CN108753672B

CN108753672B - Xylitol genetic engineering production strain and construction method and application thereof

Info

Publication number: CN108753672B
Application number: CN201810594607.5A
Authority: CN
Inventors: 吴绵斌; 王吉平; 袁新松; 林建平; 杨立荣
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-06-11
Filing date: 2018-06-11
Publication date: 2020-09-18
Anticipated expiration: 2038-06-11
Also published as: CN108753672A

Abstract

The invention discloses a xylitol genetic engineering production strain and a construction method and application thereof, belonging to the technical field of genetic engineering. The xylitol genetic engineering production strain is obtained by modifying a genome of escherichia coli W3110, wherein ptsG, xylAB and ptsF in the original escherichia coli W3110 genome are replaced by xylose reductase gene XR; also included is the substitution of at least one of pfkA, pfkB, pgi, sthA in the genome with XR. According to the invention, Escherichia coli W3110 is used as an original strain, corresponding genes in a genome are all replaced by XR, xylose reductase is efficiently expressed, xylose utilization is improved by blocking xylose metabolism and phosphorylation of xylitol, NADPH regeneration is enhanced by blocking or reducing glucose metabolism flux in an EMP (electron brain protein) path, a necessary coenzyme NADPH is provided for xylose reductase reduction xylose to generate xylitol, and the efficiency of producing xylitol by transforming xylose by genetic engineering bacteria is greatly improved.

Description

Xylitol genetic engineering production strain and construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to xylitol genetic engineering production strain and a construction method and application thereof.

Background

Xylitol is a kind of sugar alcohol containing five carbon atoms, its sweetness is equivalent to that of cane sugar, its caloric value is only about 60%, and it has the features of resisting dental caries, making metabolism independent of insulin, improving liver function, etc., and can be extensively used in the fields of food, medicine and chemical industry.

The xylitol is produced industrially mainly by hydrolyzing hemicellulose acid to obtain xylose, separating and purifying to obtain the xylose with the purity of more than 95 percent, and carrying out catalytic hydrogenation on the xylose by nickel under the conditions of high temperature and high pressure, and the process has the advantages of harsh conditions, easy pollution and higher production cost. The method for producing xylitol by a biological method does not need high-temperature and high-pressure conditions, flammable and explosive hydrogen, nickel catalysts polluting the environment, high-purity xylose and the like, and has the advantages of mild reaction conditions, safety, energy conservation and environmental friendliness, so that the method for producing xylitol by conversion by the biological method is more and more emphasized by people.

At present, microorganisms used for preparing xylitol by a fermentation method are almost all yeasts, and have natural strains and genetically engineered bacteria. Yeasts have their own advantages as xylitol producing strains, such as the ability to tolerate higher sugar concentrations, greater resistance to inhibitory factors in hemicellulose hydrolysates, and the like. However, there are problems such as potential pathogenicity, poor specificity of xylose reductase contained in yeast itself, and the like, which are unavoidable.

Coli is the best host for producing various high value-added chemicals, the research is the best at present, the gene background is clear, and the conditions for constructing the genetic engineering bacteria by using the escherichia coli are unique. Coli as a host for producing xylitol has been reported, for example, a first generation xylitol high-yield strain is constructed based on a plasmid vector system such as Supori, a plasmid vector is utilized for protein expression, and a plasmid capable of being efficiently expressed at a higher temperature is constructed through the regulation of an mRNA secondary structure; the enzyme activity is 5.68 times of that of the original strain at the temperature of 30 ℃. Eliminating the metabolite repression effect of the strain by knocking out ptsG gene of enzyme II component in a glucose phosphotransferase system, so that the strain can simultaneously transport glucose and xylose; knocking out xylA and xylB genes of xylose metabolism of the strain per se, and blocking metabolic utilization of xylose; knocking out ptsF gene of enzyme II component in fructose phosphotransferase system capable of transferring xylitol, and reducing phosphorylation of xylitol; through a series of optimization, the production efficiency of xylitol is 8.71 times of that of the original strain (research on producing xylitol by transforming hemicellulose hydrolysate with escherichia coli genetic engineering bacteria, Supeli, Zhejiang university, 2016).

Coenzymes are a general term for a large group of organic cofactors, and are essential cofactors for enzymatic redox reactions and the like. They act to transfer electrons, atoms or groups in the reaction, and coenzymes can be considered to some extent as secondary substrates in enzymatic reactions. As a key cofactor in the microbial metabolic network, the cellular metabolic function of the microorganism can be directionally changed and optimized by regulating the content of coenzyme in cells and the proportion of reduced oxidized form, so that the maximization of the metabolic flow is realized, and the method is also one of important methods for increasing target products.

Coenzyme NAD (P) H plays an important role in various enzyme-catalyzed reactions, and particularly in redox reactions, the coenzyme NAD (P) H is required to be used as electron transfer to participate in the reaction. During the synthesis of the product, a certain amount of the coenzyme is consumed. Therefore, as the reaction proceeds, the intracellular coenzyme content decreases, resulting in a decrease in catalytic efficiency. Because of the high price of coenzymes, supplementation by means of exogenous addition is impractical. Therefore, the metabolic process of the microorganism is regulated and controlled through metabolic engineering, the intracellular NADPH concentration is increased, the production efficiency can be effectively improved, the cost is reduced, and the normal operation of the biotransformation process can be better ensured. From the viewpoint of technical economy, it is important to strengthen the coenzyme regeneration cycle.

Currently, the main approach for coenzyme NADPH-based metabolic engineering is to enhance the metabolic flux of PPP. In E.coli, glucose exists mainly in two metabolic pathways, including the glycolytic pathway (EMP pathway) and the pentose phosphate pathway (PPP pathway). Wherein 2 steps of reaction in the PPP pathway can realize the regeneration of NADPH, the endogenous coenzyme NADPH is increased by over-expressing zwf and gnd genes in the two steps of reaction in the prior research, and the method can strengthen the regeneration of intracellular NADPH to a certain extent.

However, in the integrative E.coli expressing xylose reductase gene, to achieve the matching of xylose reductase enzyme activity and coenzyme regeneration rate, it is necessary to further reduce the glucose consumption rate. At present, no report on a method for realizing coenzyme NADPH regeneration strengthening and glucose utilization rate slowing by utilizing a method for blocking or reducing the glucose metabolism flux of an EMP (electron brain protein) pathway is found.

Disclosure of Invention

The invention aims to provide xylitol genetic engineering production bacteria which can greatly improve the production efficiency of xylitol.

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

according to the invention, Escherichia coli W3110 is used as an original strain, and genes influencing xylose metabolism pathways and glucose metabolism pathways in an original strain genome are replaced by using a gene replacement technology, so that the matching of xylose reductase activity and coenzyme regeneration rate is realized, and the efficiency of producing xylitol by transforming xylose by genetic engineering bacteria is further improved.

Therefore, the invention provides a xylitol genetic engineering production strain, which is obtained by modifying a genome of escherichia coli W3110, wherein ptsG, xylAB and ptsF in the original escherichia coli W3110 genome are replaced by xylose reductase gene XR; also includes the replacement of at least one of pfkA, pfkB, pgi and sthA in the genome with xylose reductase gene XR.

The Escherichia coli W3110 is purchased from German Collection of microorganisms and strains DSMZ, and is numbered DSM-5911.

The transformation of Escherichia coli W3110 of the invention comprises:

(1) within E.coli cells there is a pathway to metabolize xylose: xylose passes through xylose isomerase (xylA) to produce xylulose, xylulose is under the action of xylulokinase (xylB) to produce xylulose 5-phosphate, which is metabolized in the pentose phosphate pathway. Meanwhile, a phosphotransferase (ptsF) pathway of fructose possibly participates in the transportation of xylitol, so that the xylitol is phosphorylated while entering cells, and the phosphorylated xylitol has toxic action on the cells, so that xylAB and ptsF in a genome are replaced by a xylose reductase gene XR, the metabolism of xylose and the phosphorylation of the xylitol are blocked, the expression of the xylose reductase is increased, and the high-efficiency conversion of the xylose by engineering bacteria to produce the xylitol is facilitated.

(2) The glucose effect exists when the Escherichia coli utilizes the sugar, namely the utilization of other sugars such as xylose, arabinose and the like by the Escherichia coli is seriously inhibited when the glucose exists, so that the glucose phosphotransferase gene ptsG is replaced by the xylose reductase gene XR, the utilization rate of the genetically engineered bacteria to the glucose is reduced, and the glucose effect is eliminated.

However, in the process of reducing xylose to xylitol by xylose reductase, glucose is required as a co-substrate to provide NADPH, which is a coenzyme necessary for the reaction. Therefore, the invention replaces genes pfkA, pfkB, pgi or sthA which participate in glycolysis pathway (EMP) to block or reduce the flux of glucose metabolism in EMP pathway, and realizes PPP pathway coenzyme NADPH regeneration strengthening and glucose utilization rate slowing.

Preferably, ptsG, xylAB, ptsF, pfkA and pfkB in the original E.coli W3110 genome are all replaced with xylose reductase gene XR. Research shows that after the 5 genes are replaced by xylose reductase gene XR, the production efficiency of producing xylitol by transforming xylose by using genetically engineered bacteria reaches 1.92 g/L.

The xylose reductase gene XR is derived from Neurospora crassa, and the gene sequence is shown in NCBI accession number NCU 08384.1.

The invention also provides a construction method for constructing the xylitol genetic engineering production strain, which comprises the following steps:

(1) replacing ptsG, xylAB and ptsF in the genome of the Escherichia coli W3110 with xylose reductase gene XR to obtain a first generation of genetic engineering bacteria;

(2) at least one of pfkA, pfkB, pgi and sthA in the genome of the first generation genetically engineered bacterium is replaced with a xylose reductase gene XR.

In steps (1) and (2), gene replacement is performed by using a homologous recombination technique.

Specifically, the step (1) includes:

a. respectively constructing pTargetF plasmids for replacing ptsG, xylAB and ptsF genes and corresponding repair templates containing XR expression modules by using specific primers;

b. transforming pTargetF plasmid replacing ptsG gene and a repair template into Escherichia coli W3110 containing pCas plasmid, and screening to obtain a strain W3110 delta ptsG of which the ptsG gene in the genome is replaced by an XR expression module, wherein XR is expressed;

then, the pTargetF plasmid replacing the xylAB gene and the repair template are transformed into a strain W3110 delta ptsG: XR, and the strain W3110 delta ptsG: XR, delta xylAB: XR, with the xylAB gene in the genome replaced by an XR expression module, is obtained by homologous recombination and screening;

then, the pTargetF plasmid and the repair template replacing the ptsF gene are transformed into a strain W3110 delta ptsG, XR and delta xylAB, XR, and the strain W3110 delta ptsG, XR, delta xylAB, XR and delta ptsF, XR which are used for replacing the ptsF gene in the genome and are used as XR expression modules, is obtained through homologous recombination and screening, namely a first generation of genetic engineering bacteria;

the step (2) comprises the following steps:

d. respectively constructing pTargetF plasmids for replacing pfkA and pfkB genes and corresponding repair templates containing XR expression modules;

e. the pTargetF plasmid replacing pfkA gene and the corresponding repair template are firstly transformed into the first generation genetic engineering bacteria prepared in the step (1), XR (X-ray fluorescence) is used for screening and obtaining a strain WZ04 delta pfkA (X-ray fluorescence) with the pfkA gene replaced by an XR expression module in the genome, XR is used for transforming the pTargetF plasmid replacing pfkB gene and the corresponding repair template into a strain WZ04 delta pfkA (X-ray fluorescence) with the XR expression module, and a strain with the pfkB gene replaced by the XR expression module in the genome is screened and obtained through homologous recombination, namely the xylitol genetic engineering production bacteria.

The XR expression module comprises a promoter P43. Studies have shown that promoter P43 facilitates the expression of xylose reductase without the need for an inducer. An XR expression module using P43 as a promoter was amplified using a specific primer using pRC43M plasmid provided in patent document CN 104789586A as a template.

The invention also provides the application of the xylitol genetic engineering production strain in producing xylitol.

The application comprises the following steps: inoculating the xylitol genetic engineering production strain into a fermentation culture medium, performing fermentation culture at 30-37 ℃ for 80-90h, and keeping the concentration OD of the strain liquid in the fermentation process₆₀₀Less than 20.

The fermentation culture medium can utilize artificially prepared culture solution which takes xylose as a main component, and can also utilize hemicellulose hydrolysate as a main raw material.

Preferably, the fermentation initial conditions are: the temperature is 37 ℃, the rotating speed is 400rpm, the ventilation volume is 0.6-0.8vvm, the initial pH of a fermentation medium is about 6.5, and the dissolved oxygen is controlled at 30-35% in the culture process; when the concentration of bacterial liquid OD₆₀₀When the fermentation temperature is more than or equal to 20 ℃, feeding materials, wherein the fermentation conditions after feeding are as follows: the temperature is 30 ℃, and the dissolved oxygen is controlled to be 20-25%.

Feeding for the first time in a fed-batch mode, specifically, with OD600>20 (about 7h at 37 ℃); and (5) feeding for the second time after the glucose is completely consumed.

The feed supplement comprises the following components: hemicellulose hydrolysate or xylose mother liquor (the final concentration of xylose is 60g/L), glucose mother liquor (the final concentration of glucose is 1/2 of xylose molar concentration), and industrial-grade corn steep liquor dry powder (1/3 of xylose mass concentration).

The invention has the following beneficial effects:

according to the xylitol genetic engineering production strain provided by the invention, escherichia coli W3110 is taken as an original strain, xylAB and ptsF in a genome are replaced by xylose reductase gene XR, the metabolism of xylose and the phosphorylation of xylitol are blocked, and the utilization rate of the genetic engineering strain on xylose is increased; replacing ptsG and at least one of pfkA, pfkB, pgi and sthA in the genome with a xylose reductase gene XR, eliminating the glucose effect, simultaneously achieving the purpose of strengthening the PPP pathway NADPH regeneration by blocking or reducing the EMP pathway glucose metabolism flux, and providing a necessary coenzyme NADPH for reducing xylose by xylose reductase to generate xylitol; corresponding genes in the genome are all replaced by xylose reductase genes XR, and the xylose reductase can be efficiently expressed, so that the efficiency of producing xylitol by transforming xylose by genetic engineering bacteria is greatly improved.

Drawings

FIG. 1 shows the determination of target sites in ptsG and the design strategy of pTargetF plasmid mutation primers.

FIG. 2 is a method of constructing a repair template.

FIG. 3 is a nucleic acid electrophoresis diagram of chassis cell engineered using CRISPR technology, where M:250bp Marker, G and G: XR are fragments amplified using primers ptsG-u-F and ptsG-d-R, G: original ptsG gene, G: XR: replacing the original ptsG gene with xr; XR is the fragment amplified using primers xylAB-u-F and xylAB-d-R, AB: original xylAB gene, AB: XR: replacing the original xylAB gene with xr; XR is the fragment amplified using primers ptsF-u-F and ptsF-d-R, F: original ptsF gene, F: XR: the original ptsF gene was replaced with xr.

FIG. 4 is an analysis of the major metabolic pathway of glucose.

FIG. 5 shows the sugar and sugar alcohol concentrations in the fermentation broth after 24h shake flask fermentation.

FIG. 6 shows the result of measurement of OD600 of bacteria concentration after 24h fermentation.

FIG. 7 shows the production of Xylitol by fermentation of strain WZ31 with pure sugar, wherein Glucose is Glucose, Xylose is Xylose, and xylotol is Xylitol.

FIG. 8 is a fed-batch fermentation using WZ31 with hemicellulose hydrolysate and corn steep liquor dry powder, wherein Glucose is Glucose, Xylose is Xylose, Arabinose is Arabinose, Arabitol is Arabitol, and Xylitol is Xylitol.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Coli W3110, available from DSMZ, DSM-5911, German Collection of microorganisms and cell cultures; pTargetF plasmid addgene: # 62226; pCas plasmid addgene: # 62225; the pRC43M plasmid is derived from the patent document having the application No. 201510196843.8 and entitled "Escherichia coli genome integration vector, genetically engineered bacterium, and use for producing xylitol".

Example 1

The embodiment provides a xylitol genetic engineering production strain, which can enhance intracellular NADPH regeneration in an integrated xylose reductase expression strain and improve the production efficiency of xylitol.

The construction method of the xylitol genetic engineering production strain comprises the following steps:

1. construction of pTargetF plasmid for replacement or deletion of target Gene

Firstly, a PAM site, namely an NGG sequence, needs to be found on a target gene, a corresponding N20 sequence is determined, and a cadAspacter on a pTargetF plasmid is replaced by an N20 sequence of the target gene. Taking ptsG as an example, plasmid construction of pTargetF-ptsG as an example, pTargetF was subjected to whole plasmid mutagenesis PCR using primers N20-ptsG-F and N20-ptsG-R, and the primer design method is shown in FIG. 1.

The PCR recipe and program set up were as follows:

TABLE 1 Whole plasmid mutant PCR System

From the map of pTargetF plasmid, the total length of plasmid is 2118bp, and in order to ensure the integrity of extension during PCR, a long extension time setting is adopted in the present study.

TABLE 2 Whole plasmid PCR program set-up

The obtained PCR product is subjected to DNA nucleic acid electrophoresis verification, and whether the PCR is successful is checked by observing whether a bright band exists at about 2000 bp.

After verification, DpnI enzyme is used for digesting a PCR template, so that the positive rate of the transformant is improved. The enzymatic digestion system is as follows:

Enzyme DpnI 0.5μL

10*Buffer 1μL

PCR product 8.5. mu.L.

Adding corresponding substances according to the system, horizontally shaking and mixing, rapidly centrifuging for 20s for a short time, and placing in water bath or metal bath at 37 deg.C for 60 min. Transformation was then performed using DH5 α competence.

The obtained transformant is picked up for liquid culture, and a small amount of plasmid extraction kit is used for extracting the plasmid. Sequencing verification is carried out by using Target-check, and finally the successfully mutated plasmid pTargetF-ptsG is obtained.

2. Construction of pTsG substituted with XR Gene repair template

The construction strategy is shown in figure 2.

Using E.coli W3110 genome as template, ptsG-u-F and ptsG-u-R, ptsG-d-F and ptsG-d-R as PCR primer, proceeding conventional PCR to obtain homologous arm fragment of 500bp each at upstream and downstream of ptsG gene;

PCR was performed using pRC43M plasmid as the template and ptsG-XR-F and ptsG-XR-R as primers to obtain an XR expression module using P43 as the promoter. And carrying out DNA nucleic acid electrophoresis on the obtained PCR product and recovering the gel.

And finally, performing overlap extension PCR by using ptsG-u-F and ptsG-d-R as primers and an equal proportion mixture of upstream and downstream homology arms of ptsG and an xr expression module as a template, cutting gel and recovering fragments with corresponding lengths to obtain a repair template for replacing ptsG genes.

3. Operation of the genomic Gene replacement method

1) Heat shock method for transferring pCas plasmid

a. Wild type E.coli W3110 stored at-80 ℃ was streaked onto solid medium plates without antibody and cultured overnight at 37 ℃. Selecting single colony, culturing in liquid LB culture medium at 37 deg.C and 200rpm for about 10 hr, transferring 1mL bacterial liquid into 250mL triangular flask containing 50mL liquid LB culture medium, and growing to OD₆₀₀And (3) when the temperature reaches 0.6-0.8, carrying out ice bath on the bacterial liquid for 10min, and preparing the transformation competence according to a Takara escherichia coli competence kit.

b. Prepared E.coli W3110 competence is placed on ice, 10 μ LpCas plasmid is added under aseptic condition after melting, and placed in ice bath for 30min after mixing.

c.42 ℃ water bath or metal bath heat shock for 90s, immediately ice bath for 2 min.

d. 890. mu.L of liquid LB or resuscitating medium was added and resuscitated at 30 ℃ and 200rpm for 45 min.

e. Sucking 100 μ L of recovered bacterial liquid, and spreading on kan containing 50mg/L^ROn solid LB plates, and incubated overnight at 30 ℃.

f. And selecting a colony of the single clone to perform PCR or extract plasmid verification to obtain E.coli W3110pCas which is successfully transformed.

2) Electrotransformation of pTargetF of the corresponding replacement gene and repair template donor DNA

a. Taking Escherichia coli obtained in 1) as a starting strain, streaking on a 50mg/L kanamycin sulfate resistant plate, culturing at 30 ℃ overnight (the same antibiotics with the same concentration are required to be added in subsequent culture), selecting a single colony to be cultured in liquid LB at 30 ℃, culturing at 200rpm for 10-12h, transferring into 1mL to 50mL LB, culturing at 30 ℃ and 200rpm for 1h, adding sterilized L-arabinose with the working concentration of 0.5% to induce and express Red recombinant protein, and continuously culturing until OD is OD₆₀₀And (3) cooling the mixture for about 0.6 to 0.8 hours in ice bath for 10min to prepare electrotransformation competence.

b. The sterilized 10mL Ep tube was used, the bacterial solution was split charged, centrifuged at 4000rpm for 5min at 4 ℃ and the supernatant was discarded.

c. Resuspend with 1mL of pre-cooled sterilized 10% glycerol, centrifuge at 4000rpm for 10min at 4 ℃ and carefully discard the supernatant.

d. Repeat c step 2 times.

e. Resuspended with 100. mu.L of 10% glycerol, transferred to a sterilized 1.5mL Ep tube, and used immediately or placed in a-80 ℃ freezer for future use.

f. Prepared competence was used or previously prepared competence was taken out from-80 ℃, left on ice for 5min, and 400ng of the pTargetF plasmid constructed to replace the corresponding gene and 800ng of the repair template were added. After mixing, the mixture was transferred to a sterile 2mm electric rotor, and left on ice for 10min for electric conversion.

g. And (3) electrotransfer conditions: 2.5kV, 25 muF, 200 omega, 5ms of electric rotating time, wherein the wall and the base of the electric rotating cup are required to be wiped dry by paper towel before electric rotating, otherwise, the electric rotating cup is easy to be ignited. And the ignition cup is also triggered by overhigh salt ion residues in the extracted plasmid and the recovered repair template.

h. After the electrotransfer was completed, 1mL of liquid LB was added immediately, mixed by pipetting back and forth with a pipette, and transferred to a 2mL sterilized Ep tube. Resuscitated at 30 ℃ and 150rpm for about 3 h.

i. Centrifugally concentrating the recovered bacterial liquid, and coating the concentrated liquid on kan containing 50mg/L^RAnd 50mg/L of spec^RPlates were incubated overnight at 30 ℃.

j. Usually, the transformants are visible to the naked eye after 12 hours of cultivation. If the strain is edited many times, the growth time may be prolonged.

4. Gene replacement verification

For the positive transformant which is successfully replaced, under the condition that the sequence lengths before and after replacement are greatly different, a colony or bacterium liquid PCR amplification target band is adopted, DNA nucleic acid electrophoresis is carried out, and the sizes of the amplification bands before and after gene replacement are compared for identification. If the difference between the sizes of the genes before and after the replacement is within 200bp, the PCR product sequencing mode can be adopted for identification.

5. Similarly, the corresponding primers are used for constructing pTargetF and repair templates of other replacement genes, and xylose reductase is successfully inserted into ptsG, xylAB and ptsF regions of the genome after 3 rounds of genome editing.

The modified integrated strains WZ01, WZ02 and WZ03 for expressing xylose reductase are obtained through design. Among them, a genome diagram of WZ03(E.coli W3110, Δ ptsG: XR, Δ xylAB: XR, Δ ptsF: XR) is shown in FIG. 3.

The size of the inserted xylose reductase expression module is 1449 bp. The size of the ptsG gene is 1434bp, so the size change after the replacement is not obvious, the correctness of the result cannot be judged through a nucleic acid gel electrophoresis chart, and the Target-check primer is used for carrying out PCR product sequencing verification to successfully replace the ptsG gene with a xylose reductase expression module. The xylAB gene is 2849bp in total, is reduced by 1400bp after being replaced by XR, and is a positive transformant after being replaced by the XR; the ptsF gene is 1692bp, after replacement, the size is reduced by 243bp, and an electrophoretogram shows that the gene is positive transformation. The finally obtained WZ03 strain is an engineering bacterium with ptsG, xylAB and ptsF all replaced by expression modules of xylose reductase genes.

6. The PPP pathway in glucose metabolism can produce more NADPH as analyzed by the glucose major metabolic pathway (fig. 4), thus involving the experimental replacement of pfkA, pfkB, pgi gene and catalase sthA gene of EMP pathway with xylose reductase expression module.

The WZ03 strain is used as an original strain, and single genes of pfkA, pfkB, pgi and sthA which can enhance the regeneration capability of coenzyme NADPH are replaced to obtain strains WZ21, WZ22, WZ23 and WZ 24. And selecting engineering bacteria with better effect to carry out double replacement to obtain strains WZ31 and WZ 32.

The primers (SEQ ID NO.1-57) involved in the above experiment are shown in Table 3. The detailed strains and their genome types are shown in table 4.

TABLE 3 primer sequences

TABLE 4 strains and related genotype types

7. The shake flask fermentation experiment was carried out on the above strains under the following fermentation conditions:

(1) seed liquid preparation

Seed liquid culture: the streaked single colonies were picked into sterilized fresh liquid LB medium and cultured overnight at 37 ℃ at 200rpm to stationary phase of growth.

(2) Shake flask fermentation

Preparing a shake flask fermentation culture medium, filling 45mL of liquid in a 250mL triangular flask, inoculating 1mL of seed liquid, culturing at 37 ℃ and 200rpm for 4h, adding 5mL of sterilized mixed sugar liquid (containing 200g/L of xylose and 100g/L of glucose), carrying out shake flask fermentation at 30 ℃ and 200rpm, sampling at regular time and detecting the change condition of related parameters.

(3) Liquid phase detection method for sugar and sugar alcohol

The sample was diluted to an appropriate concentration and then filtered using a 0.22 μm filter head. Quantitative detection of xylose, glucose, arabinose, xylitol and arabitol was performed using a Dionex UltiMate 3000 high performance liquid system. A detector: corona Charged Aerosol Detector (CAD), analytical column: aminex HPX-87C (Φ 7.8 mm. times.300 mm), mobile phase: ultrapure water, flow rate: 0.6mL/min, column temperature setting: at 76 ℃.

Taking the bacterial liquid supplemented with the sugar liquid for 24 hours, extracting the intracellular oxidized coenzyme and the intracellular reduced coenzyme, and measuring the concentration by the following method:

1) placing the bacterial liquid in ice bath for 10min, centrifuging at 4 deg.C and 4000rpm for 15min, and concentrating to OD of 1mL bacterial liquid₆₀₀The coenzymes are isolated in their intracellular oxidized and reduced forms at 30.

2) Respectively taking 1mL of bacterial liquid, adding:

isolation of the oxidized form: 0.5mL of 0.3M HCl and 50mM Tricine-NaOH (pH 8.0);

isolation of the reduced form: 0.5mL of 0.3M NaOH;

3) all samples were incubated at 60 ℃ for 7min, the oxidized form was neutralized with 0.5mL of 0.3M NaOH, the reduced form was neutralized with 0.5mL of 0.3M HCl, and after neutralization 0.1mL of 1.0M Tricine-NaOH was added to each sample to maintain the pH stable.

4) Centrifuge at 13000rpm for 60min at 4 ℃. Pipette 300. mu.L of supernatant into a new Ep tube.

5) And measuring the content of the coenzyme by using the light absorption value of the reaction solution at a specific wavelength. Quantitation can be performed using 96-well plates and microplate readers. The measuring system is as follows:

oxidized form: 40 μ L of sample +40 μ L of 0.1M NaCl;

the reduced form is: 80 μ L of sample;

6) equal volumes (80. mu.L) of 2 × reaction stock (1.0M Tricine-NaOH (pH8.0), 4.2mM thiazolyl blue tetrazolium bromide (MTT), 40mM EDTA,1.67mM Phenazineethosulfate (PES), and substrate (5M ethanol for NAD determination) or 25mM glucose-6-phosphate for NADP determination) were added.

7) After mixing, the mixture was kept at 37 ℃ for 5min, and alcohol dehydrogenase at a working concentration of 10U/mL and glucose-6-phosphate dehydrogenase at a working concentration of 0.27U/mL were added (mother liquor was prepared at 10X concentration).

8) The decrease in MTT at 37 ℃ was detected using a 570nm microplate reader. Data were compared to standard curves.

The ratios of the reduced forms and oxidized forms of the coenzymes thus determined are shown in Table 5 below.

TABLE 5 ratio of reduced to oxidized coenzyme after different Gene substitutions

The results of measurements of sugars and sugar alcohols in the fermentation broth, in which the intracellular NADPH/NADP + contents were all increased by a certain amount after the substitution of the relevant gene, compared to the control group WZ03, are shown in FIG. 5.

As can be seen from the analysis of Table 5 and FIG. 5, the regeneration capacity of intracellular NADPH is enhanced after modification, more NADPH can be produced by consuming the same molar glucose, the ratio of intracellular NADPH/NADP + is increased, and the higher coenzyme concentration is maintained to provide sufficient reducing power for xylose reductase.

Growth OD of each strain₆₀₀The results of the measurement are shown in FIG. 6. The higher the coenzyme NADPH/NADP + ratio of the modified strain is under 4 copies of xylose reductase, the higher the xylitol yield of the strain is. At 5 xylose reductase copy numbers, WZ31 gave a comparable coenzyme NADPH/NADP + ratio to WZ32, but WZ31 gave higher xylitol production. With reference to fig. 6, the growth ability of the transformed strain was impaired, and the concentration of WZ32 after 24h was only about 40% of that of the original strain WZ03, which is the main reason for the low xylitol production.

Carrying out a pure sugar shake flask fermentation experiment on the strain WZ31, respectively sampling 10h, 20h and 30h after sugar addition, and determining the concentrations of sugar and sugar alcohol and OD in fermentation liquor₆₀₀The results of the measurement are shown in FIG. 7.

8. Fermenting by using hemicellulose hydrolysate and corn steep liquor dry powder, adopting WZ31 strain, and fermenting xylitol by using a 15L fermentation tank under the condition of not adding antibiotics and inducers, wherein the method comprises the following steps:

(1) first-order seed liquid culture

The preserved strain was streaked on a solid medium plate and cultured overnight at a constant temperature of 37 ℃. Single colonies were picked in liquid LB and cultured at 37 ℃ for about 12h at 200 rpm.

First-class seed liquid culture medium formula (L)^-1): 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 2% agar powder added in a solid culture medium.

(2) Second-order seed liquid culture

Transferring the primary seed liquid into a secondary seed liquid culture medium according to the volume ratio of 0.5-1%, and culturing at 37 ℃ and 200rpm for 7 h.

Secondary seed liquid culture medium formula (L)^-1): 7.5g of yeast powder, 7.5g of peptone, 10g of sodium chloride and 20g of glucose.

(3) Fermentation process control

Adjusting the pH of a fermentation culture medium to about 6.5 by using ammonia water before inoculation, inoculating the cultured secondary seed liquid according to 10-15% of the volume of the fermentation culture medium, and culturing at a temperature: the aeration rate was controlled at 0.6vvm at 37 ℃ and the initial rotation speed was controlled at 400 rpm. Regulating rotation speed and dissolved oxygen, maintaining dissolved oxygen in fermentation tank at 30-35%, sampling at certain time interval, and measuring bacterial concentration OD₆₀₀And is in OD₆₀₀And (3) feeding when the temperature is more than or equal to 20, wherein both the inoculation and feeding adopt a flame method.

The material supplementing mode comprises the following steps: fed batch

OD600>20 (about 7h at 37 ℃) and the first feed was performed. After the first feeding, the glucose was completely consumed, and the second feeding was performed.

Ingredient and final concentration of feed liquid: hemicellulose hydrolysate or xylose mother liquor (the final concentration of xylose is 60g/L), glucose mother liquor (the final concentration of glucose is 1/2 of xylose molar concentration), and industrial-grade corn steep liquor dry powder (1/3 of xylose mass concentration).

And (3) controlling conditions after feeding: controlling dissolved oxygen at 20-25%, and controlling temperature: at 30 ℃.

And (3) sampling at regular time after feeding, detecting the concentrations of glucose, xylose, arabinose, arabitol and xylitol in the fermentation process, and monitoring the growth condition of the strains in the whole engineering. The final fermentation results are shown in FIG. 8.

As can be seen from FIG. 8, 161.03g/L xylitol was obtained by feeding and fermenting in 84h batches using hemicellulose hydrolysate as substrate and corn steep liquor dry powder as carbon source, xylose, arabinose and glucose were almost completely consumed, and the by-product arabitol was only 1.63 g/L. The production efficiency of the xylitol is 1.92 g/L/h.

The above-mentioned embodiments are intended to illustrate the technical solutions and advantages of the present invention, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modifications, additions, equivalents, etc. made within the scope of the principles of the present invention should be included in the scope of the present invention.

Sequence listing

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Claims

1. A xylitol genetic engineering production strain is obtained by modifying a genome of Escherichia coli W3110, and is characterized in that ptsG, xylAB, ptsF, pfkA and pfkB in the genome of original Escherichia coli W3110 are replaced by xylose reductase gene XR, and the xylose reductase gene XR is derived from Neurospora crassa;

(2) replacing pfkA and pfkB in the genome of the first generation of genetic engineering bacteria with xylose reductase gene XR;

the step (1) comprises the following steps:

then, the pTargetF plasmid and the repair template replacing the ptsF gene are transformed into a strain W3110 delta ptsG, XR and delta xylAB, XR, and the strain W3110 delta ptsG, XR, delta xylAB, XR and delta ptsF, XR which are used for replacing the ptsF gene in the genome and are used as XR expression modules, are obtained through homologous recombination and screening, namely first generation genetic engineering bacteria;

the step (2) comprises the following steps:

e. firstly, the pTargetF plasmid replacing pfkA gene and the corresponding repair template are transformed into the first generation genetic engineering bacteria prepared in the step (1), XR (X-ray fluorescence) is used as a strain for replacing the pfkA gene in the genome with XR (X-ray fluorescence) expression module by homologous recombination, then the pTargetF plasmid replacing the pfkB gene and the corresponding repair template are transformed into the strain WZ04 delta pfkA, XR is used as a strain for replacing the pfkB gene in the genome with XR expression module by homologous recombination, and the strain for replacing the pfkB gene in the genome with XR expression module is obtained by screening, namely the xylitol genetic engineering production bacteria; the XR expression module comprises a promoter P43.

2. The xylitol genetic engineering production strain as defined in claim 1, which is used for producing xylitol.

3. The use of claim 2, comprising: inoculating the xylitol genetic engineering production strain into a fermentation culture medium, performing fermentation culture at 30-37 ℃ for 80-90h, and keeping the concentration OD of the strain liquid in the fermentation process₆₀₀Less than 20.

4. Use according to claim 3, wherein the fermentation initial conditions are: the temperature is 37 ℃, the rotating speed is 400rpm, the ventilation volume is 0.6-0.8vvm, the initial pH of a fermentation medium is 6.5, and the dissolved oxygen is controlled at 30-35% in the culture process; when the concentration of bacterial liquid OD₆₀₀When the fermentation temperature is more than or equal to 20 ℃, feeding materials, wherein the fermentation conditions after feeding are as follows: the temperature is 30 ℃, and the dissolved oxygen is controlled to be 20-25%.