CN108753672A

CN108753672A - A kind of xylitol genetic engineering production bacterium and its construction method and application

Info

Publication number: CN108753672A
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: 2018-11-06
Anticipated expiration: 2038-06-11
Also published as: CN108753672B

Abstract

The invention discloses a kind of xylitol genetic engineering production bacterium and its construction method and applications, belong to gene engineering technology field.The xylitol genetic engineering produces bacterium, is obtained through genome manipulation by Escherichia coli W3110, ptsG, xylAB and ptsF in original Escherichia coli W3110 genomes are substituted for Xylose reductase gene XR；Further include in genome at least one of pfkA, pfkB, pgi, sthA be substituted for XR.The present invention is using Escherichia coli W3110 as starting strain, corresponding gene in genome is substituted for XR, Xylose reductase is able to high efficient expression, pass through the phosphorylation of the metabolism and xylitol of blocking xylose simultaneously, improve the utilization to xylose, and by blocking or reducing the NADPH regeneration of EMP Embden Meyerbof Parnas pathway glucose metabolism flux enhancement, restores xylose generation xylitol for Xylose reductase and required coenzyme NADP 11 is provided, the efficiency of genetic engineering bacterium Bioconversion of D-xylose To Produce Xylitol is greatly improved.

Description

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

Technical field

The present invention relates to gene engineering technology fields, and in particular to a kind of xylitol genetic engineering production bacterium and its structure side Method and application.

Background technology

Xylitol is a kind of five-state controller, and sugariness is suitable with sucrose, calorie value but only its 60% or so, xylitol tool There are anti-caries tooth and metabolism not to depend on insulin, improve the features such as liver function, is widely used in food, medicine and chemical industry.

Industrial production xylitol mainly obtains xylose using hemicellulose acid hydrolysis, and purity is obtained after isolating and purifying Xylose 95% or more is made under high-temperature and high-pressure conditions with nickel catalytic hydrogenation, and this process conditions are harsh, and be easy to cause Pollution, production cost are higher.Bioanalysis production xylitol does not need high-temperature and high-pressure conditions, inflammable and explosive hydrogen, pollutes environment The xylose etc. of Raney nickel and high-purity, reaction condition is mild, energy-saving safe and environmental-friendly, so bioanalysis conversion production wood Sugar alcohol is increasingly valued by people.

The microorganism that xylitol is prepared currently used for fermentation method is nearly all saccharomycete, and existing nature strain also has gene Engineering bacteria.Saccharomycete has the advantage of oneself as microbial strain for xylitol production, for example is resistant to higher sugared concentration, to hemicellulose Inhibiting factor repellence in cellulose hydrolysate is stronger, etc..But there is also unavoidable problems, such as have potential cause a disease Property, yeast itself it is contained Xylose reductase specificity it is poor etc..

For Escherichia coli as the ideal host for producing various high valuable chemicals, current research is the most thorough, the gene back of the body Scape understands that building genetic engineering bacterium using it has advantageous condition.Xylitol has been produced using Escherichia coli as host It has been reported that, such as Su Buli constructs first generation high yield xylitol bacterial strain based on plasmid vector system, egg is carried out using plasmid vector White expression, by the adjusting of mRNA secondary structures, construct one can efficient soluble-expression at relatively high temperatures plasmid；? Under the conditions of 30 DEG C, enzyme activity is 5.68 times of starting strain.By knocking out II component of enzyme in glucose phosphotransferase system PtsG genes eliminate the catabolite repression effect of bacterial strain, bacterial strain are enable to transport glucose and xylose simultaneously；Knock-out bacterial strain itself generation The gene xylA and xylB for thanking to xylose, block the metabolism of xylose to utilize；The fructose phosphate for knocking out Transshipment Permitted xylitol shifts enzyme system The ptsF genes of II component of enzyme, reduce the phosphorylation of xylitol in system；By a series of optimization, xylitol production efficiency is Bacterium germination strain 8.71 times (" Recombinant organism convert hemicellulose hydrolysate production xylitol research ", Su Buli, 《Zhejiang University》, 2016).

Coenzyme is the general name of the organic co-factor of a major class, is confactor necessary to enzymatic oxidation reduction reaction etc.. They serve as the effect for transmitting electronics, atom or group in the reaction, and coenzyme can be regarded as in enzyme reaction to a certain extent The second substrate.As a kind of crucial co-factor in microbial metabolism network, by adjusting intracellular coenzyme content and going back The orientable cell metabolism function changed and optimize microorganism of the ratio of prototype oxidized form, to realize the maximization of metabolic fluxes, This is also one of the important method for increasing target product.

Coenzyme NAD (P) H plays an important role in all kinds of enzymic catalytic reactions, especially in redox reaction, all needs Coenzyme NAD (P) H is wanted to participate in reacting as electron transmission.During Product formation, a certain amount of coenzyme can be consumed.Therefore As the coenzyme content of the carry out intracellular of reaction is reduced, catalytic efficiency is caused to reduce.Since coenzyme is expensive, added by external source It is unpractical that the mode added, which carries out supplement,.Therefore by the metabolic process of metabolic engineering regulating and controlling microbial, intracellular NADPH is improved Concentration, can not only effectively improve production efficiency reduces cost, can also better ensure that being normally carried out for biotransformation. From the perspective of Technological Economy, it is significant to strengthen regenerating coenzyme cycle.

Currently, the metabolic engineering main method based on coenzyme NADP 11 is to enhance the metabolic flux of PPP.In large intestine bar In bacterium, glucose is primarily present two metabolic pathways, including glycolytic pathway (EMP Embden Meyerbof Parnas pathway) and the pentose phosphate pathway (ways PPP Diameter).Wherein there is the reaction of 2 steps that can realize the regeneration of NADPH in PPP approach, by being overexpressed this two-step reaction in existing research In zwf and gnd genes improve endogenous coenzyme NADP 11, this method can strengthen intracellular NADPH to a certain extent again It is raw.

But in the Escherichia coli of integrated expression Xylose reductase gene, to realize Xylose reductase enzyme activity with coenzyme again The matching of raw rate, needs the wear rate for further decreasing glucose.It is had no at present using blocking or reduces EMP Embden Meyerbof Parnas pathway grape The method of glycometabolism flux realizes that the report of glucose utilization Speed method is strengthened and slowed down in coenzyme NADP 11 regeneration.

Invention content

The purpose of the present invention is to provide a kind of xylitol genetic engineerings that can significantly promote xylitol production efficiency Produce bacterium.

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

The present invention is using Escherichia coli W3110 as starting strain, using gene replacement technology in original strain genome The gene influenced in xylose metabolism access and glucose metabolism access is replaced, to realize Xylose reductase work and regenerating coenzyme The matching of rate, and then promote the efficiency of genetic engineering bacterium Bioconversion of D-xylose To Produce Xylitol.

Therefore, the present invention provides a kind of xylitol genetic engineerings to produce bacterium, by Escherichia coli W3110 through genome manipulation It obtains, ptsG, xylAB and ptsF in original Escherichia coli W3110 genomes are substituted for Xylose reductase gene XR；Also wrap It includes at least one of pfkA, pfkB, pgi, sthA in genome and is substituted for Xylose reductase gene XR.

The Escherichia coli W3110 is purchased from Germany Microbiological Culture Collection Center DSMZ, number DSM-5911.

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

(1) there are the approach of fermenting xylose in Bacillus coli cells：Xylose generates wood by xylose isomerase (xylA) Ketose, xylulose generate X 5P under the action of Xylulokinase (xylB), and X 5P enters phosphoric acid penta Sugared approach is metabolized.Phosphotransferase (ptsF) approach of fructose may also assist in the transhipment of xylitol simultaneously, make xylose Alcohol is phosphorylated while entering cell, and the xylitol of phosphorylation is to the toxic effect of cell, therefore by xylAB in genome Be substituted for Xylose reductase gene XR with ptsF, block xylose metabolism and xylitol phosphorylation while increase xylose reduction The expression of enzyme is conducive to engineering bacteria Efficient Conversion xylose production xylitol.

(2) there are glucose effects using when sugar for Escherichia coli, i.e., in the presence of having glucose, Escherichia coli are to other sugar Such as the utilization of xylose, arabinose all can be heavily suppressed, and therefore, the present invention is by glucose phosphotransferase gene ptsG It is substituted for Xylose reductase gene XR, utilization rate of the genetic engineering bacterium to glucose is reduced, eliminates glucose effect.

But during Xylose reductase reduction xylose generates xylitol, glucose is needed to provide reaction as auxiliary substrate Required coenzyme NADP 11.Therefore, the present invention will participate in gene pfkA, pfkB, pgi or sthA progress of glycolytic pathway (EMP) It replaces, to block or reduce EMP Embden Meyerbof Parnas pathway glucose metabolism flux, realizes that grape is strengthened and slowed down in the regeneration of PPP approach coenzyme NADP 11s Sugar utilizes rate.

Preferably, ptsG, xylAB, ptsF, pfkA and pfkB in original Escherichia coli W3110 genomes are replaced At Xylose reductase gene XR.Studies have shown that after above-mentioned 5 genes are replaced by Xylose reductase gene XR, genetic engineering The production efficiency of bacterium Bioconversion of D-xylose To Produce Xylitol reaches 1.92g/L.

The Xylose reductase gene XR derives from Neuraspora crassa, and gene order is referring to NCBI accession number NCU08384.1。

The present invention also provides the construction methods that a kind of above-mentioned xylitol genetic engineering of structure produces bacterium, including following step Suddenly：

(1) ptsG, xylAB and ptsF in Escherichia coli W3110 genomes are substituted for Xylose reductase gene XR, Obtain first generation genetic engineering bacterium；

(2) pfkA in first generation genetic engineering bacterium genome, pfkB, pgi, at least one of sthA are substituted for wood Sugared reductase gene XR.

In step (1) and (2), gene replacement is carried out using homologous recombination technique.

Specifically, in step (1), including：

A. specific primer is utilized to build the pTargetF plasmids and correspondence for replacing ptsG, xylAB and ptsF gene respectively Containing XR express module recovery template；

B. the pTargetF plasmids for replacing ptsG genes and recovery template are transformed into the Escherichia coli containing pCas plasmids In W3110, through homologous recombination, screening obtains the bacterial strain W3110 △ that ptsG genes in genome are substituted for XR expression modules ptsG::XR；

The pTargetF plasmids for replacing xylAB genes and recovery template are transformed into bacterial strain W3110 △ ptsG again::In XR, Through homologous recombination, screening obtains the bacterial strain W3110 △ ptsG that xylAB genes in genome are substituted for XR expression modules::XR,△ xylAB::XR；

Then the pTargetF plasmids for replacing ptsF genes and recovery template are transformed into bacterial strain W3110 △ ptsG::XR, △xylAB::XR, through homologous recombination, screening obtains the bacterial strain W3110 △ that ptsF genes in genome are substituted for XR expression modules ptsG::XR,△xylAB::XR,ΔptsF::XR, i.e. first generation genetic engineering bacterium；

In step (2), including：

D. structure replaces the pTargetF plasmids of pfkA, pfkB gene and the corresponding reparation that module is expressed containing XR respectively Template；

E. the pTargetF plasmids for replacing pfkA genes and corresponding recovery template are first transformed into made from step (1) the In generation genetic engineering bacterium, through homologous recombination, screening obtains the bacterial strain that pfkA genes in genome are substituted for XR expression modules WZ04△pfkA::XR, then the pTargetF plasmids for replacing pfkB genes and corresponding recovery template are transformed into bacterial strain WZ04 △ pfkA::In XR, through homologous recombination, screening obtains the bacterial strain that pfkB genes in genome are substituted for XR expression modules, as described Xylitol genetic engineering produce bacterium.

Include Promoter P43 in the XR expression modules.Research shows that Promoter P43 contributes to the table of Xylose reductase It reaches, and does not need derivant.The pRC43M plasmids provided using patent document CN 104789586A are drawn as template using specificity Object is amplified expresses module by the XR of promoter of P43.

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

The application, including：The xylitol genetic engineering production bacterium is inoculated in fermentation medium, 30-37 DEG C fermented and cultured 80-90h keeps bacterial concentration OD in fermentation process₆₀₀Less than 20.

The fermentation medium can also be utilized using human configuration with xylose culture solution as main component Hemicellulose hydrolysate is primary raw material.

Preferably, fermentation primary condition：37 DEG C of temperature, rotating speed 400rpm, ventilatory capacity 0.6-0.8vvm, fermentation medium Initial pH is 6.5 or so, and incubation dissolved oxygen is controlled in 30-35%；As bacterial concentration OD₆₀₀When >=20, feed supplement, feed supplement are carried out Fermentation condition is afterwards：30 DEG C of temperature, dissolved oxygen are controlled in 20-25%.

Using batch feeding mode, specifically, OD600>20 (37 DEG C of culture 7h or so), carry out first time feed supplement；Wait for Portugal Grape sugar has completely consumed, and carries out second of feed supplement.

Feed supplement liquid ingredient, including：Hemicellulose hydrolysate or xylose mother liquid (xylose final concentration 60g/L), glucose mother liquid (the 1/2 of the final concentration of xylose molar concentration of glucose), technical grade Dried Corn Steep Liquor Powder (the 1/3 of xylose mass concentration).

The advantageous effect that the present invention has：

Xylitol genetic engineering production bacterium provided by the invention will be in genome using Escherichia coli W3110 as starting strain XylAB and ptsF be substituted for Xylose reductase gene XR, block the metabolism of xylose and the phosphorylation of xylitol, improve gene Utilization rate of the engineering bacteria to xylose；By ptsG in genome and pfkA, pfkB, pgi, at least one of sthA is substituted for Xylose reductase gene XR is reached while eliminating glucose effect by blocking or reducing EMP Embden Meyerbof Parnas pathway glucose metabolism flux Strengthen the NADPH regeneration of PPP approach, restoring xylose generation xylitol for Xylose reductase provides required coenzyme NADP 11；Genome In corresponding gene be substituted for Xylose reductase gene XR, Xylose reductase is able to high efficient expression, greatly improves gene work The efficiency of journey bacterium Bioconversion of D-xylose To Produce Xylitol.

Description of the drawings

Fig. 1 is the determination of target site and pTargetF plasmid encoding mutant PRIMER DESIGN STRATEGYs in ptsG.

Fig. 2 is the construction method of recovery template.

Fig. 3 is to use CRISPR technological transformations chassis cellular nucleic acid electrophoretogram, wherein M:250bp Marker, G and G:XR For the segment for using primer ptsG-u-F and ptsG-d-R to expand, G：Original ptsG genes, G:XR：Original ptsG genes replace It is changed to xr；AB and AB:XR is the segment expanded using primer xylAB-u-F and xylAB-d-R, AB：Original xylAB bases Cause, AB:XR：Original xylAB genes replace with xr；F and F:XR expands to obtain using primer ptsF-u-F and ptsF-d-R Segment, F：Original ptsF genes, F:XR：Original ptsF genes replace with xr.

Fig. 4 analyzes for glucose major metabolic pathways.

Fig. 5 is the concentration of sugar and sugar alcohol in zymotic fluid after shake flask fermentation for 24 hours.

Fig. 6 is bacteria concentration OD600 measurement results after fermenting for 24 hours.

Fig. 7 is that bacterial strain WZ31 produces xylitol using pure sugar fermentation, and wherein Glucose is glucose sugar, and Xylose is xylose, Xylitol is xylitol.

Fig. 8 be carry out fed batch fermentation using hemicellulose hydrolysate and Dried Corn Steep Liquor Powder using WZ31, wherein Glucose is glucose sugar, and Xylose is xylose, and Arabinose indicates that arabinose, Arabitol indicate arabite, Xylitol is xylitol.

Specific implementation mode

The present invention is further explained in the light of specific embodiments.

The E.coli W3110 used in the following example are purchased from Germany Microbiological Culture Collection Center DSMZ, and number is DSM-5911；PTargetF plasmids addgene：#62226；PCas plasmids addgene：#62225；PRC43M plasmids are from application Number it is 201510196843.8, entitled " genome of E.coli integration vector, genetic engineering bacterium and in producing xylitol Application " patent document.

Embodiment 1

The present embodiment provides a kind of xylitol genetic engineerings to produce bacterium, realizes in integrated Xylose reductase expresses bacterial strain Enhance intracellular NADPH regeneration, improves the production efficiency of xylitol.

The construction method of the xylitol genetic engineering production bacterium, includes the following steps：

1, the pTargetF plasmids of target gene are replaced or knocked out to structure

Firstly the need of a site PAM, i.e. NGG sequences is found on target gene, corresponding N20 sequences are determined, and will CadAspacer on pTargetF plasmids replaces with the N20 sequences of target gene.By taking ptsG as an example, pTargetF- is built For the plasmid construction of ptsG, designs and full plasmid is carried out to pTargetF using primer N20-ptsG-F and N20-ptsG-R and dash forward Become PCR, primer design method is as shown in Figure 1.

PCR is formulated and program setting is as follows：

1 full plasmid encoding mutant PCR system of table

By pTargetF plasmid maps it is found that plasmid total length is 2118bp, in order to ensure that PCR extends complete in the process Property, the setting of long extension of time is used in this research.

2 full plasmid PCR program of table is arranged

Obtained PCR product is subjected to DNA nucleic acid electrophoresis verifications, whether has bright wisp at the places 2000bp or so by observing Band, to examine whether PCR succeeds.

The digestion for carrying out pcr template after verification using DpnI enzymes, improves the positive rate of transformant.Enzymic digestion system is such as Under：

Enzyme DpnI 0.5μL

10*Buffer 1μL

8.5 μ L of PCR product.

Level shakes mixing after respective substance is added according to system, and rapid centrifugation 20s, is placed in 37 DEG C of water-baths or metal in short-term 60min in bath.Then it is converted using DH5 α competence.

The transformant that picking obtains carries out Liquid Culture, and plasmid is extracted using a small amount of extracts kit of plasmid.It uses Target-check carries out sequence verification, finally obtains the successful plasmid pTargetF-ptsG of mutation.

2, ptsG replaces with the structure of XR gene repair templates

Construction strategy is as shown in Figure 2.

Using E.coli W3110 genomes as template, with ptsG-u-F and ptsG-u-R, ptsG-d-F and ptsG-d- R is PCR primer, carries out the homology arm segment that Standard PCR obtains each 500bp of ptsG gene upstream and downstream；

Using pRC43M plasmids as template, ptsG-XR-F and ptsG-XR-R are that primer PCR obtains the xr using P43 as promoter Express module.Obtained PCR product is subjected to DNA nucleic acid electrophoresis and glue recycling.

It is primer, the equal proportion of ptsG upstream and downstream homology arm and xr expression modules finally to use ptsG-u-F and ptsG-d-R Mixture is template, carries out Overlap extension PCR, and gel extraction corresponds to the segment of length, obtains the reparation mould for replacing ptsG genes Plate.

3, genomic gene replacement method operates

1) heat shock method is transferred to pCas plasmids

A. the wild type E.coli W3110 for being stored in -80 DEG C are crossed in the solid medium tablets of nonreactive, overnight 37 DEG C of cultures.Picking single bacterium falls in LB liquid medium 37 DEG C, and 200rpm cultivates about 10h, and switching 1mL bacterium solutions are to equipped with 50mL In the 250mL triangular flasks of LB liquid medium, OD is grown to₆₀₀It is big according to Takara by bacterium solution ice bath 10min to 0.6~0.8 Enterobacteria competence kit preparationization turns competence.

B. the E.coli W3110 competence prepared is placed on ice, 10 μ L is aseptically added after thawing PCas plasmids, mixing are placed on ice bath and place 30min.

DEG C c.42 water-bath or metal bath heat shock 90s at, immediately ice bath 2min.

D. 890 μ L liquid LB or recovery medium is added, in 30 DEG C, 200rpm recoveries 45min.

E. the bacterium solution after 100 μ L recoveries is drawn, the kan containing 50mg/L is coated on^RSolid LB tablets on, 30 DEG C culture Overnight.

F. the bacterium colony of picking monoclonal carries out PCR or extraction plasmid verification, obtains and converts successful E.coli W3110pCas。

2) pTargetF and recovery template the donor DNA of the corresponding replacement gene of electrotransformation

A. it is starting strain by the Escherichia coli obtained in 1), lines the tablet of the kanamycin sulfate resistance of 50mg/L On, after 30 DEG C of overnight incubations (follow-up cultivation is both needed to that the antibiotic of the same race of same concentrations is added), picking single bacterium drops down onto in liquid LB, 30 DEG C, 200rpm cultivates 10-12h, and in the 1mL to 50mL that transfers in LB, 30 DEG C, after 200rpm cultivates 1h, working concentration, which is added, is The L-arabinose of 0.5% sterilizing carries out induced expression Red recombinant proteins, continues culture to OD₆₀₀To 0.6~0.8, (about 3h is left It is right), ice bath 10min carries out the preparation that electricity turns competence.

B. using 10mL Ep pipe sterilize, after bacterium solution is dispensed, 4 DEG C, 5min is centrifuged under 4000rpm, abandons supernatant.

C. it is resuspended with 10% glycerine of the precooled sterilizings of 1mL, 4 DEG C, 10min is centrifuged under 4000rpm, careful discards Supernatant.

D. step c is repeated 2 times.

E. be resuspended, be transferred in the 1.5mL Ep pipes of sterilizing with the glycerine of 100 μ L 10%, immediately using or be placed in -80 DEG C It is spare in refrigerator.

F. previously prepared competence is taken out using the competence prepared or from -80 DEG C, places 5min on ice, is added 400ng builds the pTargetF plasmids and 800ng recovery templates for replacing corresponding gene.Sterile 2mm electricity is transferred to after mixing In revolving cup, 10min is placed on ice, carries out electrotransformation.

G. electricity turns condition：2.5kV, 25 μ F, 200 Ω, electricity turn time 5ms, electricity before turning electric revolving cup wall of cup and pedestal have to It is dried with paper handkerchief, not so easily causes quick-fried cup.Salt ion is remaining excessively high in the recovery template of the plasmid and recycling that extract simultaneously It can cause quick-fried cup.

H. electricity turns after terminating, and 1mL liquid LB are added immediately, after blowing and beating mixing back and forth with liquid-transfering gun, is transferred to 2mL sterilizings Ep pipes in.Recover about 3h under 30 DEG C, 150rpm.

I. the kan containing 50mg/L will be all coated on after the bacterium solution centrifugal concentrating after recovery^RWith the spec of 50mg/L^RIt is flat Plate, 30 DEG C are incubated overnight.

J. under normal conditions, macroscopic transformant can be had by cultivating 12 hours.If bacterial strain have passed through multiple editor, Possible growth time can be extended.

4, gene replaces verification

Bacterium can be used in the case where replacement context length difference is larger for the positive transformant being replaced successfully It falls or bacterium solution PCR amplification target stripe, progress DNA nucleic acid electrophoresis, icp gene replaces the mode of front and back amplified band size To be identified.If replacing front and back gene size difference within 200bp, the mode of PCR product sequencing can be used to identify.

5, the pTargetF and recovery template that other replacement genes are similarly built using corresponding primer, by the gene of 3 wheels Group editor, is successfully inserted into the regions genome ptsG, xylAB, ptsF by Xylose reductase.

The bacterial strain WZ01, WZ02, WZ03 of improved integrated expression Xylose reductase are obtained by design.Wherein, WZ03(E.coli W3110,△ptsG:XR,△xylAB:XR,△ptsF:XR genome schematic diagram) is as shown in Figure 3.

The Xylose reductase expression block size of insertion is 1449bp.PtsG gene sizes are 1434bp, therefore after replacement Size variation is not obvious, and can not conclude its result correctness by nucleic acid gel electrophoresis figure, using Target-check primers into PtsG genes are successfully replaced with Xylose reductase and express module by row PCR product sequence verification.The total 2849bp of xylAB genes, is replaced Reduce 1400bp after being changed to XR, electrophoretogram can determine whether after replacing to be positive transformant；PtsF genes are 1692bp, are reduced after replacement 243bp, electrophoretogram are shown as positive transformants.Finally obtained WZ03 bacterial strains are that ptsG, xylAB and ptsF all replace with xylose The engineering bacteria of the expression module of reductase gene.

6, it is analyzed by glucose major metabolic pathways (Fig. 4), PPP approach can generate more in glucose metabolism NADPH, therefore be related to testing pfkA, pfkB, pgi gene and transhydrogenase sthA genes to EMP Embden Meyerbof Parnas pathway, use Xylose reductase Expression module is replaced.

Using WZ03 bacterial strains as starting strain, to gene pfkA, pfkB, the pgi of coenzyme NADP 11 power of regeneration may be enhanced, SthA carries out the replacement of individual gene, obtains bacterial strain WZ21, WZ22, WZ23, WZ24.And select wherein effect preferably engineering bacteria Double replacements are carried out, bacterial strain WZ31 and WZ32 are obtained.

Primer (SEQ ID NO.1-57) involved in above-mentioned experiment is as shown in table 3.Detailed bacterial strain and its genome class Type is as shown in table 4.

3 primer sequence of table

4 bacterial strain of table and associated genotype type

7, shake flask fermentation experiment is carried out to the above bacterial strain, fermentation condition is as follows：

(1) prepared by seed liquor

Seed liquor culture：In the fresh liquid LB culture mediums that the single bacterium colony picking of scribing line separation is extremely sterilized, in 37 DEG C, 200rpm overnight incubations are to growing stationary phase.

(2) shake flask fermentation

Medium of shaking flask fermentation is prepared, liquid amount 45mL in 250mL triangular flasks is inoculated with the seed liquor of 1mL, 37 DEG C, 200rpm culture 4h, the mixed sugar liquid (glucose containing 200g/L xyloses and 100g/L) after addition 5mL sterilizings, 30 DEG C, Shake flask fermentation is carried out under 200rpm, timing sampling simultaneously detects relevant parameter situation of change.

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

After the dilution that the sample taken is carried out to debita spissitudo, it is filtered using 0.22 μm of filtering head.It utilizes 3000 efficient liquid phase systems of Dionex UltiMate carry out xylose, glucose, arabinose, xylitol and arabite Quantitative detection.Detector：Corona Charged Aerosol Detector (CAD), analytical column：Aminex HPX-87C(Φ 7.8mm × 300mm), mobile phase：Ultra-pure water, flow velocity：0.6mL/min, column temperature setting：76℃.

The bacterium solution after adding liquid glucose for 24 hours is taken, the oxidized form and reduced coenzyme of intracellular are extracted, and carries out concentration mensuration, side Method is as follows：

1) bacterium solution is placed in 10min in ice bath, 4 DEG C, 4000rpm centrifuges 15min, is concentrated into the OD of 1mL bacterium solutions₆₀₀It is 30, It detaches intracellular oxidised form and restores the coenzyme of form.

2) bacterium solution of 1mL is taken respectively, is added：

Detach oxidised form：The Tricine-NaOH (pH8.0) of the 0.3M HCl and 50mM of 0.5mL；

Separating reducing form：The 0.3M NaOH of 0.5mL；

3) 60 DEG C of heat preservation 7min of all samples, being neutralized with the 0.3M NaOH of 0.5mL for oxidised form, restore the use of form 0.5mL 0.3M HCl are neutralized, and need that the 1.0M Tricine-NaOH of 0.1mL are added to maintain pH in each sample after neutralizing Stabilization.

4) 4 DEG C, 13000rpm centrifuges 60min.300 μ L supernatants are drawn to be transferred in new Ep pipes.

5) content of coenzyme is measured using reaction solution light absorption value at a particular wavelength.96 orifice plates and microplate reader can be used It is quantified.Measurement system is：

Oxidised form：+ 40 μ L 0.1M NaCl of 40 μ L samples；

Reduction form is：80 μ L samples；

6) 2* reaction stostes (the 1.0M Tricine-NaOH (pH8.0), 4.2mM of isometric (80 μ L) is added Thiazolyl blue tetrazolium bromide (MTT), 40mM EDTA, 1.67mM phenazine Ethosulfate (PES), and substrate (5M ethyl alcohol (for measuring NAD) or 25mM G-6-Ps (for measuring NADP).

7) 37 DEG C of constant temperature 5min after mixing are separately added into alcohol dehydrogenase and 0.27U/mL that working concentration is 10U/mL Glucose-6-phosphate dehydrogenase (G6PD) (mother liquor is prepared with 10* concentration).

8) decrement of MTT can be detected with the microplate reader of 570nm at 37 DEG C.Data reference standard curve.

The ratio of the coenzyme reduced form and oxidized form that measure is as shown in table 5 below.

The ratio of reduced form and oxidized coenzyme after 5 different genes of table are replaced

Compared to control group WZ03, the content of intracellular NADPH/NADP+ has a certain amount of raising after replacement related gene, To in zymotic fluid sugar and sugar alcohol be measured, be as a result illustrated in fig. 5 shown below.

By table 5 and Fig. 5 analyses it is found that after transformation, the power of regeneration of intracellular NADPH is strengthened, and consumption is identical Mol Glucose can generate more NADPH, while improve the ratio of intracellular NADPH/NADP+, maintain higher coenzyme dense It spends and sufficient reducing power is provided for Xylose reductase.

To the growth OD of each strain bacterial strain₆₀₀It is measured, the results are shown in Figure 6.Bacterial strain after engineered, 4 xylose reduction Under enzyme copy number, coenzyme NADP 11/NADP+ ratios are higher, and the xylitol yield of bacterial strain is also higher.5 Xylose reductase copy numbers Under, coenzyme NADP 11/NADP+ ratios of WZ31 and WZ32 are suitable, but WZ31 has higher xylitol yield.In conjunction with Fig. 6, WZ32 Since the growth ability of bacterial strain after transformation is destroyed, rear bacteria concentration only has 40% or so of starting strain WZ03 for 24 hours, this is it The main reason for xylitol yield is relatively low.

Bacterial strain WZ31 is subjected to pure sugared shake flask fermentation experiment, 10h, 20h and 30h after adding sugar are sampled respectively, surveyed Determine sugar in zymotic fluid and sugar alcohol concentration and OD₆₀₀Measurement, the results are shown in Figure 7.

8, it is fermented using hemicellulose hydrolysate and Dried Corn Steep Liquor Powder, using WZ31 bacterial strains, is not adding no antibiosis Under conditions of element and derivant, xylitol fermentation is carried out using 15L fermentation tanks, method is as follows：

(1) primary seed solution culture

The strain of preservation is lined in solid medium tablets, is incubated overnight under 37 DEG C of constant temperature.Picking single bacterium falls within liquid In body LB, 37 DEG C, 200rpm cultivates about 12h.

Primary seed solution culture medium prescription (L^-1)：Peptone 10g, yeast powder 5g, sodium chloride 10g, solid medium separately add 2% agar powder.

(2) secondary seed solution culture

By volume in 0.5~1% ratio switching primary seed solution to secondary seed liquid culture medium, 37 DEG C, 200rpm Cultivate 7h.

Secondary seed solution culture medium prescription (L^-1)：Yeast powder 7.5g, peptone 7.5g, sodium chloride 10g, glucose 20g.

(3) fermentation processes

Inoculation before the pH of fermentation medium is first adjusted to 6.5 or so with ammonium hydroxide, by fermentation medium volume 10~ The good secondary seed solution of 15% inoculated and cultured, cultivation temperature：37 DEG C, ventilatory capacity control exists in 0.6vvm, initial speed control 400rpm.Rotating speed and dissolved oxygen joint control are adjusted, holding fermentation tank dissolved oxygen is 30-35%, and it is dense that bacterium is measured by sampling in separated in time OD₆₀₀, and in OD₆₀₀Feed operation is carried out when >=20, inoculation and feed supplement are all made of flame method.

Feed profile：Batch feeding

OD600>20 (37 DEG C of culture 7h or so), carry out first time feed supplement.After first time feed supplement, glucose completely consumes It is complete, carry out second of feed supplement.

Feed supplement liquid ingredient and final concentration：Hemicellulose hydrolysate or xylose mother liquid (xylose final concentration 60g/L), glucose are female Liquid (the 1/2 of the final concentration of xylose molar concentration of glucose), technical grade Dried Corn Steep Liquor Powder (the 1/3 of xylose mass concentration).

Feed supplement postcondition controls：Dissolved oxygen control is in 20-25%, temperature control：30℃.

Timing sampling and glucose in fermentation process, xylose, arabinose and arabite and wood are detected after feed supplement The concentration of sugar alcohol monitors the growing state of bacterial strain in entire engineering.Final fermentation results are as shown in Figure 8.

As shown in Figure 8, using hemicellulose hydrolysate as substrate, Dried Corn Steep Liquor Powder eventually passes through 84h points as carbon source Fed-batch fermentation, the final xylitol for obtaining 161.03g/L are criticized, xylose, arabinose and glucose completely consume substantially, by-product Arabite is only 1.63g/L.The production efficiency of xylitol is 1.92g/L/h.

Technical scheme of the present invention and advantageous effect is described in detail in embodiment described above, it should be understood that Above is only a specific embodiment of the present invention, it is not intended to restrict the invention, it is all to be done in the spirit of the present invention Any modification, supplementary, and equivalent replacement etc., should all be included in the protection scope of the present invention.

Sequence table

<110>Zhejiang University

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

<160> 57

<170> SIPOSequenceListing 1.0

<210> 1

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

tccttcattt ggccgccgat gttttagagc tagaaatagc 40

<210> 2

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

atcggcggcc aaatgaagga actagtatta tacctaggac 40

<210> 3

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

gatcggttac tggtggaaac tgactc 26

<210> 4

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

cactcaataa agctgagctc aattgagagt gctcctgagt atggg 45

<210> 5

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

actcaggagc actctcaatt gagctcagct ttattgagtg gatga 45

<210> 6

<211> 44

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

ctccccaacg tcttacggat taccggttta ttgactaccg gaag 44

<210> 7

<211> 44

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

ggtagtcaat aaaccggtaa tccgtaagac gttggggaga ctaa 44

<210> 8

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

ggtggatggg acagtcagta aagg 24

<210> 9

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

tgcgcaagag tgcactttgc gttttagagc tagaaatagc 40

<210> 10

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

gcaaagtgca ctcttgcgca actagtatta tacctaggac 40

<210> 11

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

gatatctcga tctttttgcc agcgttc 27

<210> 12

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

cactcaataa agctgagctc attgaactcc ataatcaggt aatgc 45

<210> 13

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

acctgattat ggagttcaat gagctcagct ttattgagtg gatga 45

<210> 14

<211> 44

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 14

ggtcaggcag gggataacgt taccggttta ttgactaccg gaag 44

<210> 15

<211> 44

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 15

ggtagtcaat aaaccggtaa cgttatcccc tgcctgaccg ggtg 44

<210> 16

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 16

gagcgaaaca aacgcatttg accaaac 27

<210> 17

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 17

agcaccggtt ctggcttcat gttttagagc tagaaatagc 40

<210> 18

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 18

atgaagccag aaccggtgct actagtatta tacctaggac 40

<210> 19

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 19

gactggatga ctcgcctgcg tagtcag 27

<210> 20

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 20

cactcaataa agctgagctc tatgcctctc ctgctgtcag ttaaa 45

<210> 21

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 21

ctgacagcag gagaggcata gagctcagct ttattgagtg gatga 45

<210> 22

<211> 44

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 22

gccctgtaac acacctttta taccggttta ttgactaccg gaag 44

<210> 23

<211> 44

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 23

ggtagtcaat aaaccggtat aaaaggtgtg ttacagggca gaaa 44

<210> 24

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 24

tgcgccctac tccaccaggc gtaaa 25

<210> 25

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 25

cacaacgaat tcacagccac gttttagagc tagaaatagc 40

<210> 26

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 26

gtggctgtga attcgttgtg actagtatta tacctaggac 40

<210> 27

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 27

gtgactgacg aatcaccacg 20

<210> 28

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 28

cactcaataa agctgagctc tgatttcgga aaaaggcaga ttcct 45

<210> 29

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 29

tctgcctttt tccgaaatca gagctcagct ttattgagtg gatga 45

<210> 30

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 30

tccaaagttc agaggtagtc ccggtttatt gactaccgga agcag 45

<210> 31

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 31

tccggtagtc aataaaccgg gactacctct gaactttgga atgca 45

<210> 32

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 32

gtttgcattg ggaatcggca tc 22

<210> 33

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 33

gtcctaggta taatactagt ccaaaaagaa ctcagtgcgc 40

<210> 34

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 34

gctatttcta gctctaaaac gcgcactgag ttctttttgg 40

<210> 35

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 35

gaagaagtat tactggcgtt ccc 23

<210> 36

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 36

cactcaataa agctgagctc catttcctcc tataggctga tttca 45

<210> 37

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 37

tcagcctata ggaggaaatg gagctcagct ttattgagtg gatga 45

<210> 38

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 38

atgctggggg aatgtttttg ccggtttatt gactaccgga agcag 45

<210> 39

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 39

tccggtagtc aataaaccgg caaaaacatt cccccagcat tgggg 45

<210> 40

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 40

gactgattgc ctgccacatg atg 23

<210> 41

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 41

ctgccaacat gttcgagttc gttttagagc tagaaatagc 40

<210> 42

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 42

gaactcgaac atgttggcag actagtatta tacctaggac 40

<210> 43

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 43

gatccatttt cagccttggc ac 22

<210> 44

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 44

cactcaataa agctgagctc tagcaatact cttctgattt tgaga 45

<210> 45

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 45

aaatcagaag agtattgcta gagctcagct ttattgagtg gatga 45

<210> 46

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 46

ggcctacata tcgacgatga ccggtttatt gactaccgga agcag 45

<210> 47

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 47

tccggtagtc aataaaccgg tcatcgtcga tatgtaggcc ggata 45

<210> 48

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 48

cagaagtcgc cgcaagcgca gatatg 26

<210> 49

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 49

gaatatgcgt cgatcttccg gttttagagc tagaaatagc 40

<210> 50

<211> 40

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 50

cggaagatcg acgcatattc actagtatta tacctaggac 40

<210> 51

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 51

gttcgtctac gtcgcggaaa tg 22

<210> 52

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 52

cactcaataa agctgagctc ggtagggctt acctgttctt ataca 45

<210> 53

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 53

aagaacaggt aagccctacc gagctcagct ttattgagtg gatga 45

<210> 54

<211> 45

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 54

atggccattt cgataaagtt ccggtttatt gactaccgga agcag 45

<210> 55

<211> 46

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 55

tccggtagtc aataaaccgg aactttatcg aaatggccat ccattc 46

<210> 56

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 56

gaagcacaga cccaccagtt actg 24

<210> 57

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 57

gaagggagaa aggcggacag 20

Claims

1. a kind of xylitol genetic engineering produces bacterium, obtained through genome manipulation by Escherichia coli W3110, which is characterized in that former PtsG, xylAB and ptsF in beginning Escherichia coli W3110 genome are substituted for Xylose reductase gene XR；It further include gene At least one of pfkA, pfkB, pgi, sthA are substituted for Xylose reductase gene XR in group.

2. xylitol genetic engineering as described in claim 1 produces bacterium, which is characterized in that original Escherichia coli W3110 genes PtsG, xylAB, ptsF, pfkA and pfkB in group are substituted for Xylose reductase gene XR.

3. xylitol genetic engineering as claimed in claim 1 or 2 produces bacterium, which is characterized in that the Xylose reductase gene XR derives from Neuraspora crassa.

4. the construction method of xylitol genetic engineering production bacterium as described in claim 1, includes the following steps：

(1) ptsG, xylAB and ptsF in Escherichia coli W3110 genomes are substituted for Xylose reductase gene XR, obtained First generation genetic engineering bacterium；

(2) pfkA in first generation genetic engineering bacterium genome, pfkB, pgi, at least one of sthA are substituted for xylose also Nitroreductase gene XR.

5. construction method as claimed in claim 4, which is characterized in that in step (1) and (2), using homologous recombination technique into Row gene is replaced.

6. construction method as claimed in claim 4, which is characterized in that in step (1), including：

A. specific primer is utilized build the pTargetF plasmids of replacement ptsG, xylAB and ptsF gene respectively and corresponding contain There is the recovery template of XR expression modules；

B. the pTargetF plasmids for replacing ptsG genes and recovery template are transformed into the Escherichia coli W3110 containing pCas plasmids In, through homologous recombination, screening obtains the bacterial strain W3110 △ ptsG that ptsG genes in genome are substituted for XR expression modules::XR；

The pTargetF plasmids for replacing xylAB genes and recovery template are transformed into bacterial strain W3110 △ ptsG again::In XR, through same Source recombinates, and screening obtains the bacterial strain W3110 △ ptsG that xylAB genes in genome are substituted for XR expression modules::XR,△ xylAB::XR；

Then the pTargetF plasmids for replacing ptsF genes and recovery template are transformed into bacterial strain W3110 △ ptsG::XR,△ xylAB::XR, through homologous recombination, screening obtains the bacterial strain W3110 △ that ptsF genes in genome are substituted for XR expression modules ptsG::XR,△xylAB::XR,ΔptsF::XR, i.e. first generation genetic engineering bacterium；

In step (2), including：

D. structure replaces the pTargetF plasmids of pfkA, pfkB gene and the corresponding reparation mould that module is expressed containing XR respectively Plate；

E. the pTargetF plasmids for replacing pfkA genes and corresponding recovery template are first transformed into the first generation made from step (1) In genetic engineering bacterium, through homologous recombination, screening obtains the bacterial strain WZ04 △ that pfkA genes in genome are substituted for XR expression modules pfkA::XR, then the pTargetF plasmids for replacing pfkB genes and corresponding recovery template are transformed into bacterial strain WZ04 △ pfkA:: In XR, through homologous recombination, screening obtains the bacterial strain that pfkB genes in genome are substituted for XR expression modules, the as xylose Alcohol genetic engineering produces bacterium.

7. construction method as claimed in claim 6, which is characterized in that include Promoter P43 in the XR expression modules.

8. application of the xylitol genetic engineering production bacterium as described in any one of claims 1-3 in producing xylitol.

9. application as claimed in claim 8, which is characterized in that including：By the xylitol genetic engineering production bacterium inoculation In fermentation medium, 30-37 DEG C of fermented and cultured 80-90h keeps bacterial concentration OD in fermentation process₆₀₀Less than 20.

10. application as claimed in claim 9, which is characterized in that fermentation primary condition：37 DEG C of temperature, rotating speed 400rpm, ventilation It is 6.5 to measure 0.6-0.8vvm, the initial pH of fermentation medium, and incubation dissolved oxygen is controlled in 30-35%；As bacterial concentration OD₆₀₀≥ When 20, feed supplement is carried out, fermentation condition is after feed supplement：30 DEG C of temperature, dissolved oxygen are controlled in 20-25%.