CN108753672B - A kind of xylitol genetic engineering production bacterium and its construction method and application - Google Patents

Abstract

The invention discloses a xylitol genetic engineering production bacterium and a construction method and application thereof, belonging to the technical field of genetic engineering. The xylitol genetically engineered production bacterium is obtained from Escherichia coli W3110 through genome modification, and ptsG, xylAB and ptsF in the original Escherichia coli W3110 genome are all replaced with xylose reductase gene XR; it also includes pfkA, pfkB, At least one of pgi, sthA is replaced with XR. In the present invention, Escherichia coli W3110 is used as the starting strain, and the corresponding genes in the genome are replaced with XR, so that the xylose reductase can be expressed efficiently, and meanwhile the metabolism of xylose and the phosphorylation of xylitol are blocked, and the effect of xylose on xylose is improved. Utilize and strengthen the regeneration of NADPH by blocking or reducing the glucose metabolism flux of the EMP pathway, providing the necessary coenzyme NADPH for the reduction of xylose by xylose reductase to generate xylitol, and greatly improving the efficiency of genetically engineered bacteria to convert xylose to produce xylitol. efficiency.

Description

A kind of xylitol genetic engineering production bacterium and its construction method and application

technical field

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

Background technique

Xylitol is a five-carbon sugar alcohol whose sweetness is comparable to that of sucrose, but only about 60% of its calorie value. Pharmaceutical and chemical industries.

The industrial production of xylitol is mainly to obtain xylose by acid hydrolysis of hemicellulose. After separation and purification, xylose with a purity of more than 95% is obtained by catalytic hydrogenation of nickel under high temperature and high pressure conditions. This process condition is harsh. And it is easy to cause pollution, and the production cost is high. Biological production of xylitol does not require high temperature and high pressure conditions, flammable and explosive hydrogen, environmentally polluting nickel catalysts and high-purity xylose, etc. The reaction conditions are mild, safe, energy-saving and environmentally friendly. more and more people's attention.

At present, the microorganisms used for the preparation of xylitol by fermentation are almost all yeasts, including natural strains and genetically engineered bacteria. Yeasts have their own advantages as xylitol producing strains, such as being able to tolerate higher sugar concentrations, stronger resistance to inhibitory factors in hemicellulose hydrolyzate, and so on. However, there are also unavoidable problems, such as potential pathogenicity and poor specificity of xylose reductase contained in yeast itself.

As an ideal host for the production of various high value-added chemicals, Escherichia coli has the most thorough research and clear genetic background. It has unique conditions for constructing genetically engineered bacteria. Using Escherichia coli as a host to produce xylitol has been reported. For example, Subli et al. constructed the first generation of high-yielding xylitol strains based on the plasmid vector system. The plasmid vector was used for protein expression. A plasmid capable of high-efficiency soluble expression at higher temperature was obtained; at 30 °C, the enzyme activity was 5.68 times that of the starting strain. By knocking out the ptsG gene of the enzyme II component of the glucose phosphotransferase system, the metabolite repression effect of the strain is eliminated, so that the strain can transport glucose and xylose at the same time; Metabolic utilization of xylose; Knock out the ptsF gene of the enzyme II component of the fructose phosphotransferase system that can transport xylitol to reduce the phosphorylation of xylitol; Through a series of optimizations, the production efficiency of xylitol is the starting strain 8.71 times ("Research on the transformation of hemicellulose hydrolyzate to produce xylitol by genetically engineered Escherichia coli", Subli, "Zhejiang University", 2016).

Coenzyme is a general term for a large class of organic cofactors, which are necessary for enzymes to catalyze redox reactions. They play the role of transferring electrons, atoms or groups in the reaction, and coenzymes can be regarded as the second substrate in the enzyme reaction to a certain extent. As a key cofactor in the microbial metabolic network, by regulating the intracellular coenzyme content and the ratio of reduced oxidized forms, the cellular metabolic function of microorganisms can be directionally changed and optimized, thereby maximizing metabolic flux, which is also an increase in target products. one of the important methods.

Coenzyme NAD(P)H plays an important role in various enzyme-catalyzed reactions, especially in redox reactions, which all require coenzyme NAD(P)H as electron transfer to participate in the reaction. In the process of product synthesis, a certain amount of coenzyme will be consumed. Therefore, as the reaction proceeds, the intracellular coenzyme content decreases, resulting in a decrease in catalytic efficiency. Due to the high price of coenzymes, it is unrealistic to supplement by exogenous addition. Therefore, regulating the metabolic process of microorganisms through metabolic engineering and increasing the concentration of intracellular NADPH can not only effectively improve the production efficiency and reduce the cost, but also better ensure the normal progress of the biotransformation process. From a technical and economic point of view, it is of great significance to strengthen the regeneration cycle of coenzyme.

At present, the main method of metabolic engineering based on coenzyme NADPH is to enhance the metabolic flux of PPP. In E. coli, there are two main metabolic pathways for glucose, including the glycolysis pathway (EMP pathway) and the pentose phosphate pathway (PPP pathway). Among them, there are two steps in the PPP pathway that can achieve the regeneration of NADPH. In the existing research, the endogenous coenzyme NADPH was increased by overexpressing the zwf and gnd genes in these two steps. This method can strengthen the intracellular NADPH to a certain extent. Regeneration of NADPH.

However, in Escherichia coli expressing xylose reductase gene integratively, to achieve the matching of xylose reductase enzyme activity and coenzyme regeneration rate, it is necessary to further reduce the consumption rate of glucose. So far, there is no report on the method of enhancing the regeneration of coenzyme NADPH and slowing down the rate of glucose utilization by blocking or reducing the glucose metabolism flux of the EMP pathway.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a xylitol genetically engineered production bacterium that can greatly improve the xylitol production efficiency.

To achieve the above object, the present invention adopts the following technical solutions:

The invention uses Escherichia coli W3110 as the starting strain, and uses gene replacement technology to replace the genes affecting the xylose metabolism pathway and the glucose metabolism pathway in the genome of the original strain, so as to realize the matching of xylose reductase activity and coenzyme regeneration rate, and then Improve the efficiency of genetically engineered bacteria to convert xylose to produce xylitol.

Therefore, the present invention provides a xylitol gene-engineered production bacterium, which is obtained from Escherichia coli W3110 through genome modification, and ptsG, xylAB and ptsF in the original Escherichia coli W3110 genome are all replaced with xylose reductase gene XR; it also includes the genome At least one of pfkA, pfkB, pgi, and sthA is replaced with the xylose reductase gene XR.

The Escherichia coli W3110 was purchased from DSMZ, the German Collection of Microorganisms, and the number is DSM-5911.

The transformation of the present invention to Escherichia coli W3110 includes:

(1) There is a pathway to metabolize xylose in E. coli cells: xylose generates xylulose through xylose isomerase (xylA), and xylulose generates xylulose 5-phosphate under the action of xylulokinase (xylB). Ketose, xylulose 5-phosphate enters the pentose phosphate pathway to be metabolized. At the same time, the phosphotransferase (ptsF) pathway of fructose may also be involved in the transport of xylitol, so that xylitol is phosphorylated when it enters the cell, and the phosphorylated xylitol is toxic to cells. The xylose reductase gene XR is replaced with ptsF, which blocks the metabolism of xylose and the phosphorylation of xylitol while increasing the expression of xylose reductase, which is beneficial to the efficient conversion of xylose by engineered bacteria to produce xylitol.

(2) There is a glucose effect when Escherichia coli utilizes sugar, that is, when there is glucose, the utilization of other sugars such as xylose and arabinose by Escherichia coli will be severely inhibited. Therefore, the present invention replaces the glucose phosphotransferase gene ptsG with The xylose reductase gene XR is formed, which reduces the utilization rate of glucose by genetically engineered bacteria and eliminates the effect of glucose.

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

Preferably, ptsG, xylAB, ptsF, pfkA and pfkB in the original Escherichia coli W3110 genome are all replaced with the xylose reductase gene XR. Studies have shown that after the above five genes are replaced by xylose reductase gene XR, the production efficiency of genetically engineered bacteria to convert xylose to produce xylitol reaches 1.92g/L.

The xylose reductase gene XR is derived from Neurospora crassa, and the gene sequence is shown in NCBI accession number NCU08384.1.

The present invention also provides a construction method for constructing the above-mentioned xylitol genetically engineered production bacteria, comprising the following steps:

(1) replacing ptsG, xylAB and ptsF in the genome of E. coli W3110 with xylose reductase gene XR to obtain the first generation of genetically engineered bacteria;

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

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

Specifically, in step (1), including:

a. Use specific primers to construct pTargetF plasmids that replace the ptsG, xylAB and ptsF genes and the corresponding repair templates containing the XR expression module;

b. The pTargetF plasmid and the repair template that replace the ptsG gene are transformed into E. coli W3110 containing the pCas plasmid, and through homologous recombination, the strain W3110△ptsG::XR in which the ptsG gene in the genome is replaced with an XR expression module is obtained by screening;

Then, the pTargetF plasmid and the repair template that replaced the xylAB gene were transformed into the strain W3110△ptsG::XR, and after homologous recombination, the strain W3110△ptsG::XR,△xylAB: :XR;

Then, the pTargetF plasmid and the repair template that replaced the ptsF gene were transformed into the strain W3110△ptsG::XR,△xylAB::XR. After homologous recombination, the strain W3110△ptsG:: with the ptsF gene in the genome replaced by the XR expression module was obtained by screening. XR,△xylAB::XR,ΔptsF::XR, the first generation of genetically engineered bacteria;

In step (2), including:

D. respectively construct the pTargetF plasmid that replaces pfkA, pfkB gene and the corresponding repair template containing XR expression module;

e. First transform the pTargetF plasmid and the corresponding repair template that replace the pfkA gene into the first-generation genetically engineered bacteria obtained in step (1), and through homologous recombination, screen to obtain the bacterial strain in which the pfkA gene in the genome is replaced with an XR expression module WZ04△pfkA::XR, then the pTargetF plasmid replacing the pfkB gene and the corresponding repair template were transformed into the strain WZ04△pfkA::XR, and after homologous recombination, the strain with the pfkB gene in the genome replaced by the XR expression module was obtained by screening, That is, the xylitol genetic engineering production bacteria.

The XR expression module contains promoter P43. Studies have shown that the promoter P43 facilitates the expression of xylose reductase and does not require an inducer. Taking the pRC43M plasmid provided by the patent document CN 104789586A as a template, and using specific primers to amplify the XR expression module with P43 as the promoter.

The present invention also provides the application of the xylitol genetic engineering production bacteria in the production of xylitol.

The application includes: inoculating the xylitol genetically engineered production bacteria in a fermentation medium, fermenting and culturing at 30-37° C. for 80-90 hours, and keeping the OD ₆₀₀ of the bacterial liquid concentration less than 20 during the fermentation process.

The fermentation medium can use artificially configured culture liquid with xylose as the main component, or can use hemicellulose hydrolyzate as the main raw material.

Preferably, the initial conditions of fermentation: temperature 37°C, rotation speed 400rpm, ventilation 0.6-0.8vvm, initial pH of fermentation medium is about 6.5, and dissolved oxygen in the culture process is controlled at 30-35%; when the concentration of bacterial liquid OD ₆₀₀ ≥ 20 , feeding, the fermentation conditions after feeding are: temperature 30 ℃, dissolved oxygen controlled at 20-25%.

The batch feeding method was adopted, specifically, when OD600>20 (culturing at 37°C for about 7 h), the first feeding was performed; when the glucose was completely consumed, the second feeding was performed.

Feeding liquid ingredients, including: hemicellulose hydrolyzate or xylose mother liquor (xylose final concentration 60g/L), glucose mother liquor (glucose final concentration is 1/2 of xylose molar concentration), technical grade corn steep liquor dry powder (wood 1/3 of the sugar mass concentration).

The beneficial effects that the present invention has:

The xylitol genetically engineered production bacteria provided by the present invention takes Escherichia coli W3110 as the starting strain, and both xylAB and ptsF in the genome are replaced by xylose reductase gene XR, so as to block the metabolism of xylose and the phosphorylation of xylitol , to improve the utilization rate of xylose by genetically engineered bacteria; at least one of ptsG and pfkA, pfkB, pgi, sthA in the genome is replaced by xylose reductase gene XR, which eliminates the effect of glucose by blocking or reducing the EMP pathway glucose The metabolic flux reaches the PPP pathway to strengthen the regeneration of NADPH, which provides the necessary coenzyme NADPH for the reduction of xylose by xylose reductase to generate xylitol; the corresponding genes in the genome are replaced by the xylose reductase gene XR, and the xylose reductase is highly expressed , greatly improving the efficiency of genetically engineered bacteria to convert xylose to produce xylitol.

Description of drawings

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

Figure 2 shows the construction method of the repair template.

Figure 3 is the electrophoresis of nucleic acid in the chassis cell transformed by CRISPR technology, wherein M: 250bp Marker, G and G: XR are fragments amplified by primers ptsG-u-F and ptsG-d-R, G: original ptsG gene, G: XR: The original ptsG gene was replaced by xr; AB and AB: XR were fragments amplified using primers xylAB-u-F and xylAB-d-R, AB: the original xylAB gene, AB: XR: the original xylAB gene was replaced by xr; F and F: XR For the fragment amplified using primers ptsF-u-F and ptsF-d-R, F: original ptsF gene, F: XR: original ptsF gene replaced by xr.

Figure 4 shows the analysis of the main metabolic pathways of glucose.

Figure 5 is the concentration of sugar and sugar alcohol in the fermentation broth after 24h shake flask fermentation.

Fig. 6 is the determination result of bacterial concentration OD600 after 24h fermentation.

Figure 7 shows that strain WZ31 utilizes pure sugar to ferment xylitol, wherein Glucose is glucose, Xylose is xylose, and Xylitol is xylitol.

Figure 8 is a fed-batch fermentation using WZ31 using hemicellulose hydrolyzate and corn steep liquor dry powder, wherein Glucose is glucose, Xylose is xylose, Arabinose is arabinose, Arabitol is arabitol, and Xylitol is xylitol .

Detailed ways

The present invention will be further described below in conjunction with specific embodiments.

The E.coli W3110 used in the following examples was purchased from the German Collection of Microorganisms, DSMZ, numbered DSM-5911; pTargetF plasmid addgene: #62226; pCas plasmid addgene: #62225; pRC43M plasmid from the application number 201510196843.8, named as "Escherichia coli genome integration vector, genetic engineering bacteria and application in the production of xylitol" patent document.

Example 1

This embodiment provides a xylitol genetically engineered production bacterium, which can enhance the regeneration of intracellular NADPH in an integrated xylose reductase expressing strain and improve the production efficiency of xylitol.

The construction method of described xylitol genetic engineering production bacteria, comprises the following steps:

1. Construct the pTargetF plasmid that replaces or knocks out the target gene

First, it is necessary to find a PAM site on the target gene, that is, the NGG sequence, determine the corresponding N20 sequence, and replace the cadAspacer on the pTargetF plasmid with the N20 sequence of the target gene. Taking ptsG as an example, and the plasmid construction of pTargetF-ptsG as an example, the primers N20-ptsG-F and N20-ptsG-R were designed and used for pTargetF mutation PCR. The primer design method is shown in Figure 1.

The PCR recipe and program settings are as follows:

Table 1 Whole plasmid mutation PCR system

It can be seen from the pTargetF plasmid map that the total length of the plasmid is 2118bp. In order to ensure the integrity of the extension during the PCR process, a long extension time was used in this study.

Table 2 Whole plasmid PCR program settings

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

After verification, the PCR template was digested with DpnI enzyme to improve the positive rate of transformants. The enzymatic digestion system is as follows:

Enzyme DpnI 0.5μL

10*Buffer 1μL

PCR product 8.5 μL.

Add the corresponding substances according to the system, shake and mix horizontally, centrifuge quickly for 20s for a short time, and place in a water bath or metal bath at 37°C for 60min. DH5α competence was then used for transformation.

The obtained transformants were picked for liquid culture, and plasmids were extracted using a plasmid mini-extraction kit. Using Target-check for sequencing verification, the successfully mutated plasmid pTargetF-ptsG was finally obtained.

2. Replacement of ptsG with the construction of XR gene repair template

The build strategy is shown in Figure 2.

Using E.coli W3110 genome as a template, with ptsG-u-F and ptsG-u-R, ptsG-d-F and ptsG-d-R as PCR primers, conventional PCR was performed to obtain 500bp of homology arm fragments upstream and downstream of the ptsG gene;

Using pRC43M plasmid as template and ptsG-XR-F and ptsG-XR-R as primers, the xr expression module with P43 as promoter was obtained by PCR. The obtained PCR products were subjected to DNA nucleic acid electrophoresis and gel recovery.

Finally, using ptsG-u-F and ptsG-d-R as primers, and the equal proportion mixture of ptsG upstream and downstream homology arms and xr expression module as templates, overlap extension PCR was performed, and fragments of corresponding length were recovered by cutting the gel to obtain a repair template for replacing the ptsG gene.

3. Genome Gene Replacement Method Operation

1) Transfer to pCas plasmid by heat shock method

a. The wild-type E. coli W3110 stored at -80°C was streaked on an antibody-free solid medium plate and incubated at 37°C overnight. Pick a single colony in liquid LB medium at 37°C and cultivate at 200 rpm for about 10 hours, transfer 1 mL of bacterial solution to a 250 mL conical flask containing 50 mL of liquid LB medium, grow to OD ₆₀₀ to 0.6-0.8, and freeze the bacterial solution on ice. After bathing for 10 min, the transformation competence was prepared according to the Takara E. coli competence kit.

b. Put the prepared E.coli W3110 competent cells on ice, add 10 μL of pCas plasmid under aseptic conditions after thawing, mix well and place in an ice bath for 30 min.

c. Heat shock in water bath or metal bath for 90 s at 42°C, and immediately ice bath for 2 min.

d. Add 890 μL of liquid LB or recovery medium, and recover at 30°C and 200 rpm for 45 minutes.

e. Pipette 100 μL of the revived bacterial solution, spread it on a solid LB plate containing 50 mg/L of kan ^R , and culture at 30° C. overnight.

f. Pick a single clone for PCR or extract plasmid verification to obtain E.coliW3110pCas that has been transformed successfully.

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

a. Take the Escherichia coli obtained in 1) as the starting strain, streak it on a 50 mg/L kanamycin sulfate-resistant plate, and cultivate overnight at 30°C (the same antibiotic of the same concentration needs to be added for subsequent cultivation) , pick a single colony into liquid LB, culture at 30°C, 200rpm for 10-12h, transfer 1mL to 50mL medium LB, culture at 30°C, 200rpm for 1h, add L-arabinose with a working concentration of 0.5% sterilized Induce the expression of the Red recombinant protein, continue to culture to OD ₆₀₀ to 0.6-0.8 (about 3 hours), ice bath for 10 minutes, and prepare electrotransformation competent cells.

b. Using a sterilized 10 mL Ep tube, after dividing the bacterial liquid into a batch, centrifuge at 4°C and 4000 rpm for 5 min, and discard the supernatant.

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

d. Repeat step c twice.

e. Resuspend with 100 μL of 10% glycerol, transfer to a sterilized 1.5 mL Ep tube, use immediately or place in a -80°C refrigerator for later use.

f. Use the prepared competent cells or take out the pre-prepared competent cells from -80°C, place on ice for 5 minutes, and add 400 ng of pTargetF plasmid and 800 ng of repair template constructed to replace the corresponding gene. After mixing, it was transferred to a sterile 2mm electroporation cup and placed on ice for 10 min for electroporation.

g. Power transfer conditions: 2.5kV, 25μF, 200Ω, power transfer time 5ms, the cup wall and base of the power transfer cup must be dried with paper towels before power transfer, otherwise it will easily cause the cup to burst. At the same time, excessive salt ion residues in the extracted plasmids and recovered repair templates can also cause burst cups.

h. Immediately after electroporation, 1 mL of liquid LB was added, mixed by pipetting back and forth, and then transferred to a 2 mL sterilized Ep tube. Resuscitate for about 3h at 30°C and 150rpm.

i. After centrifuging and concentrating the recovered bacterial liquid, it was spread on a plate containing 50 mg/L of kan ^R and 50 mg/L of spec ^R , and cultured at 30°C overnight.

j. Under normal circumstances, there are visible transformants within 12 hours of culture. If the strain has undergone multiple edits, the growth time may be extended.

4. Gene replacement verification

For the positive transformants that have been successfully replaced, if the sequence lengths before and after the replacement are quite different, the target band can be amplified by colony or bacterial liquid PCR, DNA nucleic acid electrophoresis is performed, and the size of the amplified band before and after the gene replacement can be compared. to identify. If the difference in gene size before and after replacement is within 200bp, the PCR product sequencing method can be used to identify.

5. Similarly, the corresponding primers were used to construct pTargetF and repair templates of other replacement genes. After 3 rounds of genome editing, xylose reductase was successfully inserted into the ptsG, xylAB, ptsF regions of the genome.

The transformed integrated strains WZ01, WZ02 and WZ03 expressing xylose reductase were designed. Among them, the schematic diagram of the genome of WZ03 (E.coli W3110, △ptsG:XR, △xylAB:XR, △ptsF:XR) is shown in Figure 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 is not obvious after the replacement. The correctness of the result cannot be determined by the nucleic acid gel electrophoresis. The Target-check primer is used to verify the sequencing of the PCR product, and the ptsG gene is successfully replaced with the xylose reductase expression module. . The xylAB gene has a total of 2849 bp, which is reduced by 1400 bp after being replaced by XR. The electropherogram can determine that it is a positive transformant after the replacement; the ptsF gene is 1692 bp, which is reduced by 243 bp after the replacement. The WZ03 strain finally obtained is an engineering strain in which ptsG, xylAB and ptsF are all replaced with the expression module of the xylose reductase gene.

6. Through the analysis of the main metabolic pathway of glucose (Figure 4), the PPP pathway in glucose metabolism can produce more NADPH, so it involves experiments on the pfkA, pfkB, pgi genes and the transhydrogenase sthA gene of the EMP pathway, using xylose reductase The expression module is replaced.

Taking the WZ03 strain as the starting strain, the genes pfkA, pfkB, pgi and sthA, which may enhance the regeneration ability of coenzyme NADPH, were replaced by single genes to obtain strains WZ21, WZ22, WZ23 and WZ24. And select the engineering bacteria with better effect for double replacement to obtain strains WZ31 and WZ32.

The primers (SEQ ID NO. 1-57) involved in the above experiments 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 genotypes

7. The above strains were subjected to shake flask fermentation experiments, and the fermentation conditions were as follows:

(1) Preparation of seed solution

Seed liquid culture: Pick the single colonies separated by streaking into sterilized fresh liquid LB medium, and cultivate overnight at 37°C and 200 rpm to a stable growth stage.

(2) Shake flask fermentation

To prepare a shake flask fermentation medium, a 250 mL conical flask was filled with 45 mL of liquid, inoculated with 1 mL of seed solution, cultured at 37°C, 200 rpm for 4 h, and added 5 mL of sterilized mixed sugar solution (containing 200 g/L xylose and 100 g/L Glucose), shake flask fermentation at 30 °C and 200 rpm, take samples regularly and detect changes in relevant parameters.

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

The collected sample was diluted to an appropriate concentration, and then filtered using a 0.22 μm filter. Quantitative detection of xylose, glucose, arabinose, xylitol, and arabitol was performed using the Dionex UltiMate 3000 HPLC system. Detector: Corona Charged Aerosol Detector (CAD), analytical column: Aminex HPX-87C (Φ7.8mm×300mm), mobile phase: ultrapure water, flow rate: 0.6mL/min, column temperature setting: 76°C.

Take the bacterial solution after adding sugar solution for 24 hours, extract the oxidized and reduced coenzymes in the cells, and measure the concentration. The method is as follows:

1) Place the bacterial solution in an ice bath for 10 min, centrifuge at 4000 rpm for 15 min at 4°C, and concentrate to 1 mL of bacterial solution with an OD ₆₀₀ of 30 to separate the intracellular oxidized and reduced coenzymes.

2) Take 1mL of bacterial solution and add:

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

Isolated reduced form: 0.5 mL of 0.3 M NaOH;

3) All samples were incubated at 60°C for 7 min. The oxidized form was neutralized with 0.5 mL of 0.3M NaOH, and the reduced form was neutralized with 0.5 mL of 0.3M HCl. After neutralization, 0.1 mL of 1.0M Tricine should be added to each sample. -NaOH to maintain pH stability.

4) Centrifuge at 13000rpm for 60min at 4°C. Transfer 300 μL of supernatant to a new Ep tube.

5) Use the absorbance value of the reaction solution at a specific wavelength to determine the content of the coenzyme. Quantitation can be performed using a 96-well plate and a microplate reader. The measurement system is:

Oxidized form: 40 μL sample + 40 μL 0.1M NaCl;

The reduced form is: 80 μL sample;

6) Add equal volume (80 μL) of 2* reaction stock solution (1.0M Tricine-NaOH (pH8.0), 4.2mM thiazolyl blue tetrazolium bromide (MTT), 40mM EDTA, 1.67mM phenazineethosulfate (PES), and substrate (5M ethanol) (for measuring NAD) or 25 mM glucose-6-phosphate (for measuring NADP).

7) After mixing, keep the temperature at 37°C for 5 minutes, add alcohol dehydrogenase with a working concentration of 10 U/mL and glucose-6-phosphate dehydrogenase with a working concentration of 0.27 U/mL (the mother liquor is prepared at a concentration of 10*).

8) The reduction of MTT at 37°C can be detected by a microplate reader at 570 nm. Data were compared to a standard curve.

The measured ratios of the reduced and oxidized coenzymes are shown in Table 5 below.

Table 5 The ratio of reduced and oxidized coenzymes after different gene replacements

Compared with the control group WZ03, the intracellular NADPH/NADP+ content increased to a certain extent after the replacement of related genes. The sugar and sugar alcohol in the fermentation broth were determined, and the results are shown in Figure 5 below.

From the analysis in Table 5 and Figure 5, it can be seen that after the transformation, the regeneration ability of intracellular NADPH has been strengthened, and the consumption of the same mole of glucose can produce more NADPH, while increasing the ratio of intracellular NADPH/NADP+, maintaining a higher coenzyme. The concentration provides sufficient reducing power for xylose reductase.

The growth OD ₆₀₀ of each strain was measured, and the results are shown in Figure 6 . In the transformed strain, with 4 copies of xylose reductase, the higher the ratio of coenzyme NADPH/NADP+, the higher the yield of xylitol of the strain. At 5 copies of xylose reductase, the coenzyme NADPH/NADP+ ratio of WZ31 and WZ32 were comparable, but WZ31 had higher xylitol production. Combined with Figure 6, the growth ability of WZ32 was destroyed after the transformation, and the bacterial concentration after 24h was only about 40% of the starting strain WZ03, which was the main reason for its low xylitol production.

The strain WZ31 was subjected to pure sugar shake flask fermentation experiments, and samples were taken 10h, 20h and 30h after sugar supplementation, respectively, to determine the concentration of sugar and sugar alcohol in the fermentation broth and the determination of OD _600. The results are shown in Figure 7.

8. Use hemicellulose hydrolyzate and dry corn steep liquor for fermentation, use WZ31 strain, and use 15L fermenter to ferment xylitol without adding antibiotics and inducers. The method is as follows:

(1) First-class seed liquid culture

The preserved strains were streaked on solid medium plates and cultured overnight at a constant temperature of 37°C. Pick a single colony in liquid LB and culture at 37°C and 200rpm for about 12h.

First-class seed liquid medium formula (L ^-1 ): peptone 10g, yeast powder 5g, sodium chloride 10g, solid medium plus 2% agar powder.

(2) Secondary seed liquid culture

The primary seed liquid was transferred to the secondary seed liquid medium at a volume ratio of 0.5 to 1%, and cultured at 37° C. and 200 rpm for 7 h.

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

(3) Fermentation process control

Before inoculation, adjust the pH of the fermentation medium to about 6.5 with ammonia water, and inoculate the cultured secondary seed liquid according to 10-15% of the volume of the fermentation medium. Controlled at 400rpm. Adjust the speed and dissolved oxygen joint control, keep the dissolved oxygen in the fermenter at 30-35%, take samples at certain time intervals to measure the bacterial concentration OD ₆₀₀ , and carry out feeding operation when the OD ₆₀₀ ≥ 20, and the flame method is used for inoculation and feeding.

Feeding method: batch feeding

OD600>20 (cultivation at 37°C for about 7h), the first feeding was carried out. After the first feeding, the glucose was completely consumed, and the second feeding was carried out.

Feeding liquid composition and final concentration: hemicellulose hydrolyzate or xylose mother liquor (xylose final concentration 60g/L), glucose mother liquor (glucose final concentration is 1/2 of xylose molar concentration), technical grade corn steep liquor dry powder ( 1/3 of the mass concentration of xylose).

Condition control after feeding: dissolved oxygen is controlled at 20-25%, temperature control: 30°C.

After feeding, samples were taken regularly and the concentrations of glucose, xylose, arabinose, arabitol and xylitol were detected during the fermentation process, and the growth of the strains in the whole project was monitored. The final fermentation result is shown in Figure 8.

As can be seen from Figure 8, using the hemicellulose hydrolyzate as the substrate and corn steep liquor dry powder as the carbon source, 161.03g/L of xylitol was finally obtained after 84h fed-batch fermentation, and xylose, arabinose and glucose were basically the same. When completely consumed, the by-product arabitol is only 1.63g/L. The production efficiency of xylitol was 1.92 g/L/h.

The above-mentioned embodiments describe the technical solutions and beneficial effects of the present invention in detail. 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. Any modifications, additions and equivalent replacements made should be included within the protection scope of the present invention.

sequence listing

<110> Zhejiang University

<120> A kind of xylitol genetic engineering production bacterium and its construction method and application

<160> 57

<170> SIPOSequenceListing 1.0

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tccttcattt ggccgccgat gttttagagc tagaaatagc 40

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atcggcggcc aaatgaagga actagtatta tacctaggac 40

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gatcggttac tggtggaaac tgactc 26

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cactcaataa agctgagctc aattgagagt gctcctgagt atggg 45

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actcaggagc actctcaatt gagctcagct ttattgagtg gatga 45

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ctccccaacg tcttacggat taccggttta ttgactaccg gaag 44

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<212> DNA

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ggtagtcaat aaaccggtaa tccgtaagac gttggggaga ctaa 44

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ggtggatggg acagtcagta aagg 24

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tgcgcaagag tgcactttgc gttttagagc tagaaatagc 40

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gcaaagtgca ctcttgcgca actagtatta tacctaggac 40

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<212> DNA

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gatatctcga tctttttgcc agcgttc 27

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<212> DNA

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cactcaataa agctgagctc attgaactcc ataatcaggt aatgc 45

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<212> DNA

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acctgattat ggagttcaat gagctcagct ttattgagtg gatga 45

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<212> DNA

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ggtcaggcag gggataacgt taccggttta ttgactaccg gaag 44

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<212> DNA

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ggtagtcaat aaaccggtaa cgttatcccc tgcctgaccg ggtg 44

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<212> DNA

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gagcgaaaca aacgcatttg accaaac 27

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<212> DNA

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agcaccggtt ctggcttcat gttttagagc tagaaatagc 40

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<212> DNA

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atgaagccag aaccggtgct actagtatta tacctaggac 40

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<212> DNA

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gactggatga ctcgcctgcg tagtcag 27

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<212> DNA

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cactcaataa agctgagctc tatgcctctc ctgctgtcag ttaaa 45

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<212> DNA

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ctgacagcag gagaggcata gagctcagct ttattgagtg gatga 45

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<212> DNA

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gccctgtaac acacctttta taccggttta ttgactaccg gaag 44

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<212> DNA

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ggtagtcaat aaaccggtat aaaaggtgtg ttacagggca gaaa 44

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<212> DNA

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tgcgccctac tccaccaggc gtaaa 25

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<212> DNA

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cacaacgaat tcacagccac gttttagagc tagaaatagc 40

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gtggctgtga attcgttgtg actagtatta tacctaggac 40

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gtgactgacg aatcaccacg 20

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<212> DNA

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cactcaataa agctgagctc tgatttcgga aaaaggcaga ttcct 45

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<212> DNA

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tctgcctttt tccgaaatca gagctcagct ttattgagtg gatga 45

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tccaaagttc agaggtagtc ccggtttatt gactaccgga agcag 45

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<212> DNA

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tccggtagtc aataaaccgg gactacctct gaactttgga atgca 45

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<212> DNA

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gtttgcattg ggaatcggca tc 22

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gtcctaggta taatactagt ccaaaaagaa ctcagtgcgc 40

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gctatttcta gctctaaaac gcgcactgag ttctttttgg 40

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<212> DNA

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gaagaagtat tactggcgtt ccc 23

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cactcaataa agctgagctc catttcctcc tataggctga tttca 45

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<212> DNA

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tcagcctata ggaggaaatg gagctcagct ttattgagtg gatga 45

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<212> DNA

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atgctggggg aatgtttttg ccggtttatt gactaccgga agcag 45

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<212> DNA

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tccggtagtc aataaaccgg caaaaacatt cccccagcat tgggg 45

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gactgattgc ctgccacatg atg 23

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<212> DNA

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ctgccaacat gttcgagttc gttttagagc tagaaatagc 40

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<212> DNA

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gaactcgaac atgttggcag actagtatta tacctaggac 40

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<212> DNA

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gatccatttt cagccttggc ac 22

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<212> DNA

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cactcaataa agctgagctc tagcaatact cttctgattt tgaga 45

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<212> DNA

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aaatcagaag agtattgcta gagctcagct ttattgagtg gatga 45

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<212> DNA

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ggcctacata tcgacgatga ccggtttatt gactaccgga agcag 45

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<212> DNA

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tccggtagtc aataaaccgg tcatcgtcga tatgtaggcc ggata 45

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<212> DNA

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cagaagtcgc cgcaagcgca gatatg 26

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gaatatgcgt cgatcttccg gttttagagc tagaaatagc 40

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cggaagatcg acgcatattc actagtatta tacctaggac 40

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gttcgtctac gtcgcggaaa tg 22

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cactcaataa agctgagctc ggtagggctt acctgttctt ataca 45

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aagaacaggt aagccctacc gagctcagct ttattgagtg gatga 45

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atggccattt cgataaagtt ccggtttatt gactaccgga agcag 45

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tccggtagtc aataaaccgg aactttatcg aaatggccat ccattc 46

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gaagcacaga cccaccagtt actg 24

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<212> DNA

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gaagggagaa aggcggacag 20

Claims (4)

1. a xylitol genetic engineering production bacterium, obtained through genome modification by Escherichia coli W3110, is characterized in that, ptsG, xylAB, ptsF, pfkA and pfkB in the original Escherichia coli W3110 genome are all replaced by xylose reductase gene XR , the xylose reductase gene XR is derived from Neurospora crassa; The construction method of described xylitol genetic engineering production bacterium, comprises the following steps: (1) replacing ptsG, xylAB and ptsF in the genome of E. coli W3110 with xylose reductase gene XR to obtain the first generation of genetically engineered bacteria; (2) replacing pfkA and pfkB in the genome of the first-generation genetically engineered bacteria with xylose reductase gene XR; In step (1), including: a. Use specific primers to construct pTargetF plasmids that replace the ptsG, xylAB and ptsF genes and the corresponding repair templates containing the XR expression module; b. The pTargetF plasmid and the repair template that replace the ptsG gene are transformed into E. coli W3110 containing the pCas plasmid, and through homologous recombination, the strain W3110△ptsG::XR in which the ptsG gene in the genome is replaced with an XR expression module is obtained by screening; Then, the pTargetF plasmid and the repair template that replaced the xylAB gene were transformed into the strain W3110△ptsG::XR, and after homologous recombination, the strain W3110△ptsG::XR,△xylAB: :XR; Then, the pTargetF plasmid and the repair template that replaced the ptsF gene were transformed into the strain W3110△ptsG::XR,△xylAB::XR. After homologous recombination, the strain W3110△ptsG:: with the ptsF gene in the genome replaced by the XR expression module was obtained by screening. XR,△xylAB::XR,△ptsF::XR, namely the first generation of genetically engineered bacteria; In step (2), including: D. respectively construct the pTargetF plasmid that replaces pfkA, pfkB gene and the corresponding repair template containing XR expression module; e. First transform the pTargetF plasmid and the corresponding repair template that replace the pfkA gene into the first-generation genetically engineered bacteria obtained in step (1), and through homologous recombination, screen to obtain the bacterial strain in which the pfkA gene in the genome is replaced with an XR expression module WZ04△pfkA::XR, then the pTargetF plasmid replacing the pfkB gene and the corresponding repair template were transformed into the strain WZ04△pfkA::XR, and after homologous recombination, the strain with the pfkB gene in the genome replaced by the XR expression module was obtained by screening, That is, the xylitol genetically engineered production bacteria; the XR expression module includes the promoter P43. 2. the application of xylitol genetic engineering producing bacteria as claimed in claim 1 in producing xylitol. 3. application as claimed in claim 2, is characterized in that, comprises: inoculate described xylitol genetic engineering production bacteria in fermentation medium, 30-37 ℃ of fermentation culture 80-90h, keep bacteria in fermentation process The liquid concentration OD ₆₀₀ is less than 20. 4. application as claimed in claim 3, it is characterized in that, fermentation initial condition: temperature 37 ℃, rotating speed 400rpm, ventilation 0.6-0.8vvm, initial pH of fermentation medium is 6.5, and dissolved oxygen in culturing process is controlled at 30-35 %; when the concentration of bacterial liquid OD ₆₀₀ ≥ 20, feeding is carried out, and the fermentation conditions after feeding are as follows: temperature is 30°C, and dissolved oxygen is controlled at 20-25%.

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