CN116536176A - Recombinant pichia pastoris genetically engineered bacterium, construction method and application thereof - Google Patents
Recombinant pichia pastoris genetically engineered bacterium, construction method and application thereof Download PDFInfo
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- CN116536176A CN116536176A CN202310555884.6A CN202310555884A CN116536176A CN 116536176 A CN116536176 A CN 116536176A CN 202310555884 A CN202310555884 A CN 202310555884A CN 116536176 A CN116536176 A CN 116536176A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
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Abstract
The invention discloses a recombinant pichia pastoris genetically engineered bacterium, a construction method and application thereof. The recombinant pichia pastoris gene engineering bacteria are obtained by modifying PAS_FragB_0067 genes of pichia pastoris after over-expression. The invention uses pichia pastoris expression strain capable of producing xylanaseKomagataella phaffii GS115-xyn is a starting bacterium and is first over-expressedThe PAS_FragB_0067 gene is successfully constructed into recombinant bacteria 0067, the biological film forming capability of pichia pastoris is improved, and the problems of weak film forming capability, low fermentation efficiency and the like of pichia pastoris in the prior art are solved. The modified pichia pastoris genetically engineered bacteria have higher fermentation performance and stronger enzyme activity in the continuous fermentation process, and have obvious advantages in fermentation.
Description
Technical Field
The invention belongs to the technical fields of genetic engineering and fermentation engineering, and particularly relates to pichia pastoris over-expressing PAS_FragB_0067 genes, a construction method thereof and application thereof in xylanase production.
Background
Biofilm is a ubiquitous form of microorganisms that spontaneously tend to form biofilms or colonies because of their greatly improved viability under multicellular populations. The biological film can be formed on the surface of living body or non-living body, and can grow in a film-like manner, and has high resistance to antibacterial factors and various external pressures. Thus, biofilms are of great interest both in industrial and medical applications. In recent years, the research on the role of fungal biofilms in human medicine is increasing, and the research on the expression of genes in the process of forming the fungal biofilms is increasing, and the genes mainly comprise major genes related to the formation of the biofilms, genes related to the resistance to antifungal drugs, adhesion and cell wall synthesis genes and the like.
Yeast flocculation is a way of adhering cells, and has very important scientific research and industrial application value. The good flocculation property is a population protection mechanism of the yeast cells for coping with environmental stress, and provides a more effective, environment-friendly and low-cost cell separation and product clarification way for the industrial fermentation process. Many yeasts differentiate into a multicellular phenotype under adverse environmental conditions. In the Saccharomyces cerevisiae system, researchers have conducted extensive research on FLO gene family function. However, the cell adhesion process and the control mechanism of the related genes are very complex, and the influence of the FLO gene on the biological film-forming capacity of the saccharomyces cerevisiae and the fermentation effect of the saccharomyces cerevisiae still has no universal control theory. For example, paola Di Gianvito et al knockdown of FLO1 in Saccharomyces cerevisiae F6789 had no effect on flocculation ability (Sci. Rep.,2017,7 (1): 1-12); however, studies by Johan O.Westman et al show that gradual deletion of tandem repeats in FLO1 results in a corresponding decrease in different adhesion phenotypes, such as adhesion plastics and flocculation (appl. Environ. Microb.,2014,80 (22): 6908-6918).
Pichia pastoris expression system is one of the eukaryotic expression systems currently popular, has the advantages which are not possessed by many other protein expression systems, has the characteristics of strong inducible promoter, high expression, high stability, high-density fermentation, moderate glycosylation, good biological activity of the expression product, simple and convenient fermentation and purification operation method, easy industrial production and the like, and has successfully expressed many very valuable proteins. At present, few researches on biological film-forming adsorption immobilized fermentation in a pichia pastoris system are reported, and the related regulation and control mechanism of the pichia pastoris FLO family genes and biological film-forming is still yet to be explored. In the prior art, CN113046256A strengthens the biological film forming capability of pichia pastoris by constructing a strain of pichia pastoris genetic engineering bacteria over-expressing HSF1, and solves the problems that the film forming capability of the pichia pastoris is weak and the pichia pastoris cannot be used for continuous immobilized fermentation in the prior art. CN112592844A constructs a strain of Pichia pastoris genetic engineering bacteria over-expressing lectin LMAN2, enhances the biological film forming capability of Pichia pastoris, and solves the problems that the film forming capability of the Pichia pastoris is weak and the Pichia pastoris cannot be used for continuous immobilized fermentation in the prior art. However, the biological film forming mechanism of pichia pastoris is still unknown, which promotes the technical staff to continuously explore the regulation and control relation between the cell adhesion related genes and the biological film, thereby better utilizing the biological film for fixed fermentation and realizing the purposes of shortening the fermentation period and recycling the cells.
Disclosure of Invention
The invention aims to: the technical problem to be solved by the invention is to provide a recombinant pichia pastoris engineering bacterium which over-expresses PAS_FragB_0067 gene aiming at the defects of the prior art, so as to further study the film forming mechanism of pichia pastoris and the regulation and control function of myxoid protein gene PAS_FragB_0067 (related to FLO11 structural domain).
The invention also solves the technical problem of providing a construction method of the recombinant pichia pastoris genetically engineered bacterium.
The invention finally solves the technical problem of providing the application of the recombinant pichia pastoris genetically engineered bacteria.
Specifically, the invention discloses a recombinant pichia pastoris gene engineering bacterium, which is used for over-expressing a mucin-like protein gene PAS_FragB_0067 in the pichia pastoris, wherein the pichia pastoris is a pichia pastoris expression bacterium Komagataella phaffii GS-xyn capable of producing xylanase, and NCBI accession number of the flocculation gene PAS_FragB_0067 is XM_002490834.1 (SEQ ID No. 1).
The invention further provides a construction method of the recombinant pichia pastoris genetically engineered bacterium, which comprises the following steps:
(1) The PAS_FragB_0067 gene fragment is amplified by PCR by taking the genome of Pichia pastoris expression strain Komagataella phaffii GS-xyn (Pichia pastoris GS115-xyn and K.phaffii GS115-xyn all have the same meaning) as a template;
(2) Cloning the PAS_FragB_0067 gene fragment obtained in the step (1) onto an expression plasmid to obtain a recombinant plasmid;
(3) Linearizing the recombinant plasmid obtained in the step (2), introducing the linearized recombinant plasmid into pichia pastoris expression bacteria Komagataella phaffii GS-xyn, and screening to obtain recombinant pichia pastoris genetic engineering bacteria.
Preferably, the recombinant Pichia pastoris engineered bacteria are obtained by selection on a yeast extract peptone dextrose agar (YPD) resistant plate containing Zeocin. Preferably, the Zeocin concentration is 100 μg/mL.
In the step (1), the primers used for PCR amplification are a primer 1 and a primer 2, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
In step (2), the expression plasmid is preferably pGAPZ A.
The invention further discloses application of the recombinant pichia pastoris genetically engineered bacteria in cell membrane biological fermentation. The research of the application shows that through over-expressing PAS_FragB_0067 genes, the biological film forming capability of pichia pastoris is improved, the problems of weak film forming capability, low fermentation efficiency and the like of pichia pastoris in the prior art are solved, and the PAS_FragB_0067 gene can be better applied to immobilized fermentation of biological films.
In a specific application, the recombinant pichia pastoris genetically engineered bacterium 0067 can be used for fermentation production of xylanase.
Preferably, the recombinant pichia pastoris gene engineering bacteria produce xylanase by means of fixed fermentation.
Specifically, the immobilized fermentation is to add recombinant pichia pastoris genetic engineering bacterial mud into a fermentation medium containing an immobilized carrier, ferment to obtain xylanase enzyme liquid, and preferably, methanol is added in the fermentation process to induce xylanase expression.
In the process, the recombinant pichia pastoris genetically engineered bacteria mud is obtained by centrifuging recombinant pichia pastoris genetically engineered bacteria seed liquid.
The preparation method of the recombinant pichia pastoris genetically engineered bacteria seed solution comprises the following steps:
(a) Inoculating recombinant pichia pastoris gene engineering bacteria into an activation culture medium, and culturing to obtain an activation solution;
(b) Inoculating the activation solution obtained in the step (a) into a seed culture medium, and culturing to obtain the seed solution of the recombinant pichia pastoris genetically engineered bacteria.
In the step (a), the concentration of each component in the activation culture medium is 10-30g/L of peptone, 5-15g/L of yeast powder, 10-30g/L of glucose and 0.1-5mg/L of biotin; preferably 20g/L peptone, 10g/L yeast powder, 20g/L glucose, and 0.4mg/L biotin.
In step (a), the solvent of the activation medium is water.
In step (a), the culture is carried out at 28-30℃and 200-300rpm for 16-24 hours, preferably at 28℃and 250rpm for 24 hours.
In the step (b), the concentration of each component in the seed culture medium is 10-30g/L of peptone, 5-15g/L of yeast powder, 10-20g/L of amino-free yeast nitrogen source, 0.1-1g/L of dipotassium hydrogen phosphate, 1-5g/L of potassium dihydrogen phosphate and 0.1-5mg/L of biotin, the solvent is water, preferably 20g/L of peptone, 10g/L of yeast powder, 10g/L, YNB 13.4.4 g/L of glycerol, 0.3g/L of dipotassium hydrogen phosphate, 1.18g/L of potassium dihydrogen phosphate and 0.4mg/L of biotin, and the solvent is water.
Preferably, in step (b), the culturing is at 28-30℃for 16-24 hours under 200-300 rpm; preferably at 28℃and 250rpm for 24 hours.
Wherein, in the fixed fermentation, the immobilized carrier is any one or the combination of a plurality of cotton fiber fabrics, non-woven fabrics, polyester fibers, polyvinyl alcohol fibers, zeolite, bacterial cellulose films, silk, bagasse and corn stalks; preferably cotton fiber fabric.
Preferably, the immobilized carrier is pretreated, namely, the immobilized carrier is sheared into squares of 2-8cm multiplied by 2-8cm, washed and dried by pure water, soaked in ethanol for 0.5-4h, washed by pure water, and dried after being bathed in boiling water for 10-40 min; preferably, the pretreatment is to cut the immobilization carrier into squares of 4cm×4cm, wash and dry with pure water, soak in ethanol for 1h, wash with pure water, and dry with boiling water bath for 30 min.
Wherein the dosage of the immobilized carrier is 2-80g/L fermentation medium, preferably 40g/L fermentation medium.
In the xylanase-producing fermentation medium, the concentration of each component is as follows: 10-30g/L of peptone, 5-15g/L of yeast powder, 10-20g/L of amino-free yeast nitrogen source (YNB), 0.1-1g/L of dipotassium hydrogen phosphate, 1-5g/L of potassium dihydrogen phosphate and 0.1-5mg/L of biotin, wherein the solvent is water; preferably 20g/L peptone, 10g/L, YNB 13.4.4 g/L yeast powder, 0.3g/L dipotassium hydrogen phosphate, 1.18g/L potassium dihydrogen phosphate and 0.4mg/L biotin, and the solvent is water.
Further preferably, methanol is added every 24 hours during fermentation.
Still more preferably, methanol is added in an amount of 0.1-2% v/v of the fermentation medium.
Preferably, the fermentation temperature is 28-30deg.C, preferably 30deg.C.
The rotation speed of the fermentation is 200-300rpm, preferably 250rpm.
The fermentation time is 3-5d, preferably 5d.
The beneficial effects are that: the recombinant pichia pastoris gene engineering bacteria 0067 are obtained by modifying PAS_FragB_0067 genes of pichia pastoris GS115-xyn after overexpression. The PAS_FragB_0067 gene is overexpressed for the first time, the biological film forming capability of pichia pastoris is improved, and the problems of weak film forming capability, low fermentation efficiency and the like of pichia pastoris in the prior art are solved. Specifically, qRT-PCR detection shows that the PAS_FragB_0067 gene expression level in the recombinant bacteria is 3673.4 times of the PAS_FragB_0067 gene expression level in the starting bacteria; under the single immobilized fermentation condition, the maximum xylanase enzyme activity of the pichia pastoris genetic engineering bacteria can reach 4905.8U/mL, which is 1.57 times of the enzyme activity of the original bacteria GS115-xyn under the same time; by immobilized continuous fermentation, cotton fiber is used as a carrier, the enzyme production capability of the original strain is obviously reduced when the fermentation is carried out to the 3 rd batch, and the recombinant strain can stably and continuously carry out immobilized fermentation of at least 5 batches. After 5 batches of immobilized fermentation, the enzyme activity of the enzyme produced by the fermentation of the recombinant bacteria is 3.90 times of that of the starting bacteria, and the recombinant bacteria has obvious advantages in fermentation.
Drawings
FIG. 1 is a schematic diagram of the expression plasmid pGAPZ A-PAS_FragB_0067;
FIG. 2A is a PCR electrophoretogram of the target gene PAS_FragB_0067, lane M is DL5000 DNA Marker, lane 1 is the target gene PAS_FragB_0067; FIG. B is a PCR electrophoresis diagram of pGAPZ A vector fragment, lane M is DL5000 DNA Marker, and lane 1 is pGAPZ A vector fragment; FIG. C is an electrophoretogram of recombinant plasmid pGAPZ A-PAS_FragB_0067, wherein lane M is DL5000 DNA Marker, lane 1 is pGAPZ A vector fragment, and lane 2 is recombinant plasmid pGAPZ A-PAS_FragB_0067;
FIG. 3 is a schematic diagram of recombinant 0067 wherein lane M is DL5000 DNA Marker, lane 1 is 0067 negative control, lanes 2, 3 are 0067 proof gene fragment, and lane 4 is 0067 positive control;
FIG. 4 is a graph showing the results of detecting the cell climbing sheet of the starting strain GS115-xyn and the recombinant strain 0067, wherein the graph A shows the cell climbing sheet detection graph of the starting strain GS115-xyn, and the graph B shows the cell climbing sheet detection graph of the recombinant strain 0067;
FIG. 5 is a graph showing the difference between the formation of biological membranes of the starting strain GS115-xyn and the recombinant strain 0067 by using a fluorescence microscope, wherein FIG. A is a fluorescence detection graph of the starting strain GS115-xyn, and FIG. B is a fluorescence detection graph of the recombinant strain 0067;
FIG. 6 is a diagram of experimental results of semi-quantitative determination of the amount of biological membrane by using a crystal violet staining method of the starting strain and the recombinant strain;
FIG. 7 shows the relative expression level of PAS_FragB_0067 gene in recombinant bacteria;
FIG. 8 is a graph showing comparison of xylanase activity produced by single-batch fermentation immobilized on a starting strain and a recombinant strain;
FIG. 9 is a graph showing comparison of xylanase enzyme activities produced by immobilized continuous fermentation of starting bacteria and recombinant bacteria.
Detailed Description
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
EXAMPLE 1 construction of xylanase-producing recombinant Pichia pastoris GS115-xyn
The xylanase (PDB No. 3WP4_A) gene fragment from anaerobic fungus Neocallimastix patriciarum was inserted between EcoRI and Not I cleavage sites of pPIC9K plasmid, and pPIC9K-xyn plasmid was constructed, codon optimization and plasmid subcloning were performed by Souzhou Jin Weizhi Biotechnology Co. The pPIC9K-xyn plasmid was linearized using Sal I restriction enzyme, the linearized fragment was introduced into Pichia pastoris GS115 competent cells, and the transformants were grown on MD plates (MD plate medium formulation: 13.4g/L yeast basic nitrogen source, 0.4mg/L biotin, 20g/L glucose, 20g/L agar) at 30℃until single colonies developed. Single colonies on MD plates were transferred to yeast extract peptone dextrose agar (YPD) resistant plates containing 4mg/mL G418 for selection and cultured at 28-30℃for 2-3d until colonies developed.
The colony was transferred to a 250mL shaking flask containing 50mL of seed medium, and cultured at 30℃and 250rpm for 24 hours to obtain a seed solution. The seed solution was centrifuged at 4500rpm for 5min, the supernatant was discarded, and the bacterial slurry was inoculated into a fermentation medium (500 mL shaking flask, liquid loading amount 100 mL) and subjected to free fermentation at 30℃and 250rpm. The induction of xylanase expression was performed by adding 1% v/v methanol (relative to the volume of fermentation medium) every 24h during fermentation. Taking a fermentation broth sample after 72h of fermentation to detect the enzyme activity of xylanase to 3025U/mL. The Pichia pastoris GS115-xyn is successfully constructed and used for subsequent experiments.
Example 2: measurement of xylanase activity.
1. Drawing of xylose standard curve
And taking a 10mL centrifuge tube, adding each component one by one, and drawing a xylose standard curve.
Wherein, the DNS reagent comprises the following components in each liter: 7.5g of 3, 5-dinitrosalicylic acid, 14.0g of sodium hydroxide, 216.0g of potassium sodium tartrate, 5.0g of phenol, 6.0g of sodium metabisulfite and water as a solvent.
Wherein, each liter of xylose standard solution comprises the following components: 10g of xylose and water as a solvent.
Wherein, each liter of the potassium phosphate buffer solution comprises the following components: 3g of tripotassium phosphate, 11.8g of monopotassium phosphate and water as solvent.
Wherein, the xylose standard curve reaction system is shown in Table 1.
TABLE 1 xylose standard curve reaction system
According to the system, adding each component into a 10mL centrifuge tube one by one; after thoroughly mixing, the mixture was boiled in boiling water for 5min, the reaction mixture was rapidly cooled to room temperature with cold water, distilled water was added to 5mL, the mixture was zeroed with a blank, and the absorbance at 540nm was measured. And (3) taking xylose content as an ordinate and a light absorption value as an abscissa, preparing a standard curve, and fitting a regression equation.
2. Determination of xylanase Activity
Xylanase enzyme activity is determined by a 3.5-dinitrosalicylic acid (DNS) reagent method: the monosaccharides from the hydrolysis of xylan (beech) by xylanase are reacted with 3.5-dinitrosalicylic acid (DNS) reagent in color, absorbance is detected at 540nm using uv spectrophotometry, and the enzymatic activity is quantified by measuring the reducing xylose produced by the enzymatic reaction.
The measurement method is as follows: 25 mu L of an enzyme solution with proper dilution is added into 0.225mL of potassium phosphate buffer solution, then 0.5mL of xylan substrate is added for accurate reaction at 50 ℃ for 15min, 1mL of DNS reagent is added for thorough mixing and boiling for 5min, the reaction solution is rapidly cooled to room temperature by cold water, distilled water is added to 5mL, blank control without adding crude enzyme solution is used for zeroing, and the light absorption value is measured at 540 nm. The enzyme activity is defined as: under the conditions of this assay, the amount of enzyme required to release 1. Mu. Mol of reducing sugar per minute is defined as 1 enzyme activity unit (U/mL).
Example 3 an over-expression plasmid was constructed that over-expressed the PAS_FragB_0067 gene.
1. Amplification of the target Gene PAS_FragB_0067: using the extracted K.phaffii GS115-xyn genome as a template, the gene PAS_FragB_0067 was amplified with primer 1 (SEQ ID NO. 2) and primer 2 (SEQ ID NO. 3).
The PCR reaction system is shown in Table 2.
TABLE 2 PCR amplification System for target Gene PAS_FragB_0067
According to the above system, 50. Mu.L per tube.
PCR conditions: 1) Pre-denaturation at 94℃for 5min; 2) Denaturation at 98 ℃,10s; 3) Annealing at 55 ℃ for 5s; 4) Extending at 72 ℃ for 45 seconds for 35 cycles; 5) The temperature is fully 72 ℃ and 10min.
The size of the amplification product of the target gene PAS_FragB_0067 is 4251bp (SEQ ID NO. 1), and the electrophoresis chart is shown in figure 2A. The PCR product was purified by TAKARA gel recovery kit and used in the subsequent experiments.
2. Extraction of expression plasmid pGAPZ A and target fragment amplification
1) Extraction of expression plasmid pGAPZ A: coli TOP 10 glycerol bacteria harboring plasmid pGAPZ A were inoculated into 5mL of LB liquid medium (containing 25. Mu.g/mL of Zeocin) and cultured overnight at 37 ℃; collecting cells with 2.0mL centrifuge tube, centrifuging at 10000rpm for 2min, and discarding supernatant; plasmid pGAPZ A was extracted following the procedure described in the AxyPrep plasmid DNA minikit.
2) Amplification of pGAPZ A fragments
pGAPZ A fragment was amplified using the linearized pGAPZ A plasmid as template with primer 3 (SEQ ID NO. 4) and primer 4 (SEQ ID NO. 5).
The PCR reaction system is shown in Table 3.
TABLE 3 amplification PCR System of linearized plasmid pGAPZ A
According to the above system, 50. Mu.L per tube.
PCR conditions: 1) Pre-denaturation at 94℃for 5min; 2) Denaturation at 98 ℃,10s; 3) Annealing at 55 ℃ for 5s; 4) Extending at 72 ℃ for 30 seconds for 35 cycles; 5) The temperature is fully 72 ℃ and 10min. The PCR product was purified by TAKARA gel recovery kit and used in the subsequent experiments.
The amplified product of pGAPZ A fragment has a size of 2857bp (SEQ ID NO. 6) and the electrophoretogram is shown in FIG. 2B. The PCR product was purified by TAKARA gel recovery kit and used in the subsequent experiments.
3. Construction of recombinant plasmids
And (3) connecting the target gene fragment obtained in the step (1) with the pGAPZ A fragment obtained in the step (2) according to the steps of the specification of the Peasy-Basic Seamless Cloning and Assembly Kit kit to obtain the recombinant plasmid pGAPZ A-PAS_FragB_0067. The cloning reaction system is shown in Table 4, wherein the recombinant product was added to the Trans-T1 competent cells provided in the kit, and the cells were left on ice for 30 minutes, and were heat-shocked in a water bath at 42℃for 30 seconds, and immediately thereafter transferred to ice for cooling for 2 minutes.
TABLE 4 ligation reaction System
The ligation solution obtained above was transferred to 450. Mu.L of LB medium, resuscitated and plated on LB-resistant plates (containing 25. Mu.g/mL Zeocin), and cultured at 37℃for 12 hours until a distinct single colony was obtained.
The single colony of the plate is picked, inoculated in LB liquid medium (containing 25 mug/mL Zeocin) for overnight culture at 37 ℃ for 12 hours, and plasmids are extracted, and sequencing shows that the sequence is correct. The recombinant plasmid has a size of 7108bp (SEQ ID NO. 7), pGAPZ A-PAS_FragB_0067 is shown in figure 1, and the electrophoresis chart is shown in figure 2C.
Example 2: constructing a Pichia pastoris gene engineering strain which over-expresses PAS_FragB_0067 gene.
4. Transformation of recombinant plasmids
Plasmid pGAPZ A-PAS_FragB_0067 was digested tangentially with AvrII.
The linearization system of recombinant plasmid pGAPZ A-PAS_FragB_0067 is shown in Table 5.
TABLE 5 linearization System of recombinant plasmid pGAPZ A-PAS_FragB_0067
The conditions of enzyme digestion are 37 ℃ and 1h, and after the enzyme digestion reaction is finished, the enzyme digestion products are recovered by glue and used for subsequent experiments.
The linearized recombinant plasmid pGAPZ A-PAS_FragB_0067 was introduced into K.phaffii GS115-xyn competent cells, and the cells were selected on YPD plates containing 100. Mu.g/mL Zeocin, and cultured at 28-30℃for 2-3d until single colonies were grown.
5. Verification of recombinant Strain 0067
The single colony is picked, colony PCR is carried out by using a primer 5 (SEQ ID NO. 8) and a primer 6 (SEQ ID NO. 9), and whether the recombinant bacterium genome contains the Zeocin gene fragment is verified.
The PCR reaction system is shown in Table 6.
Table 6 colony PCR System of recombinant Strain 0067
According to the above system, 50. Mu.L per tube.
PCR conditions: 1) Pre-denaturation at 94℃for 5min; 2) Denaturation at 98 ℃,10s; 3) Annealing at 55 ℃ for 5s; 4) Extending at 72 ℃ for 25 seconds for 35 cycles; 5) The temperature is fully 72 ℃ and 10min. The PCR products were verified with 2.0% nucleic acid gel. The size of the PCR product of the verified gene fragment is 1482bp (SEQ ID NO. 10), and the electrophoresis chart of the verification result of the recombinant bacterium is shown in figure 3.
Example 4: and (5) film formation characterization detection of the biological film.
In fig. 4, a and B are graphs of results of observation under a 40 x microscope after a cell slide experiment of the starting bacteria GS115-xyn and the recombinant strain 0067, respectively, and it can be clearly seen that the recombinant strain has a better film forming effect than the starting bacteria, and a larger amount of biofilm is formed.
In fig. 5, a and B are the starting strain GS115-xyn and the recombinant strain 0067, respectively, and the observation result under a fluorescence microscope after the Con A staining shows that the recombinant strain shows more concentrated green than the starting strain, and the film forming effect is better.
FIG. 6 shows a semi-quantitative biofilm assay by crystal violet staining, in which 10. Mu.L of bacterial solutions of the starting strain and the recombinant strain were added to 96-well plates each containing 190. Mu.L of YPD, and the OD was measured by crystal violet staining and enzyme-labeled instrument every 24 hours when the culture was carried out for 1-3 days 570 . From the figure it can be seen that the recombinant strain 0067 has better film forming effect than GS115-xyn.
Example 5 determination of the relative expression level of PAS_FragB_0067 Gene in recombinant bacteria.
Taking a 48-hour sample of free cultured pichia pastoris recombinant bacteria, centrifugally collecting cells, sending the cells to a qing, determining the PAS_FragB_0067 gene expression quantity in the recombinant bacteria by using qRT-PCR, taking an action as an internal reference gene, and calculating the relative expression quantity of the genes by using a relative quantitative method. As a result, as shown in FIG. 7, the PAS_FragB_0067 gene expression level in the recombinant bacterium was 3673.4 times the PAS_FragB_0067 gene expression level in the starting bacterium.
EXAMPLE 6 xylanase production experiment by immobilized recombinant bacteria in single batch fermentation
1. The activating medium per liter was composed as follows: 20g of peptone, 10g of yeast powder, 20g of glucose and 0.4mg of biotin, and the solvent is water.
The seed medium per liter was composed as follows: 20g of peptone, 10g of yeast powder, 10g of glycerol, 13.4g of YNB, 0.3g of dipotassium phosphate trihydrate, 1.18g of potassium dihydrogen phosphate and 0.4mg of biotin, and the solvent is water.
The fermentation medium per liter comprises the following components: 20g of peptone, 10g of yeast powder, 13.4g of YNB, 0.3g of dipotassium hydrogen phosphate, 1.18g of monopotassium phosphate and 0.4mg of biotin, wherein the solvent is water, and a pretreated cotton fiber fabric carrier (40 g/L fermentation medium) is added, wherein the pretreatment is to cut the cotton fiber fabric into squares of 4cm multiplied by 4cm, clean and dry the cotton fiber fabric with pure water, soak the cotton fiber fabric in ethanol for 1h, clean the cotton fiber fabric with pure water, and dry the cotton fiber fabric after boiling water bath for 30 min.
2. Activating: 0067X was removed from the-80℃refrigerator, 5mL of activation medium was prepared in test tubes, inoculated in 50. Mu.L, and incubated in a shaker at 30℃for 24h at 250rpm.
And (3) switching: after activation, the mixture was poured into 250mL shake flasks containing 50mL of seed medium, and cultured at 30℃and 250rpm for 24 hours to obtain seed solutions.
Fermentation: the fermentation broth was sub-packed in 500mL shake flasks, with 100mL of liquid loading at 115℃for 20min. Centrifuging the seed solution at 4500rpm for 5min, discarding supernatant, inoculating bacterial mud into fermentation medium, and fermenting at 30deg.C and 250rpm for 4d. The induction of xylanase expression was performed by adding 1% v/v methanol (relative to the volume of fermentation medium) every 24h during fermentation.
As shown in FIG. 8, the xylanase is produced by immobilized recombinant bacteria through single fermentation, and the yield can reach 4905.8U/mL. At the same time, compared with recombinant bacteria, xylanase obtained by immobilized single fermentation of the starting bacteria has lower enzyme activity, and the enzyme activity of enzyme produced by immobilized single fermentation of the recombinant bacteria is 1.57 times of that of the starting bacteria.
Comparative example 1 starting bacterial immobilization single batch enzyme production.
The recombinant bacteria inoculated in example 6 are replaced by the starting bacteria GS115-xyn, the rest steps are the same as those of example 6, and the enzyme activity of the fermentation product is detected as shown in FIG. 8.
Example 7 comparative xylanase enzyme activities by biofilm immobilized continuous fermentation.
The experiment is divided into 5 batches of continuous fermentation, which takes 600 hours, and single batch fermentation is the same as that of example 6, cotton fiber is taken as a carrier, samples are taken every 24 hours, and the fermentation of one batch is finished when the culture is completed to 120 hours. After each batch of fermentation, the immobilized carrier is transferred to a new medium for cultivation in a sterile bench. And so on until the end of the fermentation of the 5 th batch. The immobilized continuous fermentation enzyme activity of the recombinant bacteria is shown in figure 9. By immobilized continuous fermentation, cotton fiber is used as a carrier, and when the initial strain is fermented to the 3 rd batch, the enzyme production capability is obviously reduced, and the 5 th batch of enzyme activity of the initial strain is 28.48% of that of the first batch. The recombinant strain can stably and continuously carry out at least 5 batches of immobilized fermentation, and after 5 batches of immobilized fermentation, the enzyme activity of enzyme produced by the fermentation of the recombinant strain is about 3.90 times of that of the starting strain.
Comparative example 2 enzyme production by immobilized continuous fermentation of starting bacteria.
The recombinant bacteria inoculated in example 7 are replaced by the starting bacteria GS115-xyn, the rest steps are the same as those of example 7, and the enzyme activity of the fermentation product is detected and is shown in figure 9.
The invention provides a recombinant pichia pastoris genetically engineered bacterium, a construction method thereof and a thinking and a method for increasing application of a biological film in xylanase production, particularly the method and the way for realizing the technical scheme are many, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by a person of ordinary skill in the art without departing from the principle of the invention, and the improvements and the modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. The recombinant pichia pastoris gene engineering bacteria is characterized in that the gene engineering bacteria are obtained by over-expressing flocculation genes PAS_FragB_0067 in pichia pastoris, wherein the pichia pastoris is pichia pastoris expression bacteria capable of producing xylanaseKomagataella phaffii GS115-xyn, the flocculation gene PAS_FragB_0067 NCBI accession number XM_002490834.1.
2. The construction method of recombinant pichia pastoris engineering bacteria according to claim 1, comprising the steps of:
(1) Pichia pastoris expression strainKomagataella phaffii GS115-xyn is used as a template, and PAS_FragB_0067 gene fragments are amplified by PCR;
(2) Cloning the PAS_FragB_0067 gene fragment obtained in the step (1) onto an expression plasmid to obtain a recombinant plasmid;
(3) And (3) linearizing the recombinant plasmid obtained in the step (2), then converting the linearized recombinant plasmid into pichia pastoris competent cells, screening to obtain recombinant pichia pastoris genetic engineering bacteria, and naming the recombinant pichia pastoris genetic engineering bacteria as 0067.
3. The construction method according to claim 2, wherein the recombinant pichia pastoris is obtained by screening on a yeast extract peptone glucose agar medium resistant plate containing Zeocin.
4. The use of the recombinant pichia pastoris gene engineering bacteria according to claim 1 in biomembrane immobilized fermentation.
5. The use of the recombinant pichia pastoris gene engineering bacteria according to claim 1 in the fermentative production of xylanase.
6. The use according to claim 5, wherein the recombinant pichia pastoris engineered strain produces xylanase by immobilized fermentation.
7. The use according to claim 6, wherein the xylanase enzyme solution is obtained by inoculating the bacterial sludge of the recombinant pichia pastoris genetic engineering bacteria into a fermentation medium containing the immobilized carrier, and fermenting, wherein methanol is added during the fermentation process to induce the expression of xylanase.
8. The use according to claim 7, wherein the immobilization carrier is any one or a combination of several of cotton fiber fabric, non-woven fabric, polyester fiber, polyvinyl alcohol fiber, zeolite, bacterial cellulose film, silk, bagasse and corn stover, and the amount of the immobilization carrier is 2-80g/L fermentation medium.
9. The use according to claim 7, wherein the recombinant pichia pastoris is obtained by centrifugation of seed fluid of genetically engineered bacteria; the components of the fermentation medium are as follows: 10-30 parts of peptone g/L, 5-15 parts of yeast powder g/L, 10-20 parts of yeast nitrogen source without amino groups g/L, 0.1-1 parts of dipotassium hydrogen phosphate g/L, 1-5 parts of potassium dihydrogen phosphate g/L and 0.1-5 parts of biotin mg/L, wherein the solvent is water.
10. The use according to claim 7, wherein the fermentation culture conditions are: culturing at 28-30deg.C and 200-300rpm for 3-5d, and adding methanol every 24-h in the fermentation process, wherein the addition amount of methanol is 0.1-2% of the volume of fermentation medium.
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