CN111893106A - Pichia pastoris engineering strain for heterologous expression of cellulase gene EG V and application - Google Patents

Abstract

The invention discloses a Pichia pastoris engineering bacterium for heterologous expression of a cellulase gene EG V and application thereof, wherein the engineering bacterium is Pichia pastoris-EG5, which is preserved in China general microbiological culture Collection center in 2019 and 8 months, and the preservation number is CGMCC No. 18422. The endoglycosidase gene (EG V) was obtained from Trichoderma reesei by PCR method, cloned and inserted into Pichia pastoris integrated expression vector pPIC9K to obtain pPIC9K-EG5 expression vector. The vector is introduced into pichia pastoris GS115 through electrotransformation to obtain recombinant pichia pastoris strains, and the recombinant yeast strains containing high copy integration plasmids are screened by using antibiotic G418 with different concentrations. And the optimum induction condition is determined through the primary optimization of shake flask fermentation, and the enzymatic properties of the purified recombinant protease are analyzed.

Description

Pichia pastoris engineering strain for heterologous expression of cellulase gene EG V and application

Technical Field

The invention belongs to the technical field of cellulase gene engineering, and particularly relates to construction and application of a pichia pastoris strain for heterologously expressing a cellulase gene EG V.

Background

Lignocellulose is an abundant renewable resource that can be converted into a variety of biological energy sources and chemical products. Research into the conversion of lignocellulose to fuel ethanol is receiving increasing attention. However, in the process of producing fuel ethanol by converting lignocellulose, the cost of enzyme preparation and the enzymolysis process account for about 40% of the production cost, so that the reduction of the production cost of cellulase or the integration of the enzymolysis and fermentation processes through a unified biological processing (CBP) process is an important means for improving the economy of the production of lignocellulose ethanol.

Cellulase is a complex enzyme system consisting of a plurality of enzyme systems, including endoglucanase, exoglucanase and beta-glucosidase. Most commercial cellulases on the market are secreted by Trichoderma reesei and Aspergillus niger, such as Trichoderma reesei, Trichoderma koningii and Aspergillus niger. Among them, Trichoderma reesei has strong ability to resist metabolic inhibition, and the produced cellulase has higher enzyme activity, and has become the most widely used cellulase producing strain in industry. However, the existence of a plurality of proteins in the enzyme system of trichoderma reesei results in low proportion of cellulase, and the proportion of each cellulase is mainly regulated by itself, which is difficult to control manually. The cellulase produced by expressing the exogenous gene in the yeast not only can ensure that the yeast has the capacity of directly degrading cellulose, but also can regulate and control the synthesis and secretion of the enzyme by an artificial means, thereby being beneficial to researching the enzymological properties and the interaction of the cellulase, and further improving the enzyme activity of the cellulase.

At present, recombinant protein expression systems are mainly as follows: coli, yeast, insect cells, mammalian cells, transgenic plant or animal expression systems and in vitro translation systems. Among them, eukaryotes have the characteristics of fast growth, easy culture, simple genetic operation, and the like, and can process, modify and fold the expressed protein, so that the expression system of eukaryotes is more and more emphasized and widely applied. Among them, the expression system of Pichia pastoris is the most widely used methylotrophic yeast with relatively perfect development recently. The recombinant pichia pastoris strain has strong foreign gene expression capability and high stability, can secrete cellulase protein extracellularly, and has extremely few self-secreted protein components, thereby being beneficial to the separation and purification of expressed protein.

Disclosure of Invention

The invention aims to provide a pichia pastoris engineering strain for recombinant expression of EG V enzyme, namely a pichia pastoris engineering strain is constructed by transforming cellulose endoglucanase EG V gene of Trichoderma reesei into pichia pastoris by using a technical means of gene engineering. The strain can efficiently express the cellulose endoglucanase EG V, and in order to improve the enzyme yield of the recombinant strain, the induction time, the temperature and the rotating speed of shake flask culture are researched. And the separated and purified protease is subjected to enzymatic property analysis. Finally, the strain is preserved in China general microbiological culture Collection center (CGMCC).

The technical scheme adopted by the invention is as follows:

(1) obtaining of trichoderma reesei RNA and CDNA: culturing green spores from trichoderma reesei QM9414, inoculating the green spores to an induction culture medium to obtain mycelia, and extracting total RNA and performing reverse transcription by using a kit to obtain cDNA;

(2) cloning of the target gene: designing a reasonable primer, and amplifying a target gene by PCR;

(3) constructing an expression vector: inserting an EG V gene into the downstream of a promoter AOX1 by taking a Ppic9k plasmid as a vector, wherein the 5 'end restriction site is EcoRI and the 3' end restriction site is Not I, and constructing a Ppic9k-EG V expression vector;

(4) obtaining a recombinant strain: the method comprises the following steps of successfully amplifying a target fragment by PCR (polymerase chain reaction) by taking Trichoderma reesei cDNA as a template, and successfully constructing a recombinant plasmid pPIC9K-EG V by a double enzyme digestion method; transferring the recombinant plasmid into pichia pastoris through electric transfer, and obtaining a recombinant strain through an MD plate, an MM plate, a YPD plate (containing G418 with different concentration concentrations) and genome PCR;

(5) screening of recombinant strains: YPD plates containing different concentrations of antibiotic G418 were used to screen recombinant yeast strains containing high copy integration plasmids;

(6) optimization of inducible expression conditions: the strain P.pastoris-eg4-4 was inoculated into BMMY (containing 0.5% methanol) for induction, cultured at 29 ℃ and 280rpm with shaking. Sampling for 24h, 48h, 72h, 96h, 120, 144 and 168, supplementing a certain amount of methanol for each sampling, and determining the optimal induction time by measuring enzyme activity and protein content; inoculating the recombinant strain to BMMY (containing 0.5% methanol) to perform induction at different temperatures (24, 26, 28, 30) DEG C at 280rpm, performing shaking culture, sampling at the optimal induction time, supplementing a certain amount of methanol every 24h, and determining the optimal induction temperature; inoculating the recombinant strain to a fermentation medium BMMY containing 0.5% methanol for induction at different rotating speeds, culturing at the optimal temperature, sampling at the optimal induction time, supplementing a certain amount of methanol every 24h, and determining the optimal induction rotating speed;

(7) and (3) detecting the activity of the recombinant protein: SDS-PAGE is utilized to detect whether secreted protein contains target protein and Congo red-CMC is utilized to detect whether recombinant pichia pastoris secretes active cellulose degrading enzyme

(8) Analysis of enzymatic properties: obtaining electrophoresis pure level recombinase through ammonium sulfate precipitation and nickel column purification, measuring enzyme activity under different pH conditions, and determining the optimum pH; measuring the enzyme activity of the recombinase under the conditions of the optimal pH value and different temperatures, and determining the optimal temperature; and taking substrate CMC with different concentrations under the optimal pH and the optimal temperature of the recombinase, and measuring corresponding reaction speed V to obtain the kinetic constant of the recombinase.

(9) Analysis of enzymatic properties: obtaining electrophoresis pure level recombinase through ammonium sulfate precipitation and nickel column purification, measuring enzyme activity under different pH conditions, and determining the optimum pH; measuring the enzyme activity of the recombinase under the conditions of the optimal pH value and different temperatures, and determining the optimal temperature; and taking substrate CMC with different concentrations under the optimal pH and the optimal temperature of the recombinase, and measuring corresponding reaction speed V to obtain the kinetic constant of the recombinase.

(10) Strain preservation: the preservation unit: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 26 months 8 in 2019; the number of the yeast engineering strain is as follows: CGMCC No. 18422; the classification and the designation of the pichia pastoris engineering strains are as follows: pichia pastoris (Pichia pastoris).

The invention has the advantages that:

1. according to the invention, 6 codons corresponding to histidine (His) are introduced into a primer of a gene, so that a target protein is provided with a His tag, and the purification of the target protein by a nickel column is facilitated.

2. When the pichia pastoris recombinant strain is induced and expressed, the induction expression conditions are optimized, and a proper amount of methanol is added, so that the induction effect is optimal, and the enzyme production efficiency of the recombinant strain is improved.

3. The basic enzymology property of the recombinase is determined, and the basic enzymology property of the recombinase is similar to that of the natural trichoderma reesei secretase, and the recombinase can replace the natural trichoderma reesei secretory core enzyme component to be used for cellulose substrate hydrolysis.

4. The invention realizes the heterologous expression of the cellulase protein EG V in the pichia pastoris, improves the enzyme production efficiency of the recombinant pichia pastoris in the methanol induction fermentation process, and improves the industrial production potential of the recombinant pichia pastoris.

Drawings

FIG. 1 is a diagram of PCR amplification of EG V;

FIG. 2 is a PCR-verified clone of Ppic9k-EG V;

FIG. 3 is a map of Ppic9k-EG V expression plasmid;

FIG. 4 is a linearized nucleic acid electrophoresis diagram EG V;

FIG. 5 is a genomic PCR map of EG V recombinant Pichia pastoris;

FIG. 6 shows Congo red-CMC verifying whether the inducible enzyme is EG V active;

FIG. 7 is an SDS-PAGE image of a supernatant of EG V strain fermentation broth;

FIG. 8 is a graph showing the sequencing contrast of the PCR amplified EG V.

Detailed Description

EXAMPLE 1 cloning of target Gene

1. The trichoderma reesei QM9414 spore suspension preserved in glycerin tube is taken out from a refrigerator at-80 ℃ and placed in ice (or a refrigerator at 4 ℃) for unfreezing until the spore suspension is completely melted. And (3) oscillating and mixing the spore suspension uniformly, dipping a small amount of bacterial liquid on an inoculating loop, and streaking and inoculating on a PDA culture medium plate. After sealing the plate with a sealing film, the plate was cultured in an incubator at 28 ℃. Culturing for about 5-7 days until the surface of the culture medium is covered with green spores. The plate was removed from the incubator and placed in a 4 ℃ freezer for use.

2. Inoculating fresh spore to cellulase solid for induction culture, spreading sterilized cellophane on the surface, and standing for culture until hypha grows out. After scraping hyphae from cellophane and treating with a liquid nitrogen cryo-grinder, the RNA was extracted using UNIQ-10 column total RNA extraction Kit and reverse transcription of RNA was performed using First Strand cDNA Synthesis Kit.

3. The synthesized Trichoderma reesei cDNA template is used for amplifying the gene sequence corresponding to the EG V of QM9414 by primer PCR, the amplification PCR picture is shown in figure 1, and the sequencing comparison result is shown in figure 8.

Primer: eg5-F CGGAATTCATGTACAAGGCCACCACCAC

Eg5-R TTGCGCAGTGGGTGAGGAAGACACTGGGAGGTGGTGGTGGTGGTG GTGTACPCR

PCR amplification System:

EXAMPLE 2 construction of expression vector Ppic9k-EG V

1. A double restriction enzyme system was prepared using restriction enzymes EcoRI/Not I, double restriction enzymes Ppic9k and the gene of interest eg5 as shown in the following table. Reacting for 50min at 37 ℃ in a metal bath, and recovering the product by using the recovery kit. The product after double digestion is reacted with Taq Mix enzyme (volume 1:1) in a PCR instrument at 72 ℃ for 30min, and A end connection is carried out.

A double enzyme digestion system:

2. the 5 'end EcoRI is used as an enzyme cutting site, the 3' end Not I is used as an enzyme cutting site, and the downstream of the promoter AOX1 of the Ppic9k plasmid vector is inserted to construct an expression vector Ppic9k-EG V. The expression vector map is shown in FIG. 3. The colony PCR was verified as shown in FIG. 2. And verifying the expression vector by single enzyme digestion to confirm that the construction of the expression vector Ppic9k-EG V is successful.

A connection system:

example 3 construction of recombinant Pichia pastoris

1. Restriction enzyme bglii is used for carrying out single enzyme digestion on recombinant expression plasmid Ppic9k-EG V, a linearization system is prepared, the mixture is uniform, and the temperature is increased for 1h at 37 ℃ by metal bath, so that the expression vector is changed into a linear shape from a ring shape, and the later transformation is facilitated. The linearized plasmid was verified by nucleic acid electrophoresis, see FIG. 4. And (4) transferring the strain into pichia pastoris GS115 competent cells through electrotransformation, and plating to obtain a transformant.

A linearization system:

2. the genome of the recombinant yeast transformant obtained by crude extraction of 1 by SDS thermal cracking was verified for correctness and phenotype by PCR, and the nucleic acid map of the genome PCR is shown in FIG. 5. And (3) selecting transformants on the MM plate, sequentially dotting the transformants on YPD medium plates containing G418 antibiotics with different concentrations, carrying out inverted culture at 30 ℃, observing colony morphology, and screening out multi-copy transformants.

Example 4 Induction expression and Condition optimization of Pichia pastoris recombinant strains

After primary activation of YPD plates, single colonies were inoculated into a triangular flask (25mL BMGY), incubated at 29 ℃ and 280rpm with shaking until OD600 became 2.0. The cells were collected by centrifugation at 7000rpm for 5min at 4 ℃ and the BMMY was resuspended so that OD600 became about 1.0. The collected bacterial solution was added to a 1L Erlenmeyer flask, sealed with gauze, incubated at 29 ℃ and 280rpm, and subjected to shaking culture. Methanol was added every 24h to a final concentration of 0.5%. The sample was taken 1mL, centrifuged at 12000rpm for 5min at 4 ℃ and the supernatant was stored at 4 ℃.

2. Induction time: the method is the same as that of 1 for inducing expression of the pichia pastoris recombinant strain, carrying out shaking culture at 29 ℃ and 280rpm, and sampling for 24h, 48h, 72h, 96h, 120h, 144h and 168 h.

Induction rotating speed: the procedure was as above, with sampling at 29 ℃ for optimal induction time. Rotating speed: 200rpm, 220rpm, 240rpm, 260rpm, 280 rpm.

Induction temperature: the method is the same as the above, the shaking culture is carried out at the optimal rotating speed, and the sampling is carried out at the optimal induction time. Temperature: 24 ℃, 26 ℃, 28 ℃ and 30 ℃.

Example 5 Induction of recombinant Pichia pastoris for enzyme production and verification of enzyme Activity

1. Single colonies of high-copy transformants were inoculated into 25ml of BMGY seed medium and cultured at 29 ℃ and 290rpm to

OD600 was 2 (about 16-18h), and after centrifugation for 5min, the cells were collected and resuspended in BMYY medium until OD600 became 1. The medium is covered with sterile gauze and incubated on a shaker at 29 ℃ and methanol is added to a concentration of 0.5% every 24 hours until the optimal induction time is reached. 1ml of the suspension was sampled at regular intervals, centrifuged at room temperature, and the supernatant was collected.

2. 100ul of the supernatant was added to the cavity of an agar plate containing 0.5% CMC and incubated at 50 ℃ for 1 h. Staining with 0.1% Congo red for 1h, and decolorizing with 1mol/LNacl for 40 min. Nacl can wash away the congo red which is not firmly bonded, so that transparent rings with different sizes are left, and the cellulose decomposing capacity of the large transparent rings is strong, as shown in figure 6.

3. 5% concentration fermentation is adopted; 12% separation gel, the treated sample supernatant was added, and the SDS-PAGE is shown in FIG. 7.

Embodiment 6 preservation of Pichia engineering Strain expressing cellulase Gene EG V

The preservation unit of the pichia pastoris engineering strain expressing the cellulase gene EG V is as follows: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 26 months 8 in 2019; the number of the yeast engineering strain is as follows: CGMCC No. 18422; the classification and the designation of the pichia pastoris engineering strains are as follows: pichia pastoris (Pichia pastoris).

Claims (5)

1. A construction method for heterologously expressed endoglucanase EG V protein in pichia pastoris comprises the following steps:

(1) obtaining of trichoderma reesei RNA and cDNA: culturing Trichoderma reesei QM9414 until green spores are produced, then inoculating the green spores to a solid induction culture medium (the surface of which is stuck with cellophane) to obtain mycelia, and extracting total RNA and carrying out reverse transcription by using a kit to obtain cDNA;

(2) cloning of the target gene: designing reasonable primers and carrying out PCR amplification on the EG V gene under proper conditions;

(3) constructing an expression vector: inserting an EG V gene into the downstream of a promoter AOX1 by taking a Ppic9k plasmid as a vector, wherein the 5 'end restriction site is EcoRI and the 3' end restriction site is Not I, and constructing a Ppic9k-EG V expression vector;

(4) obtaining a recombinant strain: the linearization site of the Ppic9k-EG V expression vector was defined as bglI. Realizing the linearization of an expression vector, and then transferring the expression vector into pichia pastoris through electrotransformation to obtain EG V recombinant yeast;

(5) screening of recombinant strains: YPD plates containing different concentrations of antibiotic G418 were used to screen recombinant yeast strains containing high copy integration plasmids;

(6) optimization of inducible expression conditions: the strain P.pastoris-eg2-3 was inoculated into BMMY (containing 0.5% methanol) for induction, cultured at 29 ℃ and 280rpm with shaking. Sampling for 24h, 48h, 72h, 96h, 120, 144 and 168, supplementing a certain amount of methanol for each sampling, and determining the optimal induction time by measuring enzyme activity and protein content; inoculating the recombinant strain to BMMY (containing 0.5% methanol) to perform induction at different temperatures (24, 26, 28 and 30 ℃), performing shaking culture at 280rpm, sampling at the optimal induction time, supplementing a certain amount of methanol every 24h, and determining the optimal induction temperature; inoculating the recombinant strain to a fermentation medium BMMY containing 0.5% methanol for induction at different rotating speeds, culturing at the optimal temperature, sampling at the optimal induction time, supplementing a certain amount of methanol every 24h, and determining the optimal induction rotating speed;

(7) induction of recombinant strains to produce enzymes: carrying out shake flask fermentation on the screened strains, and adding appropriate methanol to induce enzyme production;

(8) and (3) detecting the activity of the recombinant protein: detecting whether the recombinant pichia pastoris secretes active cellulose degrading enzyme or not by using a Congo red-CMC method;

SDS-PAGE detects whether the recombinant protein is the target protein and the protein expression quantity thereof;

(9) analysis of enzymatic properties: obtaining electrophoresis pure level recombinase through ammonium sulfate precipitation and nickel column purification, measuring enzyme activity under different pH conditions, and determining the optimum pH; measuring the enzyme activity of the recombinase under the conditions of optimal pH and different temperatures, and determining the optimal temperature; taking substrate CMC with different concentrations under the optimal pH and the optimal temperature of the recombinase, measuring corresponding reaction speed V, and obtaining a dynamic constant of the recombinase;

(10) preservation of the strains: the preservation unit of the pichia pastoris engineering strain expressing the cellulase gene EG V is as follows: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 26 months 8 in 2019; the number of the yeast engineering strain is as follows: CGMCC No. 18422; the classification and the designation of the pichia pastoris engineering strains are as follows: pichia pastoris (Pichia pastoris).

2. The method for constructing the EG V protein heterologously expressed in pichia pastoris according to claim 1, wherein when the recombinant strain is induced to express the enzyme, the induced expression conditions are studied: the enzyme activity of the target protein EG V in the fermentation liquor reaches the maximum enzyme activity value at 168h, 28 ℃ and 260rpm, and the enzyme activity of EG V can reach 2.95IU/mL, so that the target protein with higher yield is obtained, and the subsequent purification of the target protein is facilitated.

3. The construction method of the heterologous expression EG V protein in pichia pastoris according to claim 1, wherein a primer sequence of a target gene is optimized, codons corresponding to 6 histidines (His) are reasonably introduced, so that the expressed target protein contains histidine, and the target protein is separated and purified by a nickel column, thereby facilitating subsequent analysis of enzymatic properties of recombinant protease.

4. The method for constructing the EG V protein heterologously expressed in pichia pastoris, as claimed in claim 1, wherein the enzyme properties of EG V are measured to have the optimum pH of 4.5, the optimum temperature of 55 ℃, the Km value of about 1.6mg/mL and the Vmax value of about 99.7IU/mg, and the basic enzyme properties of the obtained recombinase are similar to those of natural trichoderma reesei secretase and can be used for cellulose substrate hydrolysis instead of the natural secretory core enzyme component of trichoderma reesei.

5. The construction method of the heterologous expression EG V protein in pichia pastoris according to claim 1, wherein heterologous expression of cellulase EG V protein in pichia pastoris is realized, a proper amount of methanol is added to induce enzyme production, the enzyme production efficiency is improved, and the industrial production potential of recombinant pichia pastoris is improved.

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