CN113913451A

CN113913451A - Construction method of pichia pastoris engineering bacteria for producing inositol

Info

Publication number: CN113913451A
Application number: CN202010653538.8A
Authority: CN
Inventors: 赵伟; 杨倩; 何庆; 段莹莹; 王健; 张雷达
Original assignee: Shandong Fuyang Biotechnology Co ltd
Current assignee: Shandong Fuyang Biotechnology Co ltd
Priority date: 2020-07-08
Filing date: 2020-07-08
Publication date: 2022-01-11
Anticipated expiration: 2040-07-08
Also published as: CN113913451B

Abstract

The invention discloses a construction method of a pichia pastoris engineering strain for producing inositol, belonging to the technical field of biological engineering. The invention discloses a construction method of a pichia pastoris engineering strain for generating inositol, which provides a new method for expressing the inositol by using pichia pastoris GS115 by knocking out a glycolysis key gene pgi and an inositol biosynthesis negative regulation gene PAS _ chr-1_0033 in pichia pastoris GS115 and overexpressing an inositol-3-phosphate synthase (ino1) gene. The invention uses the pichia pastoris as an expression host for the first time, realizes the practical technology of expressing the inositol by the pichia pastoris, adopts a high-density fermentation culture method, and finally ensures that the yield of the inositol of the engineering bacteria in a 5L fermentation tank can reach 32.3 g/L.

Description

Construction method of pichia pastoris engineering bacteria for producing inositol

Technical Field

The invention relates to the technical field of bioengineering, in particular to a construction method of a pichia pastoris engineering bacterium for producing inositol.

Background

Inositol in inositol is most well known and is known for its presence in muscle tissue. Inositol has a wide range of uses, in the feed industry as a bio-enhancer to promote growth and prevent death in livestock, and in the food industry as a dietary supplement to prevent fat deposition in humans, particularly in the cardiovascular system.

At present, the main raw materials for producing inositol in China are byproducts in the processing process of agricultural products, such as steeping liquor of corn, rice bran and the like. In order to reduce energy consumption and pollution, the preparation of inositol by biological methods is receiving more and more attention from researchers, and mainly comprises a microbial enzyme catalysis method and a microbial fermentation method. The fermentation method mainly uses mixed saccharides as raw materials, uses saccharomycetes, escherichia coli and bacillus subtilis as main production bacteria, and prepares the high-purity inositol by fermenting and secreting inositol into fermentation liquor, treating the fermentation liquor, concentrating and crystallizing.

In large-scale production, escherichia coli is easily polluted by bacteriophage, and bacillus subtilis is difficult to achieve higher thallus density, so that the saccharomycete is the most suitable bacterial strain for producing inositol through industrial fermentation. The utilization of yeast for producing inositol is based on the theoretical research on the metabolic pathway of inositol. Control of inositol biosynthesis by s.cerevisiae (ino +) inositol secreting yeast was reported in 1976. Cerevisiae found at least 3 gene sites in 1981 to repress the synthesis of key enzymes for myo-inositol biosynthesis. In 1990, the gene engineering technology is utilized to obtain Saccharomyces cerevisiae YS2 with the defect of opi1 gene of negative regulatory factor for inositol biosynthesis and multiple copies of INO1 gene, and a method for preparing inositol by utilizing the gene engineering technology is provided, but the yield of the inositol in the shake flask of YS2 is only 1.021 g/L.

Pichia pastoris is one of the most excellent exogenous gene expression systems with the most extensive application at present, and has been successfully expressedNearly 700 foreign proteins are obtained, and the biomass (OD) of the cells is 48h₆₀₀) Can reach 395. Therefore, the problem to be solved by the technical personnel in the field is to provide a construction method of a pichia pastoris engineering bacterium for producing inositol.

Disclosure of Invention

In view of the above, the invention provides a construction method of a pichia pastoris engineering bacterium for producing inositol, and the yield of the inositol is further improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

a construction method of a pichia pastoris engineering bacterium for producing inositol comprises the following specific steps:

(1) knocking out a glycolysis key gene pgi by using pichia pastoris GS115 as an initial strain to obtain a pgi gene knocked-out strain delta pgi-GS 115;

(2) knocking out a negative regulation gene PAS _ chr-1_0033 of inositol biosynthesis of the strain delta pgi-GS115 to obtain a strain delta PAS _ chr-1_0033 delta pgi-GS115 with the PAS _ chr-1_0033 gene knocked out;

(3) an inositol-3-phosphate synthase gene ino1 is overexpressed in a strain delta PAS _ chr-1_0033 delta pgi-GS115, and a pichia pastoris engineering strain delta PAS _ chr-1_0033 delta pgi-GS115(ino +) for producing inositol is constructed.

Further, the construction method of the pichia pastoris engineering bacterium for producing the inositol comprises the following specific steps:

(1) amplifying zeocin gene and pgi gene upstream and downstream 400bp sequence by PCR;

(2) connecting the 3 gene fragments by using overlapping PCR to obtain a sequence zeocin-UD;

(3) transferring the sequence zeocin-UD into a pichia pastoris GS115 competent cell through electrotransformation, and screening to obtain an engineering bacterium delta pgi-GS115 with pgi gene knocked out;

(4) amplifying 400bp sequences of the upstream and downstream of a G418 gene and an inositol biosynthesis negative regulation gene PAS _ chr-1_0033 by PCR, and connecting the sequences by adopting overlapping PCR to obtain G418-UD;

(5) transferring G418-UD into a delta pgi-GS115 competent cell in an electrotransformation mode, and screening to obtain an engineering bacterium delta PAS _ chr-1_0033 delta pgi-GS115 with a PAS _ chr-1_0033 gene knocked out;

(6) the constructed pPIC9-ino1 plasmid is transferred into a strain delta PAS _ chr-1-0033 delta pgi-GS115 through electrotransformation, and the strain delta PAS _ chr-1-0033 delta pgi-GS115(ino +) engineering strain is obtained through screening.

Further, the application of the pichia pastoris engineering bacteria for producing the inositol in preparing products containing the inositol.

The invention utilizes a bioinformatics method to search homologous protein of inositol biosynthesis transcription inhibitor gene opi1 in pichia pastoris genome, and finds 100% homology of gene PAS _ chr-1_0033 and opi1 protein through sequence comparison.

Through the technical scheme, compared with the prior art, the invention discloses a construction method of a pichia pastoris engineering bacterium for generating inositol, and provides a novel method for expressing the inositol by using pichia pastoris GS115 by knocking out a glycolysis key gene pgi and an inositol biosynthesis negative regulation gene PAS _ chr-1_0033 in the pichia pastoris GS115 and overexpressing an inositol-3-phosphate synthase (ino1) gene.

The invention uses the pichia pastoris as an expression host for the first time, realizes the practical technology of expressing the inositol by the pichia pastoris, adopts a high-density fermentation culture method, and finally ensures that the yield of the inositol of the engineering bacteria in a 5L fermentation tank can reach 32.3 g/L. At present, most of hosts for producing inositol by using a biological fermentation technology adopt escherichia coli, which is easily polluted by bacteriophage and has a long fermentation period. The Pichia pastoris engineering bacteria for producing inositol constructed by the invention can obtain higher thallus density, and simultaneously greatly reduce the influence of phage pollution on large-scale inositol production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a PCR-verified nucleic acid gel electrophoresis of a pgi gene knockout nucleic acid of Pichia pastoris GS115 of the present invention;

wherein M is a DNA Marker; CK is wild type GS115 genome PCR product; 1 is a positive clone genome PCR product obtained by colony PCR screening;

FIG. 2 is a nucleic acid gel electrophoresis diagram showing that PAS _ chr-1_0033 gene in Δ pgi-GS115 is knocked out by PCR verification according to the present invention;

wherein M is a DNA Marker; CK is wild type GS115 genome PCR product; 1-3 are 3 colony genome PCR products of resistance screening, and only 3 are positive clones obtained by colony PCR screening;

FIG. 3 is a map of a pUC57-ino1 vector of the present invention;

FIG. 4 is a map of the pPIC9 vector of the present invention;

FIG. 5 is the restriction enzyme digestion identification result of recombinant plasmid pPIC9-ino1 of the present invention;

wherein M is a DNA Marker; 1-6 are double digestion products of recombinant plasmid pPIC9-ino1 respectively;

FIG. 6 is a vector map of recombinant plasmid pPIC9-ino1 of the present invention;

FIG. 7 is a graph showing the yield of inositol in a 5L fermentor of the present invention, with an inositol peak time of 12.265 min.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 knockout of glycolytic Key Gene pgi in Pichia pastoris GS115

GS115 genome and pPIZ alpha are respectively used as templates to design and synthesize primers AP-F/R, CP-F/R, zeocin-F/R. The specific primer sequences are as follows:

AP-F：5’-ACTTAGAAACGTTGCTTTGACGTAATAGGTCACCTGTG-3’；SEQ ID NO.1；

AP-R：5’-GGCACTGGTCAACTTGGCCATTTTATCAATTGGTTGGAGTT-3’；SEQ ID NO.2；

CP-F：5’-CGAGGAGCAGGACTGAGGCGTTCTAGTTATAGAGAT-3’；SEQ ID NO.3；

CP-R：5’-CTAAACATCCCAAAGTTCAAAAACTCATTGCTGGCT-3’；SEQ ID NO.4；

zeocin-F：5’-AACTCCAACCAATTGATAAAATGGCCAAGTTGACCAGTGCC-3’；SEQ ID NO.5；

zeocin-R：5’-ATCTCTATAACTAGAACGCCTCAGTCCTGCTCCTCG-3’；SEQ ID NO.6。

and respectively carrying out PCR amplification on the DNA fragments AP and CP by using the GS115 genome as a template and the AP-F/R and the CP-F/R as primers, wherein the PCR reaction system and the reaction program are consistent. And (3) PCR reaction system: 2x Phanta Max Buffer 25. mu.l; dNTP Mix (10mM each) 1. mu.l; phanta Max Super-Fidelity DNA Polymerase 0.6. mu.l; 1 microliter of template; 0.1. mu.l of each of the upstream and downstream primers (100. mu.M); ddH₂Make up to 50. mu.l of O. Reaction procedure: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

Taking pPIZ alpha as a template and zeocin-F/R as a primer, and carrying out PCR amplification on a DNA fragment zeocin; and (3) PCR reaction system: 2x PhantaMax Buffer 25. mu.l; dNTP Mix (10mM each) 1. mu.l; phanta Max Super-Fidelity DNA Polymerase 0.6. mu.l; 100 × template 1 μ l; 0.1. mu.l of each of the upstream and downstream primers (100. mu.M); ddH₂Make up to 50. mu.l of O. Reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 1min for 30s, 30 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

The fragments AP, CP and zeocin were then ligated by overlap PCR using primers AP-F and CP-R to obtain the sequence zeocin-UD. And (3) PCR reaction system: 2x Phanta Max Buffer 25. mu.l; dNTP Mix (10mM each) 1. mu.l; phanta Max Super-Fidelity DNA Polymerase 0.6. mu.l; 1. mu.l of each of the three fragment templates; 0.1. mu.l of each of the upstream and downstream primers (100. mu.M); ddH₂Make up to 50. mu.l of O. PCR reaction procedure: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 45s, annealing at 57.5 ℃ for 45s, extension at 72 ℃ for 2min, 30 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃. To be provided withThe gene is transferred into GS115 competent cells in an electrotransformation mode, and a pgi gene is knocked out by utilizing a homologous recombination method (the nucleotide sequence of the pgi gene is shown in SEQ ID NO. 7). Resistance screening is carried out by bleomycin (zeocin, 300 mu g/mL), positive clone is screened by colony PCR, genome is extracted for PCR verification, primers are respectively AP-F and CP-R, PCR reaction system: 2x PhantaMax Buffer 10. mu.l; dNTP Mix (10mM each) 1. mu.l; phanta Max Super-Fidelity DNA Polymerase 0.6. mu.l; 1 microliter of template; 0.1. mu.l of each of the upstream and downstream primers (100. mu.M); ddH₂Make up to 20. mu.l of O. Reaction procedure: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 57.5 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃. The PCR verification result is shown in FIG. 1, and the size of the strip of the pgi gene is between 1.5k and 3k if the pgi gene is not knocked out; if knocked out, the size of the band should be around 1.2 k. The constructed new strain with the pgi gene knocked out was named as Δ pgi-GS 115.

Example 2 knockout of inositol biosynthesis negative regulator Gene PAS _ chr-1_0033 in Δ pgi-GS115

GS115 and pUG6 are respectively taken as templates to design and synthesize primers AZ-F/R, CZ-F/R, G418-F/R.

The specific primer sequences are as follows:

AZ-F：5’-CGTTAGCCAATAGTGTCCCTGCATTCTGGTTCCTCC-3’；SEQ ID NO.8；

AZ-R：5’-GTATTCTGGGCCTCCATGTCTGTTTGGCAGATTCTGTGTC-3’；SEQ ID NO.9；

CZ-F：5’-TGAATGCTGGTCGCTATACTGATTATTCTATAGATTTAGGA-3’；SEQ ID NO.10；

CZ-R：5’-TTTGGCTCGTTCGACTGCCATGGTCGTCAA-3’；SEQ ID NO.11；

G418-F：5’-GACACAGAATCTGCCAAACAGACATGGAGGCCCAGAATAC-3’；SEQ ID NO.12；

G418-R：5’-TCCTAAATCTATAGAATAATCAGTATAGCGACCAGCATTCA-3’；SEQ ID NO.13。

respectively carrying out PCR amplification on DNA fragments AZ and CZ by taking the GS115 genome as a template and AZ-F/R and CZ-F/R as primers; the PCR reaction system and the reaction procedure are consistent. And (3) PCR reaction system: 2x Phanta Max Buffer 25. mu.l; dNTP Mix (10mM each) 1. mu.l; phanta Max Super-Fidelity DNA Polymerase 0.6. mu.l; 1 microliter of template; 0.1. mu.l of each of the upstream and downstream primers (100. mu.M); ddH₂Make up to 50. mu.l of O. Reaction procedure: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30 s; annealing at 60 ℃ for 30 s; extending for 1min at 72 ℃; 30 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

PCR amplifying a DNA fragment G418 by taking pUG6 as a template and G418-F/R as a primer; and (3) PCR reaction system: 2x Phanta Max Buffer 25. mu.l; dNTP Mix (10mM each) 1. mu.l; phanta Max Super-Fidelity DNA Polymerase 0.6. mu.l; 100 × template 1 μ l; 0.1. mu.l of each of the upstream and downstream primers (100. mu.M); ddH₂Make up to 50. mu.l of O. PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min for 30s, 30 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

Then overlapping PCR is carried out by using primers AZ-F and CZ-R to connect the fragments AZ, CZ and G418, and the reaction system: 2x PhantaMax Buffer 25. mu.l; dNTP Mix (10mM each) 1. mu.l; phanta Max Super-Fidelity DNA Polymerase 0.6. mu.l; 1. mu.l of each of the three fragment templates; 0.1. mu.l of each of the upstream and downstream primers (100. mu.M); ddH₂Make up to 50. mu.l of O. PCR reaction procedure: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 45s, annealing at 62 ℃ for 45s, extension at 72 ℃ for 1min for 30s, 30 cycles; extending for 10min at 72 ℃; preserving the heat at 4 ℃ to obtain G418-UD. Transferred into a delta pgi-GS115 competent cell in an electrotransformation way, and a PAS _ chr-1_0033 gene is knocked out by utilizing a homologous recombination method (the nucleotide sequence of the PAS _ chr-1_0033 gene is shown in SEQ ID NO. 14). G418(500 mu G/mL) is used for resistance screening, positive clones are screened by colony PCR, genomes are extracted for PCR verification, primers are AZ-F and CZ-R respectively, and a PCR reaction system is as follows: 2x PhantaMax Buffer 10. mu.l; dNTP Mix (10mM each) 1. mu.l; phanta Max Super-Fidelity DNA Polymerase 0.6. mu.l; 1 microliter of template; 0.1. mu.l of each of the upstream and downstream primers (100. mu.M); ddH₂Make up to 20. mu.l of O. PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃. The PCR result is shown in FIG. 2, if PAS _ chr-1_0033 gene is amplified, the band is located between 1k and 2k, if PAS _ chr-1_0033 gene is knocked out, the band is located between 750bp and 1000bp, and only lane 3 is positive clone obtained by colony PCR screening. Nomenclature of the New strains constructedIs Δ PAS _ chr-1_0033 Δ pgi-GS 115.

Example 3 construction of Δ PAS _ chr-1_0033 Δ pgi-GS115(ino +) engineering bacteria

The ino1 gene (the nucleotide sequence of the ino1 gene is shown in SEQ ID NO.15) is completely synthesized by combining the codon bias of pichia pastoris, EcoR I and XhoI restriction enzyme sites are respectively introduced into the 5 'end and the 3' end of the gene, the synthesized gene is connected to a pUC57 vector and is named as pUC57-ino1, and the vector map is shown in figure 3.

Plasmids pUC57-ino1 and pPIC9 (map as shown in figure 4) were subjected to EcoR I and Xho I double digestion respectively; the double enzyme digestion reaction condition is water bath at 37 ℃ for 2-3 h. And (3) respectively carrying out gel recovery on the double digestion products to obtain an ino1 fragment with two ends respectively provided with EcoR I and Xho I digestion sites and a pPIC9 vector, and connecting the T4 DNA ligase at 4 ℃ overnight. The ligation products were transformed into competent cells of the large intestine DH 5. alpha. and positive clones were screened with ampicillin (50. mu.g/mL), plasmids were extracted and enzyme digestion verified, the results are shown in FIG. 5, where the plasmid enzyme digestion in lanes 2 and 6 was verified correctly. The constructed recombinant expression plasmid containing the ino1 gene was named pPIC9-ino1, and the map is shown in FIG. 6.

The constructed pPIC9-ino1 plasmid is transferred into a strain delta PAS _ chr-1-0033 delta pgi-GS115 through electrotransformation, and the strain delta PAS _ chr-1-0033 delta pgi-GS115(ino +) engineering strain is obtained through screening.

EXAMPLE 4 Shake flask culture of different strains and detection of inositol expression level

After the strains which are verified to be correct are activated, single colonies on the plate are picked, inoculated into 5mL YPD liquid culture medium together with the wild type GS115, and cultured for 18h at 30 ℃ and 220 r/min. Then inoculated into a shake flask containing 50mL of inositol-free fermentation medium (6 g/L disodium hydrogen phosphate, 6g/L potassium dihydrogen phosphate, 8g/L ammonium chloride, 3g/L magnesium sulfate, 10g/L sucrose, 20g/L glycerol, 10mg/L histidine) at an inoculation amount of 1% for secondary expansion culture. Inoculating the second-stage seed liquid to obtain thallus OD₆₀₀0.2 was inoculated into a 250mL Erlenmeyer flask containing 100mL inositol-free fermentation medium.

Inoculating different strains into a fermentation medium (6 g/L disodium hydrogen phosphate, 6g/L potassium dihydrogen phosphate, 8g/L ammonium chloride, 3g/L magnesium sulfate, 10g/L sucrose, 20g/L glycerol and 10mg/L histidine), culturing at 30 ℃ and 220r/min for 50h, centrifuging part of the fermentation liquid (10000r/min and 3min), filtering the supernatant with a 0.45 mu m filter in a sample bottle, and detecting the content of inositol by using a Shimadzu gas chromatograph GC-Plus. The column was RTX-1701(30 m.times.0.25 mm. times.0.25 μm), programmed: sample inlet temperature: 280 ℃; detector temperature: 300 ℃; carrier gas nitrogen gas: 1.06 mL/min; hydrogen flow rate: 40 mL/min; air: 400 mL/min; no flow diversion; sample introduction amount: 1 mu L of the solution; temperature programming: 0min at 120 ℃, 20min at 190 ℃ and 10min at 220 ℃. The inositol production of the different strains is shown in table 1.

TABLE 1 inositol production by different strains

Strain name	Inositol yield (g/L)
		GS115	0
△pgi-GS115	0
		△PAS_chr-1_0033△pgi-GS115	1.015
△PAS_chr-1_0033△pgi-GS115(ino+)	3.202

Example 5 Δ PAS _ chr-1_0033 Δ pgi-GS115(ino +) engineering bacteria culture in 5L fermenter

The delta PAS _ chr-1_0033 delta pgi-GS115(ino +) engineering bacteria transformant is cultured in a YPG culture medium until the light reaches the YPG culture mediumDensity value>20, the YPG culture was used as a seed bacterium in a fermenter, and the seed bacterium was added to a 5L fermenter containing 3L of a basic salt medium (dipotassium hydrogenphosphate 10g/L, dipotassium hydrogenphosphate 3g/L, sodium chloride 0.2g/L, ammonium chloride 1g/L, magnesium sulfate 0.4g/L, citric acid 1.4g/L, 20% glycerin) at a ratio of 1:20, the dissolved oxygen in the fermenter was set at 30%, and the adjustment of the dissolved oxygen was controlled in series with the stirrer of the fermenter. When the glycerol in the basic salt culture medium is exhausted, the glycerol with the volume percentage of 70 percent (700ml of glycerol, 288ml of water and 12ml of PTM1 microelement solution) is supplemented, and the glycerol is supplemented by the following method: adding 8ml/h after 0-8h, 16ml/h after 9-16h, and adding 24ml/h after 17h, when the thallus OD₆₀₀Glycerol addition was stopped when 350 was reached. After the fermentation was completed, a part of the fermentation broth was centrifuged (10000r/min, 5min), the supernatant was filtered through a 0.45 μm filter in a sample bottle, and the content of inositol was measured to be 32.3g/L using Shimadzu liquid chromatograph LC 2010Plus, and the results are shown in FIG. 7.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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<400> 13

tcctaaatct atagaataat cagtatagcg accagcattc a 41

<210> 14

<211> 1167

<212> DNA

<213> Artificial Sequence

<400> 14

atgagtaaac gaaaacttac aggggaatac tcaactgatg gtgacgaaaa cagagaagtt 60

attgccatag aagctctcaa ccaacttcga aataatgggt ccacttctcc atcagtagga 120

gtagaacagg atctagactc cgcacaattg cccccgatgg gcaaagtgga ttccaccacc 180

accaagctga tcgacaaagt tacttcatac cctattattt ctactggtat caactatatg 240

tggcaacgaa atctagtcat agcaggaaac tatatcaaag gcgaatcttc caaaaaagaa 300

gtggttacaa ccaaacgtca aaagacaact accaacgatt cgaaacctga agaaaggtcg 360

gctccatcgt acattcgtga caattccaga ttacgacacc ttcaagatct caaagacatt 420

ggaatgatta atttcaacct ggaaagcaga caaaaattgc aaatgttaat taacttttta 480

aaactgggga acaggcaact gaatcaacgt attgaaaaat tgatcgtaga gttacgtcgg 540

cagcagcgag gagacggtga cgacgacact gaaacggaaa ccattgttcc agatgcaccc 600

tcagggaaag acagtgaatc taatagatca aggtcctctt ccattacgag tgtccaaacc 660

gtctatgaag atgcaaccag cacagggttg gcatctccca atttgaacca agtcaaccat 720

aagttggacc aacacttcaa caaccagccg ctgaaccagg tgaaaaatga cattgttacc 780

actgtcaaaa gaatagttaa tgttgtatcc aagtttagtg ggtctgcatt acctgaacct 840

gcacggtcaa atgtacgaga agtactctta aaactgcctg ataactgggc aaactcagtt 900

ttacctcttg tggaaggtga aggcgctggc gactcccctg ccacagcaaa cacaactgaa 960

agatctctat tatctgaccc caacggtcgt gttcttgttt tagctcagga atcgctcgac 1020

atggtcacca aagtgatcaa gttctgtagt gacactcttg atagggcaga ccaatggaat 1080

cttaacaagc aaattgaaca aaagacccat ttgattcaca agctagaacg aattaataaa 1140

cacaaaccgg gggaaaagca agaatag 1167

<210> 15

<211> 1578

<212> DNA

<213> Artificial Sequence

<400> 15

atgaccatcc aatacacccc aaaggttcaa gttaacaccg acaaggctag atacaccgag 60

aacgagttgt tgaccgacta cacctacgag aacaccatcg ttgagaagca agctgacggt 120

acctacaacg ttaccccaac ctccttggac ttcgagttca aggttgactt gaagacccca 180

aagaccggtt tgttgttggt tggtttgggt ggtaacaacg gtaccacctt ggttggttcc 240

gttttggcta acaagcacaa catctccttc gagaccaaga ccggtatcca acaaccaaac 300

tactacggtt ccgttaccca agcttccacc gttaagttgg gtatcgactc caacggtaga 360

gacgtttacg ctccattcaa ctccttgttg ccattggttc acccaaacga cttcgttgtt 420

ggtggttggg acatctccgg tttggacttg gcttcctcca tgagaagatc ccaagttttg 480

caaccagact tggttagaaa gttggagcca tacatgaagg acatcgttcc attgccatcc 540

gtttactacc cagacttcat cgctgctaac caaaacgaga gagctgacaa ctgcttcaac 600

agagagggta aggacggtga ggtttccacc aagggtaagt ggtcccacgt tgagagaatc 660

agatccgaca tcgctgagtt caagcaaaag aacgacttgg acaaggttat cgttttgtgg 720

accgctaaca ccgagagata cgctgagttg atcccaggtg ttaacgacac cgctgagaac 780

ttgatcaagg ctatcaagaa cgaccacgag gaggtttccg cttccaccat cttcgctgtt 840

gcttgcatct tggacaagat cccatacatc aacggttccc cacaaaacac cttcgttcca 900

ggttgcatcg agttggctga gaccgagggt tccttcatcg gtggtgacga cttcaagtcc 960

ggtcaaacca agttgaagtc cgttttggct caattcttgg ttgacgctgg tatcagacca 1020

gtttccatcg cttcctacaa ccacttgggt aacaacgacg gttacaactt gtccgctcca 1080

caacaattca gatccaagga gatctccaag gcttccgttg ttgacgacat gatcgagtcc 1140

aacgagatct tgtacaacga gaagaacggt aacaccatcg accactgcat cgttatcaag 1200

tacatgaagg ctgttggtga cgacaaggtt gctatggacg agtaccactc cgagttgatg 1260

ttgggtggtc acaacaccat ctccatccac aacatctgcg aggactcctt gttggctacc 1320

ccattgatca tcgacttggt tgttatggct gagttcttgt ccagagtttc ctacaagaag 1380

aagggtgacg ctgagtacga gtccttgcac tccgttttgt ccttcttgtc ctactggttg 1440

aaggctccat tgaccagacc aggttaccaa gctatcaacg gtttgaacaa gcaaagagct 1500

ggtttggaca acttcttgag aatgttgatc ggtttgccaa cccaaaacga gttgagattc 1560

gaggagagat tgcaatag 1578

Claims

1. The construction method of the pichia pastoris engineering bacteria for producing inositol is characterized by comprising the following specific steps:

2. The construction method of the pichia pastoris engineering bacterium for producing inositol according to claim 1, wherein in the step (1) and the step (2), the pgi gene and the PAS _ chr-1_0033 gene are knocked out respectively by using a homologous recombination technology.

3. The construction method of the pichia pastoris engineered strain capable of producing inositol according to claim 1, wherein the pichia pastoris engineered strain capable of producing inositol is applied to the preparation of products containing inositol.