A method for expressing amylase in Pichia pastoris
By constructing recombinant plasmids co-expressing translation-related functional genes in Pichia pastoris, the problems of low amylase expression levels and poor enzyme suitability were solved, achieving efficient and safe amylase expression suitable for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
- Filing Date
- 2026-03-12
- Publication Date
- 2026-07-31
AI Technical Summary
In existing technologies, the expression level of amylase by microbial fermentation is low, and the adaptability of enzyme characteristics is poor, making it difficult to meet the needs of industrial production. In addition, the plant extraction method is limited by raw materials and climate, resulting in high costs and making it difficult to achieve large-scale production.
Recombinant plasmids containing translation-related functional genes were constructed in Pichia pastoris, and α-amylase was co-expressed. High-efficiency expression was achieved using the pPIC9K vector through codon optimization and signal peptide deletion. High-expression strains were obtained by screening for bleomycin, focusing on the optimization of the Pichia pastoris translation module.
It significantly increases the expression level of α-amylase, improves enzyme activity by 85.5%~92.5%, is suitable for large-scale industrial production, reduces costs, and has high compatibility and safety.
Smart Images
Figure CN121801870B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a method for expressing amylase in Pichia pastoris. Background Technology
[0002] α-Amylase (α-1,4-glycosidic-D-glycosidic hydrolase, EC 3.2.1.1) is a class of enzymes that randomly cleave the α-1,4-glycosidic bonds within starch and other polysaccharides to produce oligosaccharides of varying lengths. α-Amylase is widely used in the food, feed, and paper industries, and is one of the most widely used enzymes after proteases, accounting for approximately 30% of the entire enzyme market.
[0003] Pichia pastoris is a highly efficient host system for expressing and secreting heterologous proteins. This strain can achieve high-density fermentation (cell dry weight exceeding 150 g / L) using methanol as the sole carbon source, exhibits excellent post-translational modification capabilities, and while efficiently expressing and secreting recombinant proteins, only a very small amount of host proteins are present in the fermentation broth, making it suitable for large-scale industrial production of eukaryotic proteins. In 2006, the U.S. Food and Drug Administration (FDA) designated Pichia pastoris as a generally recognized as safe (GRAS) strain, indicating its widespread safety. Due to its unique advantages, Pichia pastoris has become a leading host in the field of protein expression. Optimization of existing Pichia pastoris protein expression systems mainly focuses on the target gene level, protein folding, transport, and degradation. This includes improving the target gene level through codon optimization, promoter replacement, and increasing copy number; reducing endoplasmic reticulum oxidative stress and promoting protein folding through overexpression of multiple molecular chaperones; enhancing the transport and secretion of target proteins through signal peptide screening; and reducing target protein degradation by knocking out the strain's own proteases, thereby increasing the expression level of target proteins. Pichia pastoris protein expression is usually achieved by using different strategies for different proteins. For example, CN110628790A (publication date: 2019.12.31) describes the construction of a pyranose oxidase gene, protein, Pichia pastoris strain, preparation and application using molecular chaperones; CN109735547A (publication date: 2019.05.10) describes a novel promoter that significantly improves the expression level of exogenous proteins in Pichia pastoris; and CN113528565A (publication date: 2021.10.22) describes a molecular chaperone expression vector and strain for secretory expression in Pichia pastoris to improve phytase.
[0004] Currently, amylase production mainly relies on two routes: microbial fermentation and plant extraction. Plant extraction is limited by raw material origin and climate conditions, and product purification is difficult and costly, making it difficult to meet industrial needs. Microbial fermentation has become the mainstream method due to its high controllability and large-scale potential; however, existing strains generally suffer from low expression levels and poor enzyme compatibility—for example, mesophilic α-amylase has an optimal temperature of 70-80℃, but its activity is reduced by more than 80% in the physiological environment of the animal digestive tract at 37-42℃, while low-temperature amylase, although highly adaptable, faces the bottleneck of low microbial expression efficiency. In existing technologies, researchers have attempted to introduce α-amylase and β-amylase genes into Pichia pastoris and achieve preliminary expression using vectors such as pPIC9K, but the enzyme activity in the fermentation broth is generally below 500 U / mL, far from meeting industrial production requirements. Furthermore, existing technologies have not addressed the strain's translation module in the modification of the Pichia pastoris protein expression system.
[0005] Therefore, it is of great significance to develop a method for efficient expression of amylase in Pichia pastoris. Summary of the Invention
[0006] To address the problems existing in the prior art, this invention provides an engineered Pichia pastoris strain capable of efficiently expressing amylase, as well as a method for its construction.
[0007] On the one hand, the present invention provides a method for expressing amylase in Pichia pastoris, which involves constructing a recombinant plasmid containing translation-related functional genes, introducing it into a Pichia pastoris strain expressing α-amylase, and co-expressing it to obtain a recombinant Pichia pastoris engineered strain.
[0008] Specifically, the translation-related functional genes are selected from at least one of the following: genes encoding protein kinases, genes encoding cytoplasmic RNA-binding proteins, and genes encoding spliceosome proteins.
[0009] Specifically, the nucleotide sequence of the gene encoding the protein kinase is shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding the cytoplasmic RNA binding protein is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the spliceosome protein is shown in SEQ ID NO.3.
[0010] Specifically, the translation-related functional genes are derived from Pichia pastoris GS115.
[0011] Specifically, the method for constructing the Pichia pastoris strain expressing α-amylase includes deleting the signal peptide of the α-amylase sequence, optimizing the codon, inserting it downstream of the signal peptide of the backbone vector, linearizing it, transferring it into host cells, and screening to obtain an initial strain expressing α-amylase.
[0012] Specifically, the host cell is Pichia pastoris cell, the signal peptide is α-factor, and the backbone carrier is pPIC9K.
[0013] Specifically, the amino acid sequence of the α-amylase is shown in SEQ ID NO.4, and the α-amylase is derived from barley.
[0014] Specifically, it also includes the steps of screening and inducing fermentation of the recombinant Pichia pastoris engineered strain, wherein bleomycin is used for screening.
[0015] On the other hand, the recombinant Pichia pastoris engineered strain constructed by the expression method described in this invention co-expresses α-amylase and translation-related functional genes, wherein the translation-related functional genes are selected from at least one gene encoding protein kinase, gene encoding cytoplasmic RNA-binding protein, and gene encoding spliceosome protein.
[0016] Compared with existing technologies, this invention has the following advantages: This invention co-expresses translation-related functional genes in Pichia pastoris expression strains, which can significantly increase the expression level of α-amylase. Compared with the control, the activity of co-expressed protein kinase α-amylase is increased by 85.5%, the activity of co-expressed cytoplasmic RNA-binding protein α-amylase is increased by 106.9%, and the activity of co-expressed spliceosome protein α-amylase is increased by 92.5%. The functional genes used in this method are derived from the host genome, exhibiting strong compatibility and high safety. It focuses on optimizing the Pichia pastoris translation module, filling a gap in existing technologies. The construction process is simple and controllable, adaptable to large-scale industrial production, and reduces costs. Attached Figure Description
[0017] Figure 1 This is a map of the basic plasmid pGAPZαA.
[0018] Figure 2 This is a map of the basic plasmid pPIC9K.
[0019] Figure 3 This is the standard curve for amylase activity detection. Detailed Implementation
[0020] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.
[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0023] Example 1: Construction of the initial strain of Pichia pastoris expressing α-amylase
[0024] The gene sequence of barley α-amylase (the amino acid sequence of α-amylase is shown in SEQ ID NO.4) was artificially synthesized by Anshengda Life Science Technology Co., Ltd. after codon optimization. It was then constructed using a seamless cloning method between the EcoRI and NotI restriction sites in plasmid pPIC9K. The pPIC9K plasmid map is shown below. Figure 2 As shown in the figure, the synthesized recombinant vector pPIC9K-amy was linearized by SacI digestion. The linearized product was purified and transformed into the host bacterium GS115 competent cells. The transformed strain was plated on MD solid medium (amino acid-free) and incubated at 30°C for 3 days to obtain transformants. The transformants were confirmed as positive transformants by colony PCR amplification of the target gene. Finally, the initial recombinant bacterium GS115 / pPIC9K-amy expressing α-amylase was obtained.
[0025] Example 2: Construction of plasmids for overexpression of translation-related functional genes
[0026] Using plasmid pGAPZαA as a template, the plasmid map is as follows: Figure 1As shown, the sequences of each functional gene (the PAS number of the gene encoding protein kinase is chr2-1_0219, the PAS number of the gene encoding cytoplasmic RNA binding protein is chr2-1_0414, and the PAS number of the gene encoding spliceosome protein is chr1-1_0383, and the nucleotide sequences of the genes they encode are shown in SEQ ID NO.1-3, respectively) were inserted between the GAP promoter and the AOX1 terminator sequence. Using the Gibson seamless cloning method, plasmid pGAPZαA was amplified using primers pGA-F / R (Table 1) as a template. PAS_chr2-1_0219, PAS_chr2-1_0414, and PAS_chr1-1_0383 gene sequences were amplified using primer pairs 0219-F / R, 0414-F / R, and 0383-F / R as templates for GS115 genome, respectively. The amplified plasmid sequences were ligated to the gene sequences, and the resulting plasmids were transformed into *E. coli* DH5α competent cells and plated on bleomycin-resistant plates. The success of ligation was checked using primers PF / R, and the sequence accuracy was verified by sequencing. The obtained plasmids were named pGAPZA-0219, pGAPZA-0414, and pGAPZA-0383, respectively.
[0027] Table 1. Primer sequences obtained for constructing functional gene overexpression plasmids
[0028]
[0029] Example 3: Construction and characterization of Pichia pastoris strains co-expressing functional genes and α-amylase
[0030] Electroporation competent cells were prepared from the strain GS115 / pPIC9K-amy constructed in Example 1. The plasmids pGAPZA-0219, pGAPZA-0414, and pGAPZA-0383, which were correctly sequenced and verified in Example 2, were extracted and linearized using AvrII restriction enzyme digestion. The linearized plasmids were purified and electroporated into GS115 / pPIC9K-amy competent cells, then plated onto solid plates containing 100 μg / mL bleomycin YPD and incubated at 30°C for 3 days until single bacteria grew. To detect whether the functional gene was successfully integrated, the transformants were subjected to colony PCR using primers PF / R from Example 2, and the sequence was sequenced to verify the correctness of the sequence. The obtained strains were named GS115 / pPIC9K-amy / pGAPZA-0219, GS115 / pPIC9K-amy / pGAPZA-0414, and GS115 / pPIC9K-amy / pGAPZA-0383, respectively. The recombinant strains were cultured in 96-well plates to characterize the effect of overexpression of the functional gene on α-amylase protein expression.
[0031] The above single colonies were inoculated into 96-well plates containing 600 µL of YPD medium and cultured for 24 hours. After 24 hours, the colonies were re-inoculated at a 5% inoculation rate into 96-well plates containing 300 µL of BMGY medium and cultured for another 24 hours. Then, 300 µL of BMMY medium was added to begin induction culture, with 1% methanol added every 24 hours. After 96 hours of induction, the supernatant of the fermentation broth was collected. Using the supernatant of the GS115 / pPIC9K-amy fermentation broth as a control, a standard curve for amylase activity detection was prepared using Solarbio's α-amylase (α-AL) activity assay kit (BC0615). Figure 3 As shown in the figure, the enzyme activity of amylase in the fermentation supernatant of the strain was detected. The amount of enzyme that produces 1 mg of reducing sugar per minute is defined as one activity unit. The enzyme activity of GS115 / pPIC9K-amy fermentation broth supernatant was 0.36 U / mL. The enzyme activity of PAS_chr2-1_0219 overexpression increased by 85.5% to 0.67 U / mL, the enzyme activity of PAS_chr2-1_0414 overexpression increased by 106.9% to 0.74 U / mL, and the enzyme activity of PAS_chr1-1_0383 overexpression increased by 92.5% to 0.69 U / mL.
[0032] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A method for expression of a starch amylase in Pichia pastoris, characterized in that, A recombinant plasmid containing translation-related functional genes was constructed and introduced into a Pichia pastoris strain expressing α-amylase. Co-expression yielded a recombinant Pichia pastoris engineered strain. The translation-related functional gene is a gene encoding a cytoplasmic RNA-binding protein, and the nucleotide sequence of the gene encoding the cytoplasmic RNA-binding protein is as shown in SEQ ID NO.2 or its degenerate sequence.
2. The expression method of claim 1, wherein, The method for constructing the Pichia pastoris strain expressing α-amylase includes deleting the signal peptide of the α-amylase sequence, optimizing the codons, inserting it downstream of the signal peptide of the backbone vector, linearizing it, transforming it into host cells, and screening to obtain the initial strain expressing α-amylase.
3. The expression method of claim 2, wherein, The host cell is Pichia pastoris cell, the signal peptide is α-factor, and the backbone carrier is pPIC9K.
4. The expression method of claim 2, wherein, The α-amylase is derived from barley.
5. The expression method of claim 4, wherein, The amino acid sequence of the α-amylase is shown in SEQ ID NO.
4.
6. The expression method of claim 1, wherein, It also includes the steps of screening and inducing fermentation of the recombinant Pichia pastoris engineered strain, wherein bleomycin is used for screening.
7. The recombinant Pichia pastoris engineered strain constructed using the expression method described in any one of claims 1-6.
8. The application of the engineered bacteria according to claim 7 in the production of α-amylase.