CN112592844B

CN112592844B - Recombinant pichia pastoris genetically engineered bacterium, construction method thereof and application thereof in xylanase production

Info

Publication number: CN112592844B
Application number: CN202110029601.5A
Authority: CN
Inventors: 应汉杰; 牛欢青; 宋佳睿; 闵志迪; 陈勇; 刘庆国; 柳东
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2021-01-11
Filing date: 2021-01-11
Publication date: 2023-08-18
Anticipated expiration: 2041-01-11
Also published as: CN112592844A

Abstract

The invention discloses a recombinant pichia pastoris genetically engineered bacterium, a construction method thereof and application thereof in xylanase production. The recombinant pichia pastoris gene engineering bacteria are used for over-expressing lectin protein gene LMAN2 in the pichia pastoris. The recombinant strain strengthens the biological film forming capability of pichia pastoris, and solves the problems that the pichia pastoris in the prior art has weak film forming capability and cannot be used for continuous immobilized fermentation. Specifically, under the free fermentation condition, the xylanase enzyme activity of the pichia pastoris genetically engineered bacteria is improved by 48.9% compared with that of the original bacteria; under the single immobilized fermentation condition, the xylanase enzyme activity of the pichia pastoris genetic engineering bacteria is improved by 38.8 percent compared with that of the original bacteria, and the recombinant bacteria can stably and continuously perform immobilized fermentation for at least seven batches to produce enzymes.

Description

Recombinant pichia pastoris genetically engineered bacterium, construction method thereof and application thereof in xylanase production

Technical Field

The invention belongs to the technical fields of genetic engineering and fermentation engineering, and in particular relates to recombinant pichia pastoris genetic engineering bacteria, a construction method thereof and application thereof in xylanase production.

Background

Xylanase is widely applied in industrial production, such as paper industry, food industry, feed industry, energy industry and the like. The main method of xylanase production is microbial fermentation. The microbial fermentation method has the advantages of wide sources of production raw materials, low cost, less environmental pollution in the production process, easy operation, safety, various strains, large-scale production and the like. Along with the maturation and development of molecular biology technology, the genetic engineering technology is used for carrying out molecular transformation on the strain, so that the strain becomes an effective means for obtaining xylanase high-yield strain at present.

The biological film is widely existed in the nature, and in the biological film forming process, the extracellular polymer secreted by the microorganism is the material basis of the biological film forming, has the characteristic of layered distribution, and plays a key role in the adhesion and aggregation characteristics of the microorganism. Intercellular adhesion between yeast cells is commonly referred to as "flocculation", and adhesion is conferred by a specific cell surface protein "adhesin" that is capable of binding specific amino acids or sugar residues on other cell surfaces or facilitating binding to abiotic surfaces. The phenomenon of yeast biofilm formation has been widely studied in candida or saccharomyces cerevisiae. There are many different yeast adhesins, such as the FLO1, FLO5, FLO9, FLO10 and FLO11 genes of the FLO gene family carried by Saccharomyces cerevisiae, the ALS and EAP genes carried by Candida albicans, the EPA genes carried by Candida glabrata, and the like. Adhesion can be classified into lectin-like adhesion (sugar-sensitive) and sugar-insensitive adhesion, LMAN2 belongs to a lectin gene, which is associated with glycoprotein transport.

Pichia pastoris is used as a eukaryotic expression system for efficiently expressing exogenous proteins, has the advantages of stable heredity, high expression efficiency, post-translational processing of proteins and the like, has important application prospect in industrial production, has very weak biological film forming capability, and is difficult to perform immobilized fermentation, in particular to perform immobilized continuous fermentation. At present, researches on biological film-forming genes in pichia pastoris and immobilized fermentation through biological film-forming adsorption are freshly reported. Therefore, the invention provides a recombinant pichia pastoris genetically engineered bacterium to effectively solve the technical problems.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing a recombinant pichia pastoris gene engineering bacterium for over-expressing an LMAN2 gene aiming at the defects of the prior art.

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.

In order to solve the first technical problem, the invention discloses a recombinant pichia pastoris gene engineering bacterium, which over-expresses lectin protein gene LMAN2 in the pichia pastoris.

Wherein the pichia pastoris is Pichia Pastoris GS115,115.

Wherein the nucleotide sequence of the lectin protein gene LMAN2 is shown as SEQ ID NO. 1.

In order to solve the second technical problem, the invention discloses a construction method of the recombinant pichia pastoris genetically engineered bacterium, which comprises the following steps:

(1) PCR amplifying LMAN2 gene fragment with Pichia pastoris genome as template;

(2) Cloning the LMAN2 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, and screening to obtain recombinant pichia pastoris genetic engineering bacteria.

In the step (1), the pichia pastoris is preferably p.pastoris GS115.

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 pGAPZαA.

In order to solve the third technical problem, the invention discloses application of the recombinant pichia pastoris genetically engineered strain in xylanase production.

Wherein, the recombinant pichia pastoris gene engineering bacteria can produce xylanase by any one of the following modes:

(i) Free fermentation: inoculating recombinant pichia pastoris genetic engineering bacterial sludge into a fermentation medium, and fermenting to obtain xylanase enzyme liquid;

(ii) And (3) fixed fermentation: inoculating recombinant pichia pastoris genetic engineering bacterial sludge into a fermentation medium containing an immobilized carrier, and fermenting to obtain xylanase enzyme liquid;

(iii) Immobilized continuous fermentation: inoculating recombinant pichia pastoris genetic engineering bacterial sludge into a fermentation medium containing an immobilized carrier, and fermenting to obtain xylanase enzyme liquid; after each batch of fermentation is finished, the fermentation broth obtained in the previous batch is replaced by a new fermentation medium, and the fermentation is repeated.

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 28-30deg.C and 200-300rpm for 16-24 hr, preferably at 30deg.C and 250rpm for 24 hr.

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, 5-15g/L, YNB-20 g/L of glycerol, 0.1-1g/L of dipotassium hydrogen phosphate, 1-5g/L of potassium dihydrogen phosphate and 0.1-5mg/L of biotin, 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.

In step (b), the solvent of the seed medium is water.

In the step (b), the culture is carried out at 28-30 ℃ and 200-300rpm for 16-24 hours; preferably at 30℃and 250rpm for 24 hours.

In the above (ii) stationary fermentation and (iii) stationary continuous fermentation, the stationary carrier cotton fiber fabric, nonwoven fabric, polyester fiber, polyvinyl alcohol fiber, zeolite, bacterial cellulose membrane, silk, bagasse and corn stover, or any one or a combination of several thereof; 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; 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.

Wherein the solvent of the fermentation medium is water.

Preferably, methanol is added during the fermentation to induce xylanase expression.

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.

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 4d.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

the invention constructs a Pichia pastoris gene engineering strain for over-expressing LMAN2 for the first time, strengthens 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. Specifically, under the free fermentation condition, the xylanase enzyme activity of the pichia pastoris genetically engineered bacteria is improved by 48.9% compared with that of the original bacteria; under the single immobilized fermentation condition, the xylanase enzyme activity of the pichia pastoris genetic engineering bacteria is improved by 38.8 percent compared with that of the original bacteria, and the recombinant bacteria can stably and continuously perform immobilized fermentation for at least seven batches to produce enzymes.

Drawings

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.

FIG. 1 is a schematic representation of the expression plasmid pGAPZαA-LMAN2.

FIG. 2A is a PCR electrophoresis diagram of a target gene LMAN2, lane M is a DL 5000DNA Marker, and lane 1 is the target gene LMAN2; FIG. B is a PCR electrophoretogram of pGAPZ alpha A vector fragment, lane M is DL 5000DNA Marker, and lane 1 is pGAPZ alpha A vector fragment; panel C is an electrophoretogram of recombinant plasmid pGAPZαA-LMAN2, wherein lane M is DL 5000DNA Marker, and lane 1 is recombinant plasmid pGAPZαA-LMAN2.

FIG. 3 is a verification diagram of recombinant GS115-LMAN2, wherein lane M is DL 1000DNA Marker, and lanes 1 and 2 are resistance gene Zeocin fragments.

Fig. 4 is a fluorescence microscopy image of p.pastoris GS115 starting bacteria and recombinant bacteria, wherein fig. a is a fluorescence microscopy image of starting bacteria p.pastoris GS115 and fig. B is a fluorescence microscopy image of recombinant strain GS115-LMAN 2.

FIG. 5 is a graph showing the experimental results of semi-quantitative determination of the amount of the biological membrane by the crystal violet staining method of the starting strain and the recombinant strain.

FIG. 6 is a graph showing comparison of xylanase activities produced by fermentation of starting and recombinant bacteria.

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 an overexpression plasmid for overexpressing the LMAN2 Gene

1. Amplification of the target Gene LMAN 2: using the extracted P.pastoris GS115 genome as a template, the gene LMAN2 was amplified with primer 1 (SEQ ID NO. 2) and primer 2 (SEQ ID NO. 3).

The PCR reaction system is shown in Table 1.

TABLE 1 target gene LMAN2 amplification PCR system

The mixture was dispensed into 25. Mu.L of each tube according to the above-mentioned system.

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 15 seconds for 35 cycles; 5) The temperature is fully 72 ℃ and 10min.

The size of the amplified product of the target gene LMAN2 is 1378bp (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 alpha A and target fragment amplification

1) Extraction of expression plasmid pGAPZalpha A: coli TOP 10 glycerol bacteria harboring plasmid pGAPZa 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; the plasmid pGAPZαA was extracted by the procedure described in the instructions of the AxyPrep plasmid DNA minikit.

2) Amplification of pGAPZ alpha A fragments

pGAPZ alpha A fragment was amplified using the linearized pGAPZ alpha 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 2.

TABLE 2 amplification PCR System of expression plasmid pGAPZ alpha A

Component (A)	Volume (mu L)	Manufacturing factories
			PrimeSTAR Max Premix(2×)	25	TAKARA BIO INC.
Primer 3 (10. Mu.M)	2	Prime organism
			Primer 4 (10. Mu.M)	2	Prime organism
Stencil (pGAPZ alpha A plasmid)	1
			Sterile water	20

PCR conditions: 1) Pre-denaturation at 94℃for 5min; 2) Denaturation at 98 ℃,10s; 3) Annealing at 50 ℃ for 15s; 4) Extending at 72 ℃ for 10 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 alpha A fragment has the size of 2857bp (SEQ ID NO. 6), and the electrophoresis chart is shown in figure 2B. The PCR product was purified by TAKARA gel recovery kit and used in the subsequent experiments.

3. Construction of recombinant plasmids

And (2) connecting the target gene fragment obtained in the step (1) with the pGAPZ alpha A fragment obtained in the step (2) according to the steps of the specification of the ClonExpII one-step cloning kit to obtain the recombinant plasmid pGAPZ alpha A-LMAN2. The ligation reaction system was shown in Table 3, and after 30min in a 37℃water bath, the mixture was immediately ice-bathed for 5min and introduced into competent cells of E.coli DH 5. Alpha. And the ligation reaction system was shown in Table 3.

TABLE 3 ligation reaction System

Component (A)	Volume (mu L)
		PCR products of pGAPZa A vector	1
Target gene LMAN2	1.2
		5*CEⅡBuffer	4
ExnaseⅡ	2
		Sterile water	11.8
Totalizing	20

The ligation solution obtained above was transferred into 100. Mu.L of E.coli DH 5. Alpha. Competent cells, resuscitated and plated on LB-resistant plates (25. Mu.g/mL Zeocin), and cultured at 37℃for 12 hours until single colonies were grown.

The single colony of the plate is picked, inoculated in LB liquid medium (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 4235bp (SEQ ID NO. 7), pGAPZalpha A-LMAN2 is shown in figure 1, and its electrophoresis diagram is shown in figure 2C.

Example 2: construction of Pichia pastoris engineering bacteria over-expressing LMAN2 gene

1. Transformation of recombinant plasmids

Plasmid pGAPZαA-LMAN2 was digested tangentially with AvrII.

The linearization system of the recombinant plasmid pGAPZalpha A-LMAN2 is shown in Table 4.

TABLE 4 linearization System for recombinant plasmid pGAPZ alpha A-LMAN2

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 pGAPZαA-LMAN2 was introduced into P.Pastois GS115 competent cells, and the cells were selected on a yeast extract peptone glucose agar medium (YPD) resistant plate (100. Mu.g/mL Zeocin) containing 100. Mu.g/mL Zeocin, and cultured at 28-30℃for 2-3d to give single colonies.

2. Verification of recombinant strain GS115-LMAN2

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 5.

TABLE 5 colony PCR System of recombinant Strain GS115-LMAN2

Component (A)	Volume (mu L)	Manufacturing factories
			PrimeSTAR Max Premix(2×)	25	TAKARA BIO INC.
Primer 5 (10. Mu.M)	2	Prime organism
			Primer 6 (10. Mu.M)	2	Prime organism
Template	1
			Sterile water	20

PCR conditions: 1) Pre-denaturation at 94℃for 5min; 2) Denaturation at 98 ℃,10s; 3) Annealing at 50 ℃ for 15s; 4) Extending at 72 ℃ for 5 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 Zeocin gene fragment is 358bp (SEQ ID NO. 10), and the electrophoresis chart of the verification result of the recombinant bacteria is shown in figure 3.

Example 3: film formation characterization detection of biological films

In FIG. 4, A and B are graphs of results of detection by a fluorescence microscope after dyeing with FITC-ConA dye solution, respectively, of the starting strain P.pastoris GS115 and the recombinant strain GS115-LMAN2, and it is obvious that the recombinant strain has a better film forming effect than the starting strain and a larger amount of formed biological film.

FIG. 5 shows a semi-quantitative biofilm assay by crystal violet staining, in which 20. Mu.L of bacterial solutions of the starting strain and the recombinant strain were added to 96-well plates each containing 200. Mu.L of YPD, and the OD570 was measured every 12 hours by crystal violet staining and an enzyme-labeled instrument when the culture was performed for 3-5 days. From the figure, it can be seen that the recombinant strain GS115-LMAN2 has better film-forming effect than p.pastoris GS115.

Example 4: determination 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 6.

TABLE 6 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 5: experiment for producing xylanase by free fermentation of recombinant bacteria

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, and the solvent is water.

2. Activating: GS115-LMAN2 was removed from the-80℃refrigerator and 5mL of the activation medium was prepared in a tube with an inoculum size of 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.

3. The enzyme activity of xylanase produced by free fermentation of recombinant bacteria is shown in figure 6.

Example 6: xylanase production experiment by single immobilized fermentation of recombinant bacteria

The fermentation broth of example 5 was added to a pretreated cotton fiber fabric support (40 g/L fermentation medium) and sterilized, the remainder of the procedure being as in example 5. The enzyme activity of xylanase produced by single immobilized fermentation of recombinant bacteria is shown in figure 6.

The pretreatment is to cut 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 in a boiling water bath for 30 min.

Comparative example 1: free enzyme production of original bacteria

The recombinant bacteria inoculated in example 5 were replaced with the starting bacteria P.pastoris GS115, and the enzyme activity of the fermentation product obtained by the detection of the rest steps in example 5 is shown in FIG. 6.

As can be seen from FIG. 6, the xylanase obtained by the free fermentation of the original bacteria has lower enzyme activity compared with the free fermentation of the recombinant bacteria, and the enzyme activity produced by the free fermentation of the recombinant bacteria is 48.9% higher than that of the original bacteria.

Comparative example 2: single immobilized enzyme production of original bacteria

The recombinant bacteria inoculated in example 6 were replaced with the original starting bacteria P.pastoris GS115, and the enzyme activity of the fermentation product obtained by the detection of the rest steps in example 6 is shown in FIG. 6.

As can be seen from FIG. 6, the enzyme activity of the enzyme produced by the single immobilized fermentation of the original recombinant bacterium is improved by 38.8% compared with that of the single immobilized fermentation of the original recombinant bacterium.

Example 7: xylanase production experiment by immobilized continuous fermentation of recombinant bacteria

In the immobilized fermentation process, recombinant bacterial thallus is adsorbed on an immobilized carrier in the first batch, fermentation liquid is poured out after shaking and culturing for 4d, the immobilized carrier adsorbed with the bacterial is left, a new 100mL fermentation medium is poured into the immobilized carrier for the second batch of fermentation, culturing for 4d, and the enzyme activity of xylanase is measured, and the enzyme activity of xylanase is shown in Table 7. The batch immobilization and continuous fermentation were followed by this procedure. The rest of the procedure is the same as in example 6.

Comparative example 3: original bacterium immobilized continuous fermentation enzyme production

The recombinant bacteria inoculated in example 7 were replaced with the original starting bacteria P.pastoris GS115, and the enzyme activities of the fermentation products obtained by the detection of the rest steps in example 7 are shown in Table 7.

As can be seen from table 7, the enzyme production ability of the original bacteria at the fourth lot of immobilized fermentation starts to be significantly reduced as compared with immobilized fermentation of the recombinant bacteria, which is capable of stably and continuously performing at least seven lots of immobilized fermentation. The number of times of continuous immobilized fermentation of the recombinant bacterium can be increased because the film forming capability of the recombinant bacterium is enhanced by over-expressing the LMAN2 gene.

TABLE 7 enzyme Activity (U/mL) of enzyme produced by immobilized continuous fermentation of starting and recombinant bacteria

Batch of	A first part	Two (II)	Three kinds of	Fourth, fourth	Five kinds of	Six kinds of	Seven pieces of
								Bacteria producing	2899.1	2174.3	1916.8	1291.4	883.6	591.3	502.4
Recombinant bacterium	4024.0	3947.7	4098.3	4172.6	4086.1	4156.2	4013.5

The invention provides a recombinant pichia pastoris genetically engineered bacterium, a construction method thereof and an application thought and a method in xylanase production, and the method for realizing the technical scheme is a plurality of methods and approaches, the above is only a preferred embodiment of the invention, and it is 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 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.

Sequence listing

<110> university of Nanjing Industrial science

<120> recombinant Pichia pastoris genetically engineered bacterium, construction method thereof and application thereof in xylanase production

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<211> 2857

<212> DNA

<213> amplification products of pGAPZaA fragment (Artificial Sequence)

<400> 6

gtctcggatc ggtacctcga gccgcggcgg ccgccagctt tctagaacaa aaactcatct 60

cagaagagga tctgaatagc gccgtcgacc atcatcatca tcatcattga gttttagcct 120

tagacatgac tgttcctcag ttcaagttgg gcacttacga gaagaccggt cttgctagat 180

tctaatcaag aggatgtcag aatgccattt gcctgagaga tgcaggcttc atttttgata 240

cttttttatt tgtaacctat atagtatagg attttttttg tcattttgtt tcttctcgta 300

cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt aggggtttgg 360

gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca gagtacagaa 420

gattaagtga gaccttcgtt tgtgcggatc ccccacacac catagcttca aaatgtttct 480

actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa 540

cacccaagca cagcatacta aattttccct ctttcttcct ctagggtgtc gttaattacc 600

cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa 660

aggcaataaa aatttttatc acgtttcttt ttcttgaaat tttttttttt agtttttttc 720

tctttcagtg acctccattg atatttaagt taataaacgg tcttcaattt ctcaagtttc 780

agtttcattt ttcttgttct attacaactt tttttacttc ttgttcatta gaaagaaagc 840

atagcaatct aatctaaggg cggtgttgac aattaatcat cggcatagta tatcggcata 900

gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg 960

ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc 1020

cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc 1080

agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc 1140

ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc 1200

gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac 1260

ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt ccgacggcgg 1320

cccacgggtc ccaggcctcg gagatccgtc ccccttttcc tttgtcgata tcatgtaatt 1380

agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg 1440

agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag 1500

aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa 1560

cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa 1620

gctggagacc aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 1680

gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 1740

aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 1800

ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 1860

cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta 1920

ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 1980

cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 2040

agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 2100

gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 2160

gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 2220

tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 2280

agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 2340

agggattttg gtcatgcatg agatcagatc ttttttgtag aaatgtcttg gtgtcctcgt 2400

ccaatcaggt agccatctct gaaatatctg gctccgttgc aactccgaac gacctgctgg 2460

caacgtaaaa ttctccgggg taaaacttaa atgtggagta atggaaccag aaacgtctct 2520

tcccttctct ctccttccac cgcccgttac cgtccctagg aaattttact ctgctggaga 2580

gcttcttcta cggccccctt gcagcaatgc tcttcccagc attacgttgc gggtaaaacg 2640

gaggtcgtgt acccgaccta gcagcccagg gatggaaaag tcccggccgt cgctggcaat 2700

aatagcgggc ggacgcatgt catgagatta ttggaaacca ccagaatcga atataaaagg 2760

cgaacacctt tcccaatttt ggtttctcct gacccaaaga ctttaaattt aatttatttg 2820

tccctatttc aatcaattga acaactattt cgaaacg 2857

<210> 7

<211> 4235

<212> DNA

<213> recombinant plasmid (Artificial Sequence)

<400> 7

agatcttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca tctctgaaat 60

atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc cggggtaaaa 120

cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct tccaccgccc 180

gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc cccttgcagc 240

aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg acctagcagc 300

ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg catgtcatga 360

gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca attttggttt 420

ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca attgaacaac 480

tatttcgaaa cgttcacacg taatcatcaa cgatgttcaa aggagctagg atcctaatag 540

ctctaatcgt ctgcattctt ttgtggagtg ctgtgtccta cactatttcc ttcttttcat 600

ctccaagtga ctccaaagtt gttaaacagg ttcaattgga tacaattttt gataatgcac 660

agatcaacgg attgctcaac aacgagatgc ccaaccttct caaagtcaac aagaaactcc 720

tacaggactt tagcatttca aagccatgga cctttgatca aattactgaa aaatttcaca 780

tcagaggagc ttctaccatg attgcaaggg ataagattag actggtcagg gattctcctc 840

aacaagttgg gctgattcaa tccaccaaac agttggaggt ttcgaaatat gagtctacct 900

cgctggagat tgacttcaat atcaactttg atgacgatgc gaagaaacgt ctcagtaaga 960

aactttacgg agacggtgta ggtgtttggt tgagtgaaaa tgaactacac tcaggcgccg 1020

ccttaggtgt tgatgataca actttcaagg gtgttgctgt gttgattgat acttatcaaa 1080

attctccaag tgcctctaaa ctgggcaaat tccctcgtgt gtctattcaa tattctcaag 1140

gtggtggtta catatatgat aagggtcaag atggaaagtc aaactatgaa ctcggttatt 1200

gtaacttgaa tctgaaatac ttgaattctc cttcaaaaat gagaataacc tatttgaaag 1260

attcagggta cctttctttg gactttggag ccaagaaaaa tggcatgcct ggctccgaat 1320

ggtttaactg tttcactaaa gagggtgtgg ttttgcctga aaaagtttac ttagcattga 1380

gttctgaatg tggcgcatta catcacaact cagacatcct gggaattgaa tggaacgctt 1440

taacggacga acatggtgac gtgctgtcat ctatcagcga cctttcgaat atactaaatg 1500

acgagtatat cgatgagtat gttcaaaacg aaagcgaaaa tgaagtcaag gaaagaattg 1560

ccaagcaacg ccatagaagg ctgacaaaag ataaacgtcc agaaccagag gcttctgaaa 1620

gaaggaaaac agctcaacga ctaaggaagg cagaggaaag gctgcgaagg cagcatcgtg 1680

aaaataacct aaacaaatat ggctatgagt caaggttgac atactttctt caaacagtat 1740

ggcttctatt aaaatggaca tttattatca taagcttttt gctagtgggt tttatattgt 1800

acaaccgaat ccgcaagctg aaaaagaaaa ctggcggatt cattgtatag gtttgatgta 1860

tgtataggtt gtctcggatc ggtacctcga gccgcggcgg ccgccagctt tctagaacaa 1920

aaactcatct cagaagagga tctgaatagc gccgtcgacc atcatcatca tcatcattga 1980

gttttagcct tagacatgac tgttcctcag ttcaagttgg gcacttacga gaagaccggt 2040

cttgctagat tctaatcaag aggatgtcag aatgccattt gcctgagaga tgcaggcttc 2100

atttttgata cttttttatt tgtaacctat atagtatagg attttttttg tcattttgtt 2160

tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt 2220

aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca 2280

gagtacagaa gattaagtga gaccttcgtt tgtgcggatc ccccacacac catagcttca 2340

aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac 2400

cacttcaaaa cacccaagca cagcatacta aattttccct ctttcttcct ctagggtgtc 2460

gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct 2520

tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaat tttttttttt 2580

agtttttttc tctttcagtg acctccattg atatttaagt taataaacgg tcttcaattt 2640

ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc ttgttcatta 2700

gaaagaaagc atagcaatct aatctaaggg cggtgttgac aattaatcat cggcatagta 2760

tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc 2820

cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct 2880

cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac 2940

cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg 3000

ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg 3060

ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc 3120

cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt 3180

ccgacggcgg cccacgggtc ccaggcctcg gagatccgtc ccccttttcc tttgtcgata 3240

tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg 3300

aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 3360

tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 3420

acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 3480

taatttgcaa gctggagacc aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 3540

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 3600

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 3660

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 3720

ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca 3780

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 3840

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 3900

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 3960

cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 4020

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 4080

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 4140

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 4200

actcacgtta agggattttg gtcatgcatg agatc 4235

<210> 8

<211> 20

<212> DNA

<213> primer 5 (Artificial Sequence)

<400> 8

atggccaagt tgaccagtgc 20

<210> 9

<211> 20

<212> DNA

<213> primer 6 (Artificial Sequence)

<400> 9

ccacgaagtg cacgcagttg 20

<210> 10

<211> 358

<212> DNA

<213> Zeocin

<400> 10

atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc 60

gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt 120

gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac 180

aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag 240

gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag 300

ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtgg 358

Claims

1. The application of the recombinant pichia pastoris gene engineering bacteria in xylanase production is characterized in that the recombinant pichia pastoris gene engineering bacteria are used for over-expressing lectin protein gene LMAN2 in the pichia pastoris; the pichia pastoris is Pichia Pastoris GS; the nucleotide sequence of the lectin protein gene LMAN2 is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein the construction method of the recombinant pichia pastoris gene engineering bacteria comprises the following steps:

(1) PCR amplifying LMAN2 gene fragment with Pichia pastoris genome as template;

3. The use according to claim 1, wherein the recombinant pichia pastoris engineered strain produces xylanase by episomal fermentation.

4. The use according to claim 1, wherein the recombinant pichia pastoris engineered strain produces xylanase by means of immobilized fermentation.

5. The use according to claim 1, wherein the recombinant pichia pastoris engineered strain produces xylanase by immobilized continuous fermentation.

6. The use according to claim 4 or 5, characterized in that the amount of immobilized carrier is 2-80g/L fermentation medium.

7. The use according to claim 1, wherein the xylanase-producing fermentation medium comprises the following concentrations of the components: 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 monopotassium hydrogen phosphate and 0.1-5mg/L of biotin.