between genes PAS_chr3_1156 and PAS_chr3_0154 on chromosome 3 of the Pichia pastoris or between genes PAS_chr4_0465 and PAS_chr4_0467 on chromosome 4; The nucleotide sequence of the lipase gene is shown in SEQ ID NO.
1.
2. The genetically engineered bacterium according to claim 1, characterized in that: The copy number of the lipase gene is 1-5.
3. The genetically engineered bacterium according to claim 2, characterized in that: The α-MF signal peptide gene of Pichia pastoris is inserted into the genome of the Pichia pastoris; the nucleotide sequence of the α-MF signal peptide gene is shown in SEQ ID NO.3; the expression product of the α-MF signal peptide gene is connected to the N-terminus of the expression product of the lipase gene.
4. The genetically engineered bacterium according to claim 3, characterized in that: The genetically engineered bacteria are also inserted with a gene encoding a molecular chaperone Pdi and / or a gene encoding a molecular chaperone Sbh1.
5. The genetically engineered bacterium according to claim 4, characterized in that: The gene encoding the molecular chaperone Pdi and the gene encoding the molecular chaperone Sbh1 are inserted into the genetically engineered bacteria; the copy number of the gene encoding the molecular chaperone Pdi is 2, and the copy number of the gene encoding the molecular chaperone Sbh1 is 1.
6. The method for preparing a genetically engineered bacterium according to claim 5, characterized in that: The following steps are involved: (1) Four copies of the lipase gene with an α-MF signal peptide were constructed between the PAS_chr3_1156 and PAS_chr3_0154 genes using the CRISPR-Cas9 method; (2) Using the pAO815 plasmid as the original expression vector, two copies of the coding gene fragment of the molecular chaperone Pdi with a GAP promoter and terminator and one copy of the coding gene fragment of the molecular chaperone Sbh1 with a GAP promoter and terminator were inserted to construct a molecular chaperone combination gene expression vector, which was then inserted into the His4 integration site of the strain in (1) by homologous recombination to obtain a genetically engineered bacterium.
7. A method for producing lipase, characterized in that: The genetically engineered bacteria according to any one of claims 1 to 5 or the genetically engineered bacteria prepared by the preparation method according to claim 6 is used as a fermentation bacterium to carry out fermentation to produce lipase.
8. The method according to claim 7, characterized in that During the fermentation process, OD 600 The temperature is 450~500℃ and the fermentation time is 100~150h.
9. The method according to claim 7, characterized in that: During the fermentation process, after the first rebound of dissolved oxygen, glycerol is added; after the second rebound of dissolved oxygen, methanol is added.