CN117247851A - Strain for improving methanol utilization efficiency of pichia pastoris as well as preparation method and application thereof - Google Patents

Strain for improving methanol utilization efficiency of pichia pastoris as well as preparation method and application thereof Download PDF

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CN117247851A
CN117247851A CN202310570767.7A CN202310570767A CN117247851A CN 117247851 A CN117247851 A CN 117247851A CN 202310570767 A CN202310570767 A CN 202310570767A CN 117247851 A CN117247851 A CN 117247851A
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strain
badfd
utilization efficiency
pichia pastoris
plasmid
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杨艳坤
李康
杨少杰
白仲虎
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Jiangnan University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01046—Formaldehyde dehydrogenase (1.2.1.46)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Abstract

The invention discloses a strain for improving methanol utilization efficiency of pichia pastoris, and a preparation method and application thereof, wherein the strain comprises the following components: the strain is a strain with the Badfd gene knocked out. The invention provides a preparation method and application of a bacterial strain for improving methanol utilization efficiency of a K.phaffi GS115 bacterial strain. The provided strain can relieve the strain growth environmental pressure caused by excessive formaldehyde and formic acid accumulation, the modified engineering strain can enter a stable period faster than the original strain, and the OD 600 Higher than the original strain.

Description

Strain for improving methanol utilization efficiency of pichia pastoris as well as preparation method and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a method for improving methanol utilization efficiency of pichia pastoris and a preparation method and application thereof.
Background
Pichia pastoris (Komagataela phafi, K.phafi) GS115 is a methyl nutrition type microorganism, and methanol can be used as a carbon source to produce high-value organic matters, so that grain crisis and energy crisis can be effectively relieved. Chinese patent document CN 114634946A (application No. 202210181077.8) discloses construction of Pichia pastoris genetic engineering bacteria and application thereof in improving methanol assimilation rate and fixing carbon dioxide. Taking K.phaffi GS115 as an original strain, knocking out a Formate Dehydrogenase (FDH) gene of a methanol catabolism pathway to reduce the loss of carbon atoms, and knocking out a das gene to block the methanol catabolism pathway of the K.phafi GS115-FDH strain; the mis1, gcv2, gcv3 are over expressed to construct a new methanol assimilation pathway; the fld and fgh genes of the methanol catabolism pathway are positioned in peroxisomes to further improve the utilization rate of methanol.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide a strain for improving the methanol utilization efficiency of pichia pastoris.
In order to solve the technical problems, the invention provides the following technical scheme: a strain that increases methanol utilization efficiency of pichia pastoris, comprising: the strain for improving the methanol utilization efficiency of the pichia pastoris is a strain for knocking out the Badfd gene.
As a preferable scheme of the strain for improving the methanol utilization efficiency of pichia pastoris, the invention comprises the following steps: the Badfd gene knockout process strain contains SX1628-71sg-Badfd plasmid, and the sequence of the SX1628-71sg-Badfd plasmid is shown in SEQ ID No. 1.
Another object of the present invention is to provide a method for preparing a strain that improves methanol utilization efficiency of Pichia pastoris.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of a strain for improving methanol utilization efficiency of pichia pastoris comprises the following steps:
preparing a plasmid: knocking out the Badfd gene from the prepared plasmid, and preparing to obtain a plasmid with the Badfd gene knocked out;
preparing a strain: and transforming the prepared homologous arm fragments of the knockout Badfd gene and plasmids of the knockout Badfd gene into the competence of the GS115 strain to prepare the GS 115-delta Badfd strain.
As a preferred scheme of the preparation method of the strain for improving the methanol utilization efficiency of pichia pastoris, the invention comprises the following steps: in preparing the plasmid, the prepared plasmid is plasmid SX1628-71sgXG.
As a preferred scheme of the preparation method of the strain for improving the methanol utilization efficiency of pichia pastoris, the invention comprises the following steps: the prepared strain was GS115.
As a preferred scheme of the preparation method of the strain for improving the methanol utilization efficiency of pichia pastoris, the invention comprises the following steps: preparation of plasmid As preparation of plasmid SX1628-71sgXG, PCR was performed using the complementary sequence of the first 6 bases of the sg fragment, the HH sequence, and the sg fragment, and after completion of PCR, the template was removed, and the fragments SX1628-71sg-Badfd-1 and SX1628-71sg-Badfd-2 were recovered, and the SX1628-71sg-Badfd was obtained by homologous recombination, and the strain carrying the SX1628-71sg-Badfd was cultured to obtain the SX1628-71sg-Badfd plasmid.
As a preferred scheme of the preparation method of the strain for improving the methanol utilization efficiency of pichia pastoris, the invention comprises the following steps: in the preparation of the strain, the homologous arm fragments of the Badfd gene are knocked out by taking the 3 'end and the 5' end of the Badfd gene as templates.
As a preferred scheme of the preparation method of the strain for improving the methanol utilization efficiency of pichia pastoris, the invention comprises the following steps: in preparing the strain, the constructed homologous arm fragment of the knockout Badfd gene is introduced into competent cells of the GS115 strain.
As a preferred scheme of the preparation method of the strain for improving the methanol utilization efficiency of pichia pastoris, the invention comprises the following steps: the GS 115-. DELTA.Badfd strain was able to grow in an environment in which methanol was used as a carbon source.
As a preferred scheme of the preparation method of the strain for improving the methanol utilization efficiency of pichia pastoris, the invention comprises the following steps: the GS 115-. DELTA.Badfd strain was able to grow in an environment where methanol was the only carbon source or the main carbon source.
The invention has the beneficial effects that:
in order to further explore a method for improving the utilization rate of methanol and improve the OD (optical density) of a strain in the condition that methanol is taken as a sole carbon source, taking into consideration that the excessive metabolism of methanol can cause the growth environment pressure of the strain, knocking out the Badfd genes of bifunctional enzymes (Bifunctional enzyme with alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase) of alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase, and verifying engineering strains.
The invention discloses a strain construction flow for improving the methanol utilization efficiency of a K.phaffi GS115 strain and application thereof. The prior art means can not be comprehensive, and the invention can complement and perfect the blank of the technology for improving the utilization rate of the methanol.
The existing CRISPR technology in the laboratory is utilized to delete the Badfd gene, thereby reducing the strain growth environmental pressure caused by excessive formaldehyde and formic acid accumulation, the modified engineering strain can enter the stabilization period faster than the original strain, and the OD 600 Higher than the original strain.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a schematic diagram of the structure of an artificially modified plasmid SX1628-71 sg-Badfd;
FIG. 2 is an electrophoresis diagram of SX1628-71sg-Badfd-2 fragment;
FIG. 3 is a graph of an alignment of SX1628-71sg-Badfd sequencing results;
FIG. 4 is a diagram of Badfd knockout homology arm construction electrophoresis;
FIG. 5 is a diagram of SX1628-71sg-Badfd mediated Badfd knockout electrophoresis;
FIG. 6 is a graph of the growth of the GS 115-. DELTA.Badfd strain versus the GS115 strain;
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The sources of the biological raw materials adopted in the embodiment of the invention are as follows:
the sources of biological materials are shown in table 1.
TABLE 1
The culture medium used in the embodiment of the invention is as follows:
(1) LLBZ liquid culture medium (g/L) comprises yeast extract 5, peptone 10, sodium chloride 5, bleomycin final concentration 25 μg/ml, pH 7.0;
(2) LLBZ solid culture medium (g/L) comprises yeast extract 5, peptone 10, sodium chloride 5, agar 20, bleomycin final concentration 25 μg/ml, and pH 7.0;
(3) YPDSZ liquid medium (g/L): yeast extract 10, peptone 20, sorbitol 182, glucose 20, bleomycin final concentration 100 μg/ml, pH 7.0;
(4) YPDSZ solid medium (g/L): yeast extract 10, peptone 20, sorbitol 182, glucose 20, agar 20, bleomycin final concentration 100 μg/ml, pH 7.0.
(5) YPDS liquid medium (g/L): yeast extract 10, peptone 20, sorbitol 182, glucose 20, ph 7.0;
(6) MMH liquid medium (g/L): YNB (yeast nitrogen source alkali contains no amino acid and ammonium sulfate) 13.4, histidine 0.04,
methanol 6% (V/V);
example 1
The purpose of this example was to construct a SX1628-71sg-Badfd plasmid:
1) The plasmid SX1628-71sg XG is taken as a template, a primer is designed to construct the SX1628-71sg-Badfd plasmid by PCR, and the plasmid can be taken as a knockout plasmid to knock out the Badfd gene. PCR is carried out on the primers 71-ad-sg1-F and 71-ad-sg1-R to obtain a SX1628-71sg-Badfd-1 fragment, the fragment consists of a complementary sequence of the front 6 bases of the sg fragment, an HH sequence and the sg fragment, a PCR system is shown in a table 1, and a PCR program is shown in a table 2; the primers SX1628-71sgXG-F and SX1628-71sgXG-R were subjected to PCR to obtain SX1628-71sg-Badfd-2 fragment (5751 bp), the PCR system was shown in Table 3, the PCR procedure was shown in Table 2, the DpnI elimination template was used after the PCR was completed, the system of the DpnI elimination template was shown in Table 4, and the procedure was shown in Table 5. Then, the fragment SX1628-71sg-Badfd-2 is subjected to nucleic acid electrophoresis, the electrophoresis diagram is shown in figure 2, agarose gel strips with correct molecular weight are cut, then gel recovery is carried out, and the concentration is measured for later use. The SX1628-71sg-Badfd plasmid sequence in the specification is shown as seq ID No. 1; the gene Badfd sequence is shown in seq ID No. 2.
TABLE 1 construction of PCR reaction System from SX1628-71sg-Badfd-1 fragment
TABLE 2 construction of PCR reaction program for SX1628-71sg-Badfd-1 fragment
Note that: denaturation and annealing performed for 30 cycles in table 2;
TABLE 3 construction of PCR System from SX1628-71sg-Badfd-2 fragment
TABLE 4 Dpn I reaction System
TABLE 5 Dpn I reaction procedure
2) The fragment SX1628-71sg-Badfd-1PCR product and the fragment SX1628-71sg-Badfd-2 were subjected to homologous recombination to construct a SX1628-71sg-Badfd plasmid, the recombination system is shown in Table 6, and the recombination procedure is shown in Table 7. JM109 was competent after the recombination was completed, and the plates were plated on LLBZ plates after incubation at 200rpm for 1 hour at 37℃on a shaker, and the plates were allowed to stand overnight at 37 ℃.
TABLE 6 homologous recombination System
TABLE 7 homologous recombination procedure
3) The single colony of the plate is selected and cultured in 3mL LLBZ liquid culture medium, and the liquid culture medium is placed in a shaking table at 37 ℃ and 200rpm for 16 hours, then plasmid sequencing is extracted, the sequencing result is shown in figure 3, and the plasmid with correct sequencing is preserved and placed at-20 ℃.
Example 2
The purpose of this example was to knock out construction of the Badfd gene 1) knockout homology arm in the GS115 strain. The GS115 genome is used as a template to construct a Badfd-knockout homology arm fragment. PCR was performed using primers ad-TY-F1 and ad-TY-R1 to obtain Badfd-knockout homology arm fragment 1 (1000 bp upstream of Badfd gene), the PCR system was shown in Table 8, and PCR was performed using primers ad-TY-F2 and ad-TY-R2 to obtain Badfd-knockout homology arm fragment 2 (1000 bp downstream of Badfd gene), the PCR system was shown in Table 9, and the PCR procedure was shown in Table 2; and (3) performing nucleic acid electrophoresis after the PCR is finished, wherein the electrophoresis result is shown in fig. 4 (a), performing gel recovery after cutting the band with the correct molecular weight, constructing homologous arm fragments through a fusion PCR program after measuring the concentration, performing nucleic acid electrophoresis after the PCR is finished, and performing gel recovery after cutting the band with the correct molecular weight, and placing the band at-20 ℃ for standby after measuring the concentration.
TABLE 8 PCR System for Badfd-knockout homology arm fragment 1
TABLE 9 PCR System for Badfd-knockout of homology arm fragment 2
TABLE 10 Badfd-knockout homology arm fragment 1 and fragment 2 fusion system
TABLE 11 Badfd-knockout homology arm fragment PCR System
2) Preparation of competence of GS115 strain. Taking out the GS115 strain from a refrigerator at the temperature of minus 80 ℃, thawing the GS115 strain at room temperature, placing the strain on an ultra-clean workbench, absorbing 200 mu L of bacterial liquid in a sterile environment, coating the bacterial liquid on YPDS culture medium for 16 hours, preparing competence, and placing the prepared competence on the refrigerator at the temperature of minus 80 ℃ for standby.
3) Mu.g of the Badfd knockout homologous fragment and the SX1628-71sg-Badfd plasmid constructed in example 1 were each transformed into GS115 competent, cultured at 30℃for 1.5h, and then plated on YPDSZ medium for 2d. 4) The single colony of the plate in step 3 of example 2 was picked up and cultured in YPDSZ liquid medium for 2d, then the genome was extracted, deletion verification of Badfd gene was performed by PCR, primers ad-QCTYBYZ-F and ad-QCTYBZ-R were used as primers for verification of homologous arm of deletion of Badfd gene, and primers ad-NBYZ-F and ad-NBYZ-R were used as primers for internal verification of deletion of Badfd gene, the PCR system was as shown in Table 12, the PCR procedure was as shown in Table 13, and the nucleic acid electrophoresis pattern was as shown in FIG. 5. The strain that was verified to be correct was deposited at-80 ℃.
Table 12 Gly gene deletion verification PCR system
TABLE 13 PCR reaction procedure
Note that: denaturation and annealing was performed for 30 cycles;
example 3
This example compares the growth status of the GS 115-. DELTA.Badfd strain with that of the GS115 strain
1) The GS 115-delta Badfd strain and the GS115 strain are taken out from a refrigerator at the temperature of minus 80 ℃ and are thawed at room temperature, and 100 mu L of bacterial liquid is respectively inoculated into a triangular flask containing 25mL of YTDS liquid culture medium for 1-2d.
2) Taking 5mL of the bacterial liquid in the step (1) of the example 3, placing the bacterial liquid in a sterilized 50mL centrifuge tube for centrifugation (6000 rpm,10 min), discarding the supernatant in a sterile environment, re-suspending the bacterial liquid by 5mL of MMH culture medium, centrifuging again, repeating the step for 3 times, taking 500 mu L of bacterial liquid, and detecting OD 600 Values.
3) Separately, 25mL of sterilized MMH medium was dispensed into 6 sterilized 100mL triangular shake flasks, and the bacterial cell OD detected in step (2) of example 3 was used 600 Value it was added to 25mL MMH medium to bring its initial OD 600 A value of 0.1; culturing 6 shake flasks in shaking table (30 deg.C, 200 rpm), sampling every 1-2d at the early stage, and detecting OD 600 Values. The growth of the GS 115-. DELTA.Badfd strain and the GS115 strain in the medium with methanol as the sole carbon source is shown in FIG. 6.
TABLE 14 primer sequence listing
As can be seen from fig. 6, the GS115- Δbadfd strain prepared in my invention can exhibit better adaptation and growth ability in a medium using methanol as a sole carbon source, and the GS115- Δbadfd strain provided in my invention is a strain having better adaptation and growth ability with respect to methanol as a sole carbon source, and can be conveniently used in a use environment using methanol as a sole carbon source or a main carbon source by itself or subsequent development.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.

Claims (10)

1. A strain for improving methanol utilization efficiency of pichia pastoris, which is characterized in that: the strain for improving the methanol utilization efficiency of the pichia pastoris is a strain for knocking out the Badfd gene.
2. The strain for improving methanol utilization efficiency of pichia pastoris according to claim 1, wherein: the Badfd gene knockout process strain contains SX1628-71sg-Badfd plasmid, and the sequence of the SX1628-71sg-Badfd plasmid is shown in SEQ ID No. 1.
3. A preparation method of a strain for improving methanol utilization efficiency of pichia pastoris is characterized by comprising the following steps: the method comprises the following steps:
preparing a plasmid: knocking out the Badfd gene from the prepared plasmid, and preparing to obtain a plasmid with the Badfd gene knocked out;
preparing a strain: and transforming the prepared homologous arm fragments of the knockout Badfd gene and plasmids of the knockout Badfd gene into the competence of the GS115 strain to prepare the GS 115-delta Badfd strain.
4. The method for preparing the strain for improving methanol utilization efficiency of pichia pastoris according to claim 3, wherein the method comprises the steps of: in the preparation of the plasmid, the prepared plasmid is plasmid SX1628-71sgXG.
5. The method for preparing the strain for improving methanol utilization efficiency of pichia pastoris according to claim 3, wherein the method comprises the steps of: the prepared strain was GS115.
6. The method for preparing the strain for improving methanol utilization efficiency of pichia pastoris according to claim 3 or 4, wherein the method comprises the following steps: the preparation plasmid is prepared by taking a plasmid SX1628-71sgXG as a template, carrying out PCR by using a complementary sequence of the front 6 bases of the sg fragment, an HH sequence and the sg fragment, eliminating the template after the PCR is finished, recovering the fragments SX1628-71sg-Badfd-1 and SX1628-71sg-Badfd-2, carrying out homologous recombination to obtain SX1628-71sg-Badfd, and culturing the strain loaded with the SX1628-71sg-Badfd to obtain the SX1628-71sg-Badfd plasmid.
7. The method for preparing the strain for improving methanol utilization efficiency of pichia pastoris according to claim 3 or 5, wherein the method comprises the following steps: in the preparation strain, the homologous arm fragments of the Badfd gene are knocked out by taking the 3 'end and the 5' end of the Badfd gene as templates.
8. The method for preparing the strain for improving methanol utilization efficiency of pichia pastoris according to claim 3 or 5, wherein the method comprises the following steps: in the preparation strain, the constructed homologous arm fragment of the knockout Badfd gene is introduced into competent cells of the GS115 strain.
9. An application of a strain for improving methanol utilization efficiency of pichia pastoris, which is characterized in that: the GS115- ΔBadfd strain can grow in an environment where methanol is used as a carbon source.
10. The use of the strain for improving methanol utilization efficiency of pichia pastoris according to claim 9, wherein: the GS115- ΔBadfd strain can grow in an environment where methanol is the sole carbon source or the main carbon source.
CN202310570767.7A 2023-05-19 2023-05-19 Strain for improving methanol utilization efficiency of pichia pastoris as well as preparation method and application thereof Pending CN117247851A (en)

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