CN118931747A - Engineering strain of Kluyveromyces marxianus for efficient synthesis of inositol and its application - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to Kluyveromyces marxianus engineering bacteria for efficiently synthesizing inositol and application thereof. The Kluyveromyces marxianus engineering bacteria for efficiently synthesizing inositol is obtained by sequentially knocking out a glucose-6-phosphate isomerase PGI gene, a glucose-6-phosphate dehydrogenase ZWF1 gene, an inositol oxygenase MIOX5 gene and an inositol transporter ITR2 gene in a Kluyveromyces marxianus FIM1 delta ura3 strain, and then transferring into vectors pUKDN-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu for co-expressing the trypanosoma brucei inositol phosphate synthase TbIPS gene and the escherichia coli inositol monophosphate EcIMP gene. The invention also discloses a method for efficiently preparing inositol, namely, the Kluyveromyces marxianus engineering strain is used as a fermentation strain, glucose, glycerol, yeast extract, peptone and other components are used as a culture medium, inositol is produced by culture and fermentation, and the yield of the inositol in the supernatant can reach 80.65g/L.

Description

Kluyveromyces marxianus engineering bacteria for efficiently synthesizing inositol and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to Kluyveromyces marxianus engineering bacteria for synthesizing inositol and application thereof.

Background

Inositol was isolated from muscle extracts as early as 1850 (Bizzarri et al, eur Rev Med Pharmacol sci.2014, 18:1896-1903), has multiple functions in organisms (MICHELL ET al, biochem Soc symp.2007, 74:223-46), has attracted clinician attention in the past twenty years (Dinicola et al, int J Mol Sci,2021, 22:10575), and various studies have shown that inositol or derivatives thereof can affect embryonic development, insulin signaling, stress response, and the like (Thomas et al, ANGEW CHEM INT ED,2016, 55:1614). The human body itself has an endogenous inositol synthesis pathway, endogenous inositol can be synthesized by glucose-6-phosphate, which is isomerised by inositol-3-phosphate synthase (Inositol-phosphate synthase, IPS) to inositol-3-phosphate (Wong et al, neurochem,1987, 48:1434-1442), which is then dephosphorylated by inositol monophosphatase (Inositol monophosphatase, IMP) to form inositol (Loewus et al, J Biol chem.1980, 255:11710-11712.). However, it is still necessary to ingest about 1g of inositol (Dinicola et al., S Int J Mol Sci,2017, 18:2187.) from cereals, legumes, nuts on a daily basis, especially when the endogenous inositol of the human body is insufficient to meet the physiological needs of the human body. Based on the needs and various functions of the human body, inositol is widely used in the fields of foods, feeds, medicines, cosmetics, industry and the like.

Inositol production can be classified into chemical acid hydrolysis, in vitro enzyme synthesis, and biological fermentation. Chemical acid hydrolysis mainly uses phytate as a raw material, inositol is extracted by a pressurized acid hydrolysis treatment mode (Li et al, biotechnol Appl biochem.2022, 69:1101-1111), but a large amount of waste liquid is generated in the process, and the step process is relatively complicated. The method for synthesizing the in vitro enzyme can combine purified enzymes and coenzymes from different sources, realizes one-pot biological cascade reaction (You et al, process biochem.2017, 52:106-14), has controllable Process and high yield, and easily separated products, has obvious advantages compared with a chemical acid hydrolysis method, but has high production cost compared with a biological fermentation method, and meanwhile, the enzyme has instability. The biological fermentation method is to produce inositol by different microorganism hosts, and has the advantage of green sustainable property. In recent years, hosts for producing inositol by a representative biological fermentation method mainly include E.coli (young et al, microb Cell face.2020, 19:109) and Pichia pastoris (Zhang et al, microb Cell face.2022, 21:112), and E.coli can achieve high titer, but it is not a food-safe strain, which limits application prospects. In pichia pastoris, the titer of inositol is significantly lower than in escherichia coli, so that a microbial host for producing inositol by biological fermentation still has room for further excavation and improvement.

Kluyveromyces marxianus is an unconventional yeast, is the fastest growing yeast (Groeneveld et al., FEBS J,2009, 27:254-270) and has high temperature resistance (Lane et al, anton Leeuw Int J G,2011, 100:507-519), and can utilize various carbon sources such as inulin, xylose, lactose and the like (Fonseca et al, appl Microbiol Bioechnol,2008,79 (3): 339-354), and is GRAS organism to obtain QPS certification, and is recognized as a food safety strain by the Ministry of health of China (Wu et al, J Biotechnol,2020, 320:11-16). And complete whole genome sequencing in 2012 (Jeong et al, eukaryotic Cell,2012, 11:1584-1585). The physiological and metabolic characteristics of Kluyveromyces marxianus make the Kluyveromyces marxianus have wide application in biotechnology. As a broad class of Crabtree negative yeasts, it favors respiration rather than fermentation, and therefore does not require the transfer of ethanol metabolism. Wild type yeasts have been used to produce aromatic compounds, which can synthesize ethyl acetate at rates higher than 2g L ^-1h^-1. In 2017, a gene editing tool for Kluyveromyces marxianus was developedEt al Biotechnol Biofuels,2017, 10:164) provide the basis for multiple complex genetic and metabolic engineering, thus increasingly being used for the production of proteins and various economically valuable products.

Disclosure of Invention

The invention aims to provide a Kluyveromyces marxianus metabolic engineering strain for efficiently synthesizing inositol and application thereof.

The Kluyveromyces marxianus metabolic engineering strain for efficiently synthesizing inositol is constructed by the following steps:

(1) The Kluyveromyces marxianus FIM1 delta ura3 strain is obtained by utilizing a Crispr gene editing technology, and a glucose-6-phosphate isomerase PGI gene (Genbank accession number: QGN 13438.1) and a glucose-6-phosphate dehydrogenase ZWF1 gene (Genbank accession number: QGN 16990.1) of a genome are knocked out, so that the Kluyveromyces marxianus strain is constructed and obtained and is marked as FIM1 delta ura3 delta PGI delta ZWF1;

(2) Knocking out inositol oxygenase MIOX5 gene (Genbank accession number: QGN 14246.1) in Kluyveromyces marxianus strain FIM1Δura3 Δpgi Δzwf1, and constructing to obtain Kluyveromyces marxianus chassis strain, namely FIM1Δura3 Δpgi Δzwf1 Δ MIOX5;

(3) Knocking out an inositol transporter ITR2 gene (Genbank accession number: QGN 17849.1) in a Kluyveromyces marxianus chassis strain FIM1Δura3 ΔpgiΔzwf1 Δ miox5, and constructing and obtaining the Kluyveromyces marxianus chassis strain, namely FIM1Δura3 ΔpgiΔzwf1 Δ miox5 Δ ITR2;

(4) Constructing a trypanosoma brucei Trypanosoma brucei inositol phosphate synthase TbIPS gene expression frame by using an inulase promoter Pinu and a TEF terminator Ttef; constructing an escherichia coli ESCHERICHIA COLI gene inositol monophosphate EcIMP gene expression frame by using a TEF promoter Ptef and an inulase terminator Tinu; sequentially inserting TbIPS gene expression frame and EcIMP gene expression frame into a Kluyveromyces marxianus expression vector pUKDN, constructing and obtaining a co-expression plasmid of inositol phosphate synthase TbIPS and inositol monophosphate enzyme EcIMP, and marking the co-expression plasmid as pUKDN115-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu;

(5) Transforming the expression plasmid pUKDN-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu obtained by the construction into the Kluyveromyces marxianus strain FIM 1. DELTA. Ura 3. DELTA. Pgi. DELTA. Zwf 1. DELTA. miox5 described in step (2) or the Kluyveromyces marxianus strain FIM 1. DELTA. Ura 3. DELTA. Pgi. DELTA. Zwf 1. Miox. DELTA. itr2 described in step (3); screening and respectively obtaining:

the Kluyveromyces marxianus engineering strain FIM1Δura3ΔpgiΔzwf1Δ miox 5/pUKDN-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu is named as Kluyveromyces marxianus JC-3;

Kluyveromyces marxianus engineering strain FIM1 delta ura3 delta pgi delta zwf1 delta miox delta itr2/pUKDN115-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu is named as Kluyveromyces marxianus JC-4.

The Kluyveromyces marxianus FIM1 delta URA3 strain is obtained by knocking out uracil synthase gene URA3 on genome of Kluyveromyces marxianus FIM1 strain (collection number: CGMCC No. 10621) preserved in China general microbiological culture Collection center.

The kluyveromyces marxianus expression vector pUKDN in the step (3) contains the kluyveromyces marxianus autonomous replication sequence, an inulinase promoter, an inulinase terminator, a screening marker gene URA3 and other elements.

In Kluyveromyces marxianus strain FIM 1. DELTA.ura 3, the glucose-6-phosphate isomerase PGI gene was knocked out, and the purpose was to block intracellular isomerization of glucose-6-phosphate to fructose-6-phosphate, thereby entering the EMP pathway (Embden-Meyerhof-PARNAS PATHWAY).

In Kluyveromyces marxianus strain FIM1Δura3, the glucose-6-phosphate dehydrogenase ZWF1 gene is knocked out, and the purpose is to block the oxidation of glucose-6-phosphate to 6-phosphogluconate in cells, thereby entering the pentose phosphate pathway (pentose phosphate pathway).

Further, in Kluyveromyces marxianus FIM 1. DELTA.ura 3. DELTA.pgi. DELTA.zwf1 strain, the myo-inositol oxygenase MIOX5 gene was knocked out for the purpose of preventing the produced myo-inositol from being oxidatively degraded.

In the Kluyveromyces marxianus strain FIM1Δura3ΔpgiΔzwf1, the ITR2 gene of the inositol transporter is knocked out, and the purpose of the gene is to block the transport of inositol into cells to be metabolized and utilized.

In order to synthesize inositol in kluyveromyces marxianus cells, the invention provides a method for coexpression of inositol phosphate synthase TbIPS and inositol monophosphate EcIMP, which comprises the steps of constructing a TbIPS gene expression frame and a EcIMP gene expression frame in series on a pUKDN115 vector, and then introducing kluyveromyces marxianus.

The nucleotide sequence of the inositol phosphate synthase TbIPS is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2.

The nucleotide sequence of the inositol monophosphate enzyme EcIMP is shown in SEQ ID No.3, and the amino acid sequence is shown in SEQ ID No. 4.

The invention also provides a method for efficiently synthesizing inositol by using the Kluyveromyces marxianus metabolic engineering strain (comprising Kluyveromyces marxianus JC-3 and JC-4), specifically, the Kluyveromyces marxianus metabolic engineering strain is used as a fermentation strain, and glucose, glycerol, yeast extract, peptone and other components are used as a culture medium, and inositol is produced by culture and fermentation.

The specific operation is as follows: kluyveromyces marxianus JC-3 or JC-4 was inoculated into 50mL of YPD medium containing 2% glycerol and continuously cultured at 30℃and 220rpm for 168 hours. Wherein 1mL of 50% glycerol was added to the culture for 96 hours, and 1mL of 50% glucose was added to glucose every 24 hours. Fermenting for 144-168 h to obtain high yield inositol: 29.1g/L to 38.9g/L.

Specifically, the yield of inositol can reach 29.1g/L after 144h fermentation, and the yield of inositol is 38.9g/L when the fermentation is completed for 168 h.

In a preferred mode, kluyveromyces marxianus JC-3 or JC-4 containing YPD medium of 2% glycerol is inoculated into a fermentation tank of the YPD medium of 2% glycerol in an amount of 10%, 50% glucose is fed as a first carbon source, 50% glycerol is fed as a second carbon source, continuous feed fermentation is carried out, and under the condition of continuous feed fermentation, the strain is fermented at high density for 168-192 hours, so that high-yield inositol is obtained: 59.68g/L-80.65g/L.

Concretely, the strain is fermented for 168 hours at high density, the inositol yield can reach 59.68g/L, and the sugar alcohol conversion rate can reach 56.65%; high-density fermentation is carried out for 192h, and the inositol yield is 80.65g/L.

The Kluyveromyces marxianus engineering strain provided by the invention can efficiently utilize glucose to synthesize inositol, has a wide application prospect, can realize large-scale fermentation production of inositol, has obvious advantages in yield, and has good application value.

Drawings

FIG. 1 shows a diagram of the metabolic engineering lines of Kluyveromyces marxianus for synthesizing inositol engineering bacteria.

FIG. 2 is a plasmid map of Kluyveromyces marxianus recombinant co-expressed myo-inositol synthetases TbIPS and EcIMP.

FIG. 3 shows the growth curves (a) and inositol yield curves (b) of the engineered Kluyveromyces marxianus synthetic inositol strains JC-1, JC-2 and JC-3, respectively, shake flask fermented in YPD of 5% glucose and 2% glycerol.

FIG. 4 shows the growth curve (a) and the inositol yield curve (b) of the engineered Kluyveromyces marxianus synthetic inositol strains JC-3 and JC-4 shake flask fermented in YPD of 2% glucose and 2% glycerol.

FIG. 5 shows the bacterial growth and inositol yield of the Kluyveromyces marxianus engineering strain JC-4 fermentation tank by continuous feed fermentation.

Detailed Description

Example 1 construction of Kluyveromyces marxianus FIM 1. DELTA.ura 3. DELTA.pgi Strain

In the synthesis process of inositol, glucose-6-phosphate is catalyzed by inositol phosphate synthase IPS to generate inositol phosphate, and then the inositol monophosphate IMP catalyzes the hydrolysis of the phosphate to generate inositol. In order to increase the efficiency of synthesis of intracellular inositol in kluyveromyces marxianus, it is necessary to block the downstream metabolism of glucose-6-phosphate in the kluyveromyces marxianus glycolysis pathway and direct the flow of glucose metabolism into the inositol synthesis pathway. Glucose-6-phosphate isomerase (Phosphoglucose isomerase, PGI) is an enzyme that iso-forms glucose-6-phosphate into fructose-6-phosphate, and is also a key enzyme for glycolysis. Therefore, the invention uses CRISPR CAS gene editing technology to knock out glucose-6-phosphate isomerase PGI of Kluyveromyces marxianus FIM1 delta ura3 strain. And synthetic gRNA primers PGIg-F (tcatggcaactgagttgtccgca) and PGIg-R (aactgcggacaactcagttgcca), the gRNA targeting the 35-55bp position of the PGI cds region. The gRNA plasmid and the Donor sequence required for knocking out the PGI gene are constructed, wherein the Donor sequence is composed of a total of 1000bp sequences which are 500bp respectively at the upstream and downstream of the cds region of the PGI gene. Simultaneously converting the gRNA plasmid and the donor sequence into a FIM1 delta ura3 strain, obtaining clone which is used for successfully knocking out the PGI gene through uracil deficiency plate screening and PCR verification, and obtaining the strain FIM1 delta ura3 delta PGI through 5-fluoroorotic acid plate screening of the strain which loses the gRNA plasmid.

Example 2 construction of Kluyveromyces marxianus FIM 1. DELTA.ura 3. DELTA.pgi. DELTA.zwf1 Strain

The glucose-6-phosphate dehydrogenase ZWF1 gene was further knocked out on the basis of the strain FIM 1. DELTA.ura 3. DELTA.pgi. Firstly, constructing a gRNA plasmid and a donor sequence required by knocking out a ZWF1 gene by a basic molecular cloning technology, wherein the gRNA primer is a 667-687bp position of a ZWF1g-F (tcatttggtacagaaggccgcgg) and a ZWF1g-R (aacccgcggccttctgtaccaaa) targeted ZWF1 cds region. The Donor sequence consists of 500bp each upstream and downstream of the cds region of the ZWF1 gene. Meanwhile, the gRNA plasmid and the donor sequence are simultaneously transformed into the FIM1 delta ura3 delta PGI strain, cloning of successfully knocking out the ZWF1 gene is obtained through uracil deficiency plate screening and PCR verification, and then the strain losing the gRNA plasmid is screened through a 5-fluoroorotic acid plate, and after PGI and ZWF1 genes are simultaneously knocked out, glucose metabolism of Kluyveromyces marxianus is completely blocked theoretically, so that glycerol with the final concentration of 2% is needed to be added as a second carbon source in screening and subsequent culture to maintain cell growth. The Kluyveromyces marxianus strain FIM1Δura3ΔpgiΔzwf1 was obtained.

Example 3 construction of Kluyveromyces marxianus FIM 1. DELTA.ura 3. DELTA.pgi. DELTA.zwf1. DELTA. miox5 Strain

The fim1Δura3ΔpgiΔzwf1 strain has blocked downstream shunt of glucose-6-phosphate, further blocking downstream degradation pathways of inositol. Further knocking out inositol oxygenase MIOX5 gene on the basis of FIM1 Deltaura 3 Deltapgi Deltazwf 1 strain, constructing gRNA plasmid and donor sequence required for knocking out MIOX5 gene by basic molecular cloning technology, and the gRNA primer is as follows: MOIX5g-F: TCAAGTCATTATTCATAGTGATA; MIOX5g-R: AACTATCACTATGAATAATGACT, targeting the 66-86bp position of the MIOX5 gene cds region. The Donor sequence consists of a total of 1000bp of sequences each 500bp upstream and downstream of the cds region of the MIOX5 gene. Simultaneously converting the gRNA plasmid and the donor sequence into a FIM1 delta ura3 delta pgi delta zwf1 strain, obtaining clone which is successfully knocked out miox through uracil deficiency plate screening and PCR verification, and obtaining the strain FIM1 delta ura3 delta pgi delta zwf1 delta miox through 5-fluoroorotic acid plate screening of the strain which loses the gRNA plasmid.

Example 4 construction of Kluyveromyces marxianus FIM 1. DELTA.ura 3. DELTA.pgi. DELTA.zwf1. DELTA. miox. DELTA. itr2 Strain

The fim1Δura3ΔpgiΔzwf1Δ miox5 strain has blocked downstream branching of glucose-6-phosphate and downstream breakdown pathways of inositol. Further blocking the transport pathway of inositol into the cell to prevent the cell from transporting inositol in the system to intracellular utilization. Therefore, on the basis of the FIM1 Deltaura 3 Deltapgi Deltazwf1 Delta miox5 strain, the inositol transporter ITR2 gene is further knocked out, and the gRNA plasmid and the donor sequence required for knocking out ITR2 are constructed by a basic molecular cloning technology, wherein the gRNA primers are as follows: ITR2g-F: TCAGGTTGCTGGTAGACTAGTTA; ITR2g-R: AACTAACTAGTCTACCAGCAACC, targeting the 480-500bp position of the ITR2 gene cds region. The Donor sequence consists of 500bp each upstream and downstream of the ITR2 gene cds region. Simultaneously converting the gRNA plasmid and the donor sequence into the FIM1 delta ura3 delta pgi delta zwf1 delta miox strain, obtaining clone for successfully knocking out the ITR2 gene through uracil deficiency plate screening and PCR verification, and obtaining the strain FIM1 delta ura3 delta pgi delta zwf1 delta miox delta ITR2 through 5-fluoroorotic acid plate screening of the strain losing the gRNA plasmid.

Example 5 construction of Kluyveromyces marxianus inositol Synthesis pathway Strain

Inositol phosphate synthase TbIPS and inositol monophosphate EcIMP required for inositol synthesis are derived from trypanosoma brucei and escherichia coli, respectively, and are codon optimized and synthesized without changing the amino acid sequence. The TbIPS and EcIMP fragments can be obtained by PCR amplification, and since TbIPS and EcIMP are required to be expressed simultaneously, 2 sets of expression elements are required to regulate the expression of TbIPS and EcIMP, respectively. The TEF promoter TEFpr and the TEF terminator TEFt were obtained from the PCR amplification on plasmids stored in the laboratory, the four fragments of TEFt, TEFpr, ecIMP, tbIPS were fused into fragments EcIMP-TEFt-TEFpr-TbIPS by the overlap PCR technique, and the fused fragments were inserted into Kluyveromyces marxianus expression vector pUKDN115 for expression to obtain plasmids pUKDN-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu.

The constructed pUKDN-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu plasmids are respectively transformed into Kluyveromyces marxianus chassis cells FIM1 delta ura3 delta pgi, FIM1 delta ura3 delta pgi delta zwf1 delta miox5, FIM1 delta ura3 delta pgi delta zwf1 delta miox5 delta itr to obtain Kluyveromyces marxianus engineering bacteria FIM1 delta ura3 delta pgi/pUKDN-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu, and the obtained Kluyveromyces marxianus engineering bacteria are named JC-1; kluyveromyces marxianus engineering bacteria FIM1 delta ura3 delta pgi delta zwf1/pUKDN115-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu, named JC-2; kluyveromyces marxianus engineering bacteria FIM1 delta ura3 delta pgi delta zwf1 delta miox/pUKDN-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu, named JC-3; kluyveromyces marxianus engineering bacterium FIM1 delta ura3 delta pgi delta zwf1 delta miox delta itr2/pUKDN115-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu, and is named as JC-4.

Example 6 shake flask fermentation of JC-1, JC-2, JC-3 to myo-inositol

Strains JC-1, JC-2, JC-3 were streaked on YPD plates containing 2% glycerol, respectively, and activated overnight at 30 ℃. 3-5 clones were picked and inoculated into 50mL of YPD medium containing 5% glucose and 2% glycerol and cultured at 30℃for 168h under 220 rpm. Cell density and inositol production were recorded every 24h samples. The results showed (fig. 3) that the cell densities of fermentation to 168h, jc-1, jc-2, jc-3 were OD ₆₀₀＝46.51,OD₆₀₀＝44.32,OD₆₀₀ = 44.68, respectively. The yields of myo-inositol from JC-1, JC-2 and JC-3 were 8.23g/L,11.57g/L and 14.68g/L, respectively. The yield of JC-2 is increased by 40.5% compared with JC-1, and the yield of JC-3 is increased by 26.9% compared with JC-2.

Example 7 production of inositol by fermentation of Kluyveromyces marxianus JC-4 in shake flask

Kluyveromyces marxianus strain JC-3, JC-4 was streaked on YPD plates containing 2% glycerol, respectively, and activated overnight at 30 ℃. 3-5 clones were picked and inoculated into 50mL of YPD medium containing 2% glucose and 2% glycerol and cultured at 30℃for 168h under 220 rpm. And 1mL of 50% glycerol was added at 96h of cultivation, and 1mL of 50% glucose was added every 24h up to 144h of cultivation. The results showed (FIG. 4) that JC-3, after lowering the initial glucose, had a cell density of OD ₆₀₀ = 89 and an inositol yield of 29.1g/L at fermentation time 168h, which was 98.2% higher than in YPD medium with 5% glucose and 2% glycerol. When the JC-4 strain is fermented for 168 hours, the cell density is OD ₆₀₀ =103, the inositol yield is 38.9g/L, and the yield is improved by 33.9% compared with JC-3.

Example 8 continuous fed-batch fermentation of Kluyveromyces marxianus JC-4 to myo-inositol

Strain JC-4 was streaked onto YPD plates containing 2% glycerol and activated overnight at 30 ℃. 3-5 single clones were picked and inoculated into 150mL YPD medium containing 2% glycerol and cultured for 48h. And transferring the strain JC-4 with the initial inoculation amount of 10 percent into a fermentation tank of YPD culture medium with 2 percent of glycerol, feeding 50 percent of glucose as a first carbon source, feeding 50 percent of glycerol as a second carbon source, and carrying out continuous feed fermentation. The process maintains dissolved oxygen constant by 30%, the concentration of the system glycerol is 1% -3%, and the concentration of glucose is 1% -2%. The results show (FIG. 5) that JC-4 produces 59.68g/L inositol when fermented 168; the inositol yield from fermentation to 192h is 80.65g/L, and the sugar alcohol conversion rate is about 56.65%.

Claims (4)

1. A Kluyveromyces marxianus metabolic engineering strain for efficiently synthesizing inositol is characterized by being constructed by the following steps:

(1) Knocking out glucose-6-phosphate isomerase PGI gene and glucose-6-phosphate dehydrogenase ZWF1 gene of genome in Kluyveromyces marxianus FIM1 delta ura3 strain by using Crispr gene editing technology, and constructing and obtaining the Kluyveromyces marxianus strain which is marked as FIM1 delta ura3 delta PGI delta ZWF1;

(2) Knocking out inositol oxygenase MIOX5 gene in Kluyveromyces marxianus strain FIM1Δura3 Δpgi Δzwf1, and constructing to obtain Kluyveromyces marxianus chassis strain which is marked as FIM1Δura3 Δpgi Δzwf1 Δ MIOX5;

(3) Knocking out an inositol transporter ITR2 gene in a Kluyveromyces marxianus chassis strain FIM1Δura3ΔpgiΔzwf1 Δ miox5, and constructing and obtaining the Kluyveromyces marxianus chassis strain which is marked as FIM1Δura3ΔpgiΔzwf1 Δ miox5 Δ ITR2;

(4) Constructing a trypanosoma brucei Trypanosoma brucei inositol phosphate synthase TbIPS gene expression frame by using an inulase promoter Pinu and a TEF terminator Ttef; constructing an escherichia coli ESCHERICHIA COLI gene inositol monophosphate EcIMP gene expression frame by using a TEF promoter Ptef and an inulase terminator Tinu; sequentially inserting TbIPS gene expression frame and EcIMP gene expression frame into a Kluyveromyces marxianus expression vector pUKDN, constructing and obtaining a co-expression plasmid of inositol phosphate synthase TbIPS and inositol monophosphate enzyme EcIMP, and marking the co-expression plasmid as pUKDN115-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu;

(5) Transforming the expression plasmid pUKDN-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu obtained by the construction into the Kluyveromyces marxianus strain FIM 1. DELTA. Ura 3. DELTA. Pgi. DELTA. Zwf 1. DELTA. miox5 described in step (2) or the Kluyveromyces marxianus strain FIM 1. DELTA. Ura 3. DELTA. Pgi. DELTA. Zwf 1. Miox. DELTA. itr2 described in step (3); screening and respectively obtaining:

the Kluyveromyces marxianus engineering strain FIM1Δura3ΔpgiΔzwf1Δ miox 5/pUKDN-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu is named as Kluyveromyces marxianus JC-3;

Kluyveromyces marxianus engineering strain FIM1 delta ura3 delta pgi delta zwf1 delta miox delta itr2/pUKDN115-Pinu-EcIMP-Ttef-Ptef-TbIPS-Tinu, named Kluyveromyces marxianus JC-4;

Wherein:

The kluyveromyces marxianus expression vector pUKDN contains a kluyveromyces marxianus autonomous replication sequence, an inulase promoter, an inulase terminator and a screening marker gene URA3 element;

The Kluyveromyces marxianus FIM1 delta ura3 strain is a Kluyveromyces marxianus FIM1 strain which is preserved in China general microbiological culture Collection center, and has the preservation number: CGMCC No.10621, and knocking out uracil synthase gene URA3 on genome;

The glucose-6-phosphate isomerase PGI gene, genbank accession number: QGN13438.1; the glucose-6-phosphate dehydrogenase ZWF1 gene, genbank accession number: QGN16990.1; the myo-inositol oxygenase MIOX5 gene, genbank accession No.: QGN14246.1; the inositol transporter ITR2 gene, genbank accession No.: QGN17849.1;

The nucleotide sequence of the inositol phosphate synthase TbIPS is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2; the nucleotide sequence of the inositol monophosphate enzyme EcIMP is shown in SEQ ID No.3, and the amino acid sequence is shown in SEQ ID No. 4.

2. A method for synthesizing inositol by using the kluyveromyces marxianus metabolic engineering strain as claimed in claim 1, which is characterized in that the kluyveromyces marxianus metabolic engineering strain is used as a fermentation strain, glucose, glycerol, yeast extract and peptone components are used as culture mediums, and inositol is produced by culture and fermentation.

3. The method for synthesizing inositol according to claim 2, characterized by the following specific operations: inoculating Kluyveromyces marxianus metabolic engineering strain into 50mL YPD culture medium containing 2% glycerol, and culturing at 30deg.C and 220rpm for 168h; and 1mL of 50% glycerol was added when the culture was carried out for 96 hours, and 1mL of 50% glucose was added every 24 hours when the culture was carried out for 96 hours, and fermentation was carried out for 144 hours to 168 hours, to obtain inositol with high yield.

4. The method for synthesizing inositol according to claim 2, characterized by the following specific operations: inoculating Kluyveromyces marxianus metabolic engineering strain containing YPD medium of 2% glycerol, inoculating 10% of the strain into a fermentation tank of YPD medium of 2% glycerol, feeding 50% glucose as a first carbon source, feeding 50% glycerol as a second carbon source, performing continuous feed fermentation, and performing high-density fermentation for 168-192 h under the continuous feed fermentation condition to obtain high-yield inositol.