CN104593308A - Genetic engineering strain, construction method and application in xylitol production - Google Patents

Abstract

The invention discloses a genetic engineering strain, a construction method and application thereof in xylitol production. The genetic engineering strain includes Escherichia coli and an expression vector transferred into the Escherichia coli. An XR gene is inserted to the downstream of the expression vector's promoter, the base sequence of the XR gene is shown as SEQ ID No.4, the promoter is Trc promoter, the 5' end of the XR gene has RBS from pET30a, and at least one of ptsG, xylA, xylB and ptsF genes in the Escherichia coli is inhibited or knocked out. Through translation initiation rate screening on xylose reductase gene and selection of appropriate RBS and promoter, the XR gene in the genetic engineering strain can obtain high efficiency expression at 30DEG C, and no inclusion body is generated. By knockout of the ptsG gene, the glucose effect is eliminated, and through knockout of the xylA, xylB and ptsF genes, xylose metabolism and xylitol phosphorylation are blocked.

Description

A kind of genetic engineering bacterium and construction process thereof and producing the application in Xylitol

Technical field

The invention belongs to genetically engineered and biological technical field, particularly relate to a kind of genetic engineering bacterium and construction process thereof and producing the application in Xylitol

Background technology

Current industrial production Xylitol mainly obtains with nickel catalysis xylose hydrogenation under high-temperature and high-pressure conditions, and this technique needs condition harsh, and easily pollutes, and is more and more subject to people's attention so biological process transforms production Xylitol.The microorganism of preparing Xylitol for fermentation method is nearly all yeast, and existing natural bacterial classification, also has genetic engineering bacterium.Yeast has the advantage of oneself as microbial strain for xylitol production, such as can tolerate higher sugared concentration, comparatively strong to the supressor resistivity in hemicellulose hydrolysate, etc.But also there is unavoidable problem, be mainly reflected in: (1) at present main yeast producing Xylitol is candida tropicalis, it has potential pathogenic, and is not suitable for the production of food; (2) the Xylose reductase specificity that yeast itself is contained is poor, higher catalytic efficiency is had to wood sugar and pectinose, when so utilizing hemicellulose hydrolysate to transform production Xylitol, the generation of a large amount of arabitol must be had, cause downstream separation difficulty, increase production cost; (3) when utilizing other recombination yeast to produce Xylitol as yeast saccharomyces cerevisiae, because it does not have narrow spectrum xylose transport albumen, wood sugar uptake rate is comparatively slow so production efficiency is lower.

Publication number is that the Chinese patent literature of CN101497904A discloses a kind of method of simultaneously producing Xylitol and pectinose, the method utilizes candiyeast fermentation hemicellulose hydrolysate coproduction pectinose and Xylitol, this technique is conceived to the comprehensive utilization of resource, there is good application prospect, but the content of pectinose only has about 1/10th of Xylose Content in large absolutely number hemicellulose hydrolysate, cause pectinose content in fermentation mother liquor not high, and pectinose and Xylitol need again the sepn process of a relative complex, the production cost of Xylitol can be added like this.

Intestinal bacteria are the most thorough as current research, genetic background the most clearly bacterial strain, utilize it to build genetic engineering bacterium and have advantageous condition, it has culture condition simple, fast growth, the plurality of advantages such as it is very simple that yeast is compared in genetic manipulation, and protein expression level is high, and in safety issue, U.S. FDA also have approved with e. coli k12 is the production of recombinant bacterium for biomedical product of starting strain.But intestinal bacteria also have certain defect when expressing protein, be namely easy at relatively high temperatures form inclusion body.

Publication number is the United States Patent (USP) of PCT/US2011/021277 when utilizing colibacillus engineering strain to carry out fed-batch cultivation, and utilize 160g wood sugar can produce 156g Xylitol, concentration reaches 136g/L, and throughput rate is 1.92g/L/h; When hemicellulose hydrolysate after utilizing detoxification ferments, utilize 50.6g wood sugar can produce 46g Xylitol, throughput rate is 0.56g/L/h.Although the research of document report has been the highest level that current recombination bacillus coli produces Xylitol, also do not give full play to the potentiality that Escherichia coli fermentation produces Xylitol, its concentration of substrate, production concentration and production efficiency can't match in excellence or beauty with Yeast Phase.

Due in fermenting process, even if do not have inclusion body to produce in the early stage, but also can produce inclusion body along with time lapse, along with albumen and inclusion body are constantly assembled, not have activated target protein to get more and more.The existing colibacillus engineering strain containing Xylose reductase gene, for preventing producing a large amount of inclusion bodys in fermenting process, generally need fermenting at a lower temperature, as carried out shake flask fermentation at about 26 DEG C, carrying out ferment tank at about 18 DEG C.But colibacillary cellar culture temperature, at 37 DEG C, is fermented at such lower temperatures, and vitro growth rates will reduce, the catalytic efficiency of Xylose reductase also can be subject to low temperature effect simultaneously.Therefore it is vital for building the fermentative production of expression vector for Xylitol not having inclusion body to produce at relatively high temperatures.

Summary of the invention

The invention provides a kind of genetic engineering bacterium, utilize this genetic engineering bacterium to produce Xylitol, can production efficiency be increased substantially, reduce production cost.

A kind of genetic engineering bacterium, comprise intestinal bacteria and proceed to colibacillary expression vector, the promotor downstream of described expression vector is inserted with Xylose reductase gene, and the base sequence of described Xylose reductase gene is as shown in SEQ ID No.4.

The present invention is that two codons (first and third codon after initiator codon) in the Xylose reductase gene of NCU08384.1 carry out same sense mutation to NCBI accession number, acquisition has the Xylose reductase gene of different translation efficiency (the translation initiation rate), and therefrom have chosen the Xylose reductase gene (its translation efficiency is 6689.5) shown in SEQ ID No.4, this gene can obtain high expression under the comparatively high temps of 30 DEG C, and does not almost have inclusion body to produce.

If this Xylose reductase gene is expressed under the strong promoters such as T7, fermenting process still has more inclusion body and produces after proceeding to the middle and later periods, causes enzyme to be lived and reduces rapidly.Therefore in the present invention, as preferably, described promotor is Trc promotor, and 5 ' end band of described Xylose reductase gene has ribosome bind site, and the base sequence of described ribosome bind site is:

5’-AGAAGGAGATATACAT-3’。

Test finds, under the initiation of this ribosome bind site and Trc promotor, Xylose reductase gene still can obtain high expression under the comparatively high temps of 30 DEG C, and does not almost have inclusion body to produce.

As preferably, described expression vector is pTrc99a.PTrc99a carries Trc promotor, need not Trc promotor be connected in expression vector extraly.

In addition, the present invention also transforms intestinal bacteria, comprising:

(1) elimination of glucose effect:

Intestinal bacteria have a strict hierarchical relationship when utilizing sugar, particularly when there being glucose to exist, intestinal bacteria all can be subject to serious suppression to other sugar utilizations as wood sugar, pectinose etc., namely there is glucose effect.But Xylose reductase reduction wood sugar generates in the process of Xylitol, needs the coenzyme NADP 11 that glucose provides reaction required as auxiliary substrate.

For eliminating the glucose effect of genetic engineering bacterium of the present invention, in described intestinal bacteria, glucose phosphotransferase gene (ptsG) is suppressed or knock out.After ptsG is knocked, the utilize speed of genetic engineering bacterium to glucose significantly reduces, and no matter in low glucose concentrations or under high glucose concentration, xylose and glucose all can be utilized simultaneously, and the speed that utilizes of wood sugar exceedes glucose, glucose effect is eliminated.

(2) metabolic pathway optimization:

The approach of fermenting xylose is there is: wood sugar generates xylulose through xylose isomerase (xylA) in Bacillus coli cells, xylulose generates X 5P under the effect of xylulokinase (xylB), and X 5P enters phosphopentose pathway and fallen by metabolism.Phosphotransferase (ptsF) approach of fructose also may take part in the transhipment of Xylitol simultaneously, be phosphorylated while making Xylitol enter cell, and the Xylitol of phosphorylation is to the toxic effect of cell, thus block the metabolism of wood sugar and the phosphorylation of Xylitol particularly important to engineering bacteria Efficient Conversion xylose production Xylitol.

Therefore, in described intestinal bacteria, in xylose isomerase gene and xylulokinase gene, at least one is suppressed or knock out; In described intestinal bacteria, fructose phosphate transferase gene is suppressed or knock out.

After xylose isomerase gene, xylulokinase gene or fructose phosphate transferase gene are knocked, the utilize speed of genetic engineering bacterium to wood sugar increases substantially, and significantly increases the throughput rate of Xylitol; And knock out by ptsG the growth defect brought and also substantially obtain recovery.

Present invention also offers the construction process of described genetic engineering bacterium, comprise the following steps:

(1) glucose phosphotransferase gene knockout carrier, xylose isomerase gene knockout carrier, xylulokinase gene knockout carrier, fructose phosphate transferase gene knockout carrier is built respectively, and get at least one transformation of E. coli competent cell in four kinds of knockout carriers, obtain the intestinal bacteria having knocked out at least one in glucose phosphotransferase gene, xylose isomerase gene, xylulokinase gene and fructose phosphate transferase gene;

(2) Xylose reductase gene of 5 ' end band ribosome bind site is connected to the Trc promotor downstream of pTrc99a, obtains expression vector;

(3) by the intestinal bacteria that described expression vector step of converting (1) obtains, described genetic engineering bacterium is obtained.

Present invention also offers described genetic engineering bacterium and produce the application in Xylitol.

Described application comprises:

Described genetic engineering bacterium is seeded in nutrient solution and is cultured to OD ₆₀₀be 0.6 ~ 1.0, add inductor and carry out abduction delivering, after completing, from nutrient solution, separation and purification obtains Xylitol.

As preferably, in 1L, consisting of of described nutrient solution: glucose 30-100g/L, yeast extract paste 3-8g/L, peptone 5-10g/L, sodium-chlor 2-10g/L, potassium primary phosphate 6-15g/L, Sodium phosphate dibasic 3-10g/L, ammonium chloride 1-5g/L, magnesium sulfate 1-3g/L, wood sugar 80-300g/L;

Or, consisting of of described nutrient solution: yeast extract paste 3-8g/L, peptone 5-10g/L, sodium-chlor 2-10g/L, potassium primary phosphate 6-15g/L, Sodium phosphate dibasic 3-10g/L, ammonium chloride 1-5g/L, magnesium sulfate 1-3g/L, containing the hemicellulose hydrolysate 0.1 ~ 0.4L/L of 20%-50% wood sugar.

As preferably, abduction delivering 40 ~ 120h at 26 ~ 30 DEG C.

The nutrient solution of main component that what genetic engineering bacterium of the present invention both can utilize human configuration with wood sugar is, also can utilize hemicellulose hydrolysate for main material production Xylitol.When utilizing this genetic engineering bacterium to produce Xylitol, wood sugar is almost all converted to Xylitol, transformation efficiency is more than 0.95 gram (Xylitol)/gram (wood sugar), and productive rate can reach more than 1.4 grams of (Xylitol)/liters per hours, and Xylitol concentration can reach more than 173 grams per liters.

Compared with prior art, beneficial effect of the present invention is:

(1) the present invention is by carrying out translation efficiency screening to Xylose reductase gene, and have selected suitable ribosome bind site and promotor, make Xylose reductase gene in genetic engineering bacterium can obtain high expression under the comparatively high temps of 30 DEG C, and almost do not have inclusion body to produce;

(2) glucose phosphotransferase gene has been knocked out in genetic engineering bacterium of the present invention, after ptsG gene is knocked, the utilize speed of genetic engineering bacterium to glucose significantly reduces, xylose and glucose all can be utilized simultaneously, and the speed that utilizes of wood sugar exceedes glucose, on the basis ensureing coenzyme NADP 11 supply, ensure that effective conversion of Xylitol;

(3) xylose isomerase gene and/or xylulokinase gene has been knocked out in genetic engineering bacterium of the present invention, block thalline itself to the metabolism of wood sugar, knock out fructose phosphate transferase gene, block the xylitol phosphate of cell interior, thus increase substantially genetic engineering bacterium speed is utilized to wood sugar, the throughput rate of Xylitol significantly increases, and knocks out the growth defect brought also substantially obtain recovery by ptsG.

Accompanying drawing explanation

Fig. 1 is the construction strategy figure of genetic engineering bacterium of the present invention;

Wherein, Expression system represents expression system, Promoter represents promotor, XR represents Xylose reductase, Transcription represents and transcribes (Strong, Medium, Weak represents that transcriptional level is strong successively, medium, weak), translation represents translation, Ribosome represents rrna, Secondary structure represents (mRNA) secondary structure, RBS represents ribosome bind site, AUG is initiator codon, Vector represents that (Amp represents ampicillin resistance gene to carrier, ori represents replicon, Terminator represents terminator, Ptrc represents Trc promotor), Strain improvement represents strain improvement, Optimal plasmid represents optimum plasmid, D-Xylose represents D-wood sugar, xylA represents xylose isomerase, D-Xylulose represents D-xylulose, xylB represents xylulokinase, D-Xylulose-5-P represents 5-phosphoric acid-xylulose, Glucose represents glucose, and ptsG represents glucose phosphotransferase, and glf represents glucose transporter, Glucose-6-phosphate represents G-6-P, Pyruvate represents pyruvic acid, and Xylitol represents Xylitol, and ptsF represents fructose phosphate transferring enzyme, Process improvement represents optimizing fermentation, and Hemicellulosic hydrolysate represents hemicellulose hydrolysate, Detoxification detoxification, and Bioreactor represents bio-reactor, and Crystallization represents crystallization,

Fig. 2 a is the plasmid construct schematic diagram will obtained in XR gene clone to pTRc99a;

Fig. 2 b is the plasmid construct schematic diagram XR gene clone of band His-tag label obtained to pET-30a (+);

Fig. 2 c is the plasmid construct schematic diagram will obtained in the XR gene clone of different translation efficiency to pET-30a (+);

Fig. 2 d is the plasmid construct schematic diagram XR gene clone with the RBS site deriving from pET-30a (+) obtained to pTRc99a;

Fig. 2 e is the secondary structure of the mRNA that recombinant expression vector pET-30a-xr transcribes;

Fig. 2 f is the secondary structure of the mRNA that recombinant expression vector pTRc99a-xr transcribes;

Fig. 3 a is the SDS-PAGE figure of different engineering strain abduction delivering at 18 DEG C;

Fig. 3 b is the SDS-PAGE figure of different engineering strain abduction delivering at 30 DEG C;

In Fig. 3 a and Fig. 3 b, swimming lane 1 is albumen marker, cleer and peaceful precipitation in the expression that swimming lane 2 and 3 is respectively engineering bacteria HK005, cleer and peaceful precipitation in the expression that swimming lane 4 and 5 is respectively engineering bacteria HK006, cleer and peaceful precipitation in the expression that swimming lane 6 and 7 is respectively engineering bacteria HK007, cleer and peaceful precipitation in the expression that swimming lane 8 and 9 is respectively engineering bacteria HK008;

Fig. 3 c be engineering bacteria HK004, HK009 at different temperatures abduction delivering SDS-PAGE figure;

Wherein, swimming lane 1 is albumen marker, swimming lane 2 and 3 is respectively cleer and peaceful precipitation in the expression of engineering bacteria HK004 at 30 DEG C, swimming lane 4 and 5 is respectively cleer and peaceful precipitation in the expression of engineering bacteria HK009 at 30 DEG C, swimming lane 6 and 7 is respectively cleer and peaceful precipitation in the expression of HK004 at 18 DEG C, and swimming lane 8 and 9 is respectively cleer and peaceful precipitation in the expression of engineering bacteria HK009 at 18 DEG C;

Fig. 4 is the plasmid map of pTRc99a-rbs-xr6600;

Fig. 5 is the nucleic acid electrophoresis figure of different knock-out bacterial strain primers designed amplification;

Wherein, M is DNA Marker, swimming lane 1 is intestinal bacteria W3110 (containing ptsG), swimming lane 2 is engineering bacteria HK101 (having knocked out ptsG), swimming lane 3 is intestinal bacteria W3110 (containing xylA, xylB), swimming lane 4 is engineering bacteria HK301 (having knocked out xylA, xylB), and swimming lane 5 is intestinal bacteria W3110 (containing ptsF), and swimming lane 6 is engineering bacteria HK401 (having knocked out ptsF);

Fig. 6 a is the fermentation results figure of wild-type E.coli K-12W3110;

Fig. 6 b is the fermentation results figure of engineering bacteria HK102;

Fig. 6 c is the fermentation results figure of engineering bacteria HK302;

Fig. 6 d is the fermentation results figure of engineering bacteria HK402;

Wherein, Concentration (g/L) indicated concentration (g/L), Time (h) represents fermentation time (h);

Fig. 7 a is that engineering bacteria HK402 utilizes half fiber hydrolysate to produce the fermentation results figure of Xylitol in shaking flask;

Fig. 7 b is the fermentation results figure that engineering bacteria HK402 utilizes pure sugar batch fermentation in 5L fermentor tank;

Fig. 7 c is the fermentation results figure that engineering bacteria HK402 utilizes pure sugared fed-batch fermentation in 15L fermentor tank;

Fig. 7 d is the fermentation results figure that engineering bacteria HK402 utilizes hemicellulose hydrolysate to ferment in 5L fermentor tank;

Wherein, Arabinose represents pectinose, and Arabinitol represents arabitol, Enzyme activity (10 ³u/L) represent that enzyme lives (10 ³u/L);

Fig. 8 is the typical curve of muscovado (glucose, wood sugar, pectinose, each 10g/L of Xylitol);

Wherein, peak 1 represents glucose (7.455 indicate peak time, lower same), and peak 2 represents wood sugar, and peak 3 represents pectinose, and peak 4 represents Xylitol;

Fig. 9 is the concentration that engineering bacteria HK402 utilizes each sugar in fermented liquid when starting containing the fermentation culture abduction delivering of 100g/L wood sugar; Wherein, peak 3 represents glucose, and peak 4 represents wood sugar.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.

As shown in Figure 1, concrete building process comprises the construction strategy of the present embodiment genetic engineering bacterium:

1, the screening of suitable for translation efficiency gene

According to NCBI accession number NCU08384.1, the complete synthesis Xylose reductase gene (XR) deriving from Neuraspora crassa.

With the Xylose reductase gene of above-mentioned synthesis for template, design same sense mutation primer, to obtain the mRNA with different secondary structure, and then screening has the XR same sense mutation gene of suitable for translation efficiency.

Wherein, the upstream primer of first pair of same sense mutation primer is M-XR3700-P1:

5 '-GGAATTC cATATGgTACCGGCTATCAAGCTCAACTC-3 ' (underscore part is NdeI restriction enzyme site);

The upstream primer of second pair of same sense mutation primer is M-XR5500-P1:

5’-GGAATTC CATATGGTACCGGCAATCAAGCTCAACTC-3’；

The upstream primer of the 3rd pair of same sense mutation primer is M-XR6600-P1:

5’-GGAATT CCATATGGTGCCTGCGATCAAGCTCAACTC-3’；

The upstream primer of the 4th pair of same sense mutation primer is M-XR9600-P1:

5’-GGAATT CCATATGGTTCCTGCGATCAAGCTCAACTC-3’；

The downstream primer of four pairs of same sense mutation primers is XR-P4:

5 '-CCG gAATTCcTAACCGAAAATCCAGAGGTTCTC-3 ' (underscore part is EcoRI restriction enzyme site);

Four pairs of same sense mutation primers increase respectively and obtain XR same sense mutation gene: M-XR3700 (SEQ ID No.2, wherein, after initiator codon, first codon GTT sports GTA, second codon CCT sports CCG), M-XR5500 (SEQ ID No.3, wherein, after initiator codon, first codon GTT sports GTA, second codon CCT sports CCG, 3rd codon GCT sports GCA), M-XR6600 (SEQ ID No.4, wherein, first codon GTT sudden change GTG after initiator codon, 3rd codon GCT sports GCG), M-XR9600 (SEQ ID No.5, wherein, 3rd codon GCT sports GCG).

Adopt upstream primer XR-P3 and downstream primer XR-P4 to increase Xylose reductase gene (SEQ ID No.1, does not suddenly change), the base sequence of XR-P3 is simultaneously:

5’-GGAATTC CATATGGTTCCTGCTATCAAGCTCAA-3’。

PCR reaction system is 50 μ L, wherein PrimerSTAR Max DNA Polymerase 25 μ L, each 1.5 μ L of upstream and downstream primer (10 μMs), template ((50ng/ml) 1 μ L, ddH ₂o 21 μ L;

PCR response procedures is: 95 DEG C of denaturation 2min; 98 DEG C of sex change 10s, Tm anneal 15s, and 72 DEG C extend 5s/kb, 30 circulations; 72 DEG C extend 5min, 4 DEG C of insulations.

RBS Calculator v1.1 is utilized to calculate the translation efficiency (see table 1) of different XR same sense mutation gene respectively.

PCR primer is after electrophoresis checking is correct, glue reclaims purifying, and utilize NdeI and EcoRI to Xylose reductase gene (XR), Xylose reductase gene (XR-His-tag) with histidine-tagged, M-XR3700, M-XR5500, M-XR6600, M-XR9600 and pET-30a (+) carrier respectively enzyme cut, enzyme connects with T4 ligase enzyme after cutting, respectively construction recombination plasmid; And by recombinant plasmid transformed competent escherichia coli cell DH5 α, the bacterium colony that picking ampicillin plate grows, extract plasmid, through enzyme cut qualification and order-checking after, by recombinant plasmid correct for checking called after respectively: pET-30a-xr, pET-30a-tag-xr, pET-30a-xr3700, pET-30a-xr5500, pET-30a-xr6600, pET-30a-xr9500, plasmid is proceeded to BL21 (DE3), build the genetic engineering bacterium containing corresponding recombinant plasmid, respectively called after HK003, HK004, HK005, HK006, HK007, HK008.

XR gene clone is entered in pTrc99a, pBAD24 simultaneously, build corresponding genetic engineering bacterium HK001, HK002, in contrast.The structure of each recombinant plasmid is as shown in Fig. 2 a, 2b, 2c, 2d, 2e, 2f.

LB (5gl is inoculated in after activated for each genetic engineering bacterium ^-1yeast extract paste, 10gl ^-1peptone, and 10gl ^-1naCl) in substratum, at 18 DEG C, 30 DEG C, cultivate 15h respectively, cultivated rear centrifugal collecting cell, use potassium phosphate buffer (pH 7.4) resuspended, adjustment cell suspension OD ₆₀₀broken born of the same parents after 2.0, the enzyme adopting the absorbance method of NADPH at 340nm place to detect Xylose reductase is respectively lived, and detected result is in table 1 and Fig. 3.

The each genetic engineering bacterium of table 1 at different temperatures abduction delivering enzyme live and expressing quantity

Note: a: the base sequence of each goal gene, b:TIR represents translation efficiency, c: thalline cultivates the protein content of acquisition at 30 DEG C, d:ND represents and does not detect, e: genetic engineering bacterium HK007 is cultured to 30h, f:SEQ ID No.7 is the base sequence of the SEQ ID No.4 that 5 ' end band has RBS sequence, RBS is AGAAGGAGATATACAT.

From table 1 and Fig. 3, XR gene, the translation efficiency in pTrc99a and pBAD24 is all lower, goal gene do not express or expression amount very low.

When taking pET-30a as expression vector, when translation efficiency is lower, goal gene do not express or expression amount very low, when translation efficiency improves, under low temperature (18 DEG C), expression amount has a certain amount of increase, but at relatively high temperatures (30 DEG C), along with the increase of translation efficiency, the amount of inclusion body also adds accordingly, particularly having a large amount of inclusion body to produce at relatively high temperatures with histidine-tagged goal gene, inclusion body generation can make activated target protein significantly reduce; When efficiency of serving as interpreter is 6689.5, under genetic engineering bacterium high temperature, produce inclusion body hardly, so select to make the goal gene that M-XR 6600 is follow-up test.

And, promotor due to pET-30a (+) carrier is T7 strong promoter, when genetic engineering bacterium HK007 is cultured to 30h (middle and later periods), still has a lot of inclusion body to produce, cause enzyme to be lived to reduce rapidly, therefore need to select suitable promotor.

2, the optimization in promotor and RBS site

Design primer XR-P1 and XR-P2 amplification M-XR:

XR-P1:5 '-CCG gAATTCaTGGTTCCTGCTATCAAGCTCAA-3 ' (underscore part is EcoRI restriction enzyme site);

XR-P2:5 '-CCC aAGCTTcTAACCGAAAATCCAGAGGTTCTC-3 ' (underscore part is HindIII restriction enzyme site);

PCR reaction system response procedures is the same.

PCR primer is after electrophoresis checking is correct, glue reclaims purifying, be cloned into the Trc promotor downstream of carrier pTrc99a, by recombinant vectors transformation of E. coli competent cell DH5 α, the bacterium colony that picking ammonia benzyl flat board grows, extract plasmid, cut after qualification through enzyme, by recombinant plasmid called after pTrc99a-xr correct for checking, plasmid is proceeded to BL21 (DE3) and build the genetic engineering bacterium called after HK001 containing corresponding recombinant plasmid.

The enzyme adopting above-mentioned identical method to detect genetic engineering bacterium HK001 is lived and expressing quantity.But detected result shows, and M-XR is not expressed.

Therefore, take pET-30a-xr6600 as template, design primer XR-RBS-P1 and XR-RBS-P2 further, increases:

XR-RBS-P1：5’-CCGGAATTCCCCCTCTAGAAATAATTTTG-3’；

XR-RBS-P2：5’-TGGAAGCTTCTAACCGAAAATCCAGAG-3’；

PCR reaction system response procedures is the same.

PCR primer is after electrophoresis checking is correct, glue reclaims purifying, be cloned into the Trc promotor downstream of carrier pTrc99a, by recombinant vectors transformation of E. coli competence DH5 α, the bacterium colony that picking ammonia benzyl flat board grows, extract plasmid, cut after qualification through enzyme, by recombinant plasmid called after pTrc99a-rbs-xr6600 (plasmid map is shown in Fig. 4) correct for checking, plasmid is proceeded to W3110 and build the genetic engineering bacterium called after HK009 containing corresponding recombinant plasmid.

As can be seen from Table 1, utilize the RBS site from carrier pET-30a (+), M-XR 6600 obtains effective expression on pTrc99a, do not have complete abduction delivering at low temperatures, at high temperature obtain comparatively high expression level, and do not have inclusion body to produce, make genetic engineering bacterium HK009 be more suitable for fermenting at relatively high temperatures.

3, the knocking out of Host Strains metabolic pathway key gene

Gene knockout basic step knocks out system (Datsenko and Wanner, 2010) according to the RED/ET of standard and carries out, specific as follows:

(1) the knocking out of ptsG gene

1. the clone of ptsG homology arm Cm gene is with

With plasmid pKD3 for template, utilize primer Del-ptsG-P1 and Del-ptsG-P2 (homology arm of this upstream and downstream primer 5 ' end containing 50bp ptsG upstream and downstream) to increase, obtain the chloramphenicol resistance gene (Cm) that FRT site is contained at two ends:

Del-ptsG-P1：

5’-ATGTTTAAGAATGCATTTGCTAACCTGCAAAAGGTCGGTAAATCGCTGATGGAGCGATTGTGTAGGCTGGA-3’；

Del-ptsG-P2：

5’-TTAGTGGTTACGGATGTACTCATCCATCTCGGTTTTCAGGTTATCGGATTTACTTAACGGCTGACATGGGA-3’；

PCR reaction system response procedures is the same.

Amplified production carries out DNA gel electrophoretic analysis, cuts the nucleic acid fragment of glue recovery with correct target stripe.

2. the induction of Red recombination system and the preparation of competent cell

By transforming the intestinal bacteria W3110 having pKD46, in 30 DEG C of overnight incubation; Next day is seeded to 100mLLB substratum (ampicillin concentration is for 50mg/mL) with 1:50, and 30 DEG C are cultured to OD ₆₀₀when=0.25, add L-arabinose to 5mmol/L, induction 1h (OD ₆₀₀can not more than 0.6), Exo, Bet and Gam tri-albumen on pKD46 are given full expression to.Precooling 10min, 4000r/min on ice, 4 DEG C of centrifugal 10min, abandon substratum; With precooling 10% glycerine centrifuge washing 3 times, concentrated 100 times of competent cells becoming 1mL, often pipe 100 μ L.

3. electricity transforms

The band ptsG homology arm Cm gene fragment that clone obtains is about 500ng and adds competent cell, mixing, proceed to 0.2cm and shock by electricity in cup, do electricity conversion with Bio-Rad electroporation.Electric shock condition: 200 Ω, 25 μ F, shock voltage 2.5kV, the electric shock time is 4 ~ 5ms, adds rapidly the LB liquid nutrient medium of 1mL, 150r/min after electric shock, cultivates 1h, is applied to paraxin flat board (chloramphenicol concentration is 34 μ g/mL) afterwards for 37 DEG C; Identify by PCR method the Chloramphenicol-resistant clones grown after cultivating 12h.

4. FLP site specific recombination

PCP20 is proceeded to Chloramphenicol-resistant clones, and after 30 DEG C of cultivation 8h, bring up to 42 DEG C and spend the night, thermal induction FLP recombinase is expressed, and plasmid is also lost gradually.Dip bacterium liquid with transfering loop and draw plate on antibiotic-free substratum, choose on single bacterium colony point to chlorampenicol resistant flat board of growing and corresponding not containing on antibiotic flat board, what do not grow is deleted by FLP recombinase for chloramphenicol resistance gene.The clone doing the disappearance of PCR chloramphenicol resistance with primers designed ptsG-check1 and ptsG-check2 carries out identifying (see Fig. 5).

ptsG-check1：5’-CACCCATACTCAGGAGCACTCTCA-3’；

ptsG-check2：5’-CCTTAGTCTCCCCAACGTCTTACG-3’；

PCR reaction system and response procedures the same.To identify that the clone designation that correct ptsG has been knocked is HK101.

(2) the knocking out of xylA and xylB gene

1. the clone of the Cm gene of xylA upstream homology arm and xylB downstream homology arm is with

With W3110 genome for template, utilize primer Del-xylA-P1 and Del-xylA-P2 to increase, obtain the upstream homology arm of xylA gene:

Del-xylA-P1：5’-GGTCACCAACGACTTTAATTTTTCC-3’；

Del-xylA-P2：

5’-GACGTGTAATGCTGCAATCTATTGAACTCCATAATCAGGTAATGCC-3’；

PCR reaction system and response procedures the same.

Amplified production carries out DNA gel electrophoretic analysis, cuts the nucleic acid fragment of glue recovery with correct target stripe.

With plasmid pKD3 for template, utilize primer xylAB-FRT-P1 and xylAB-FRT-P2 to increase, obtain Cm gene:

xylAB-FRT-P1：

5’-ACCTGATTATGGAGTTCAATAGATTGCAGCATTACACGTCTTGA-3’；

xylAB-FRT-P2：

5’-GGTCAGGCAGGGGATAACGTGCAATTTTCAGTGACACAGGAACA-3’；

PCR reaction system and response procedures the same.

Amplified production carries out DNA gel electrophoretic analysis, cuts the nucleic acid fragment of glue recovery with correct target stripe.

With W3110 genome for template, utilize primer Del-xylB-P1 and Del-xylB-P2 to increase, obtain the downstream homology arm of xylB gene:

Del-xylB-P1：

5’-CCTGTGTCACTGAAAATTGCACGTTATCCCCTGCCTGACC-3’；

Del-xylB-P2：5’-GCACCGCTACTGCACCAAAC-3’；

PCR reaction system and response procedures the same.

Amplified production carries out DNA gel electrophoretic analysis, cuts the nucleic acid fragment of glue recovery with correct target stripe.

With the downstream homology arm of the upstream homology arm of xylA gene, Cm gene, xylB gene for template, utilize primer Del-xylA-P1, Del-xylB-P2 to carry out over-lap PCR, obtain the Cm gene of band xylA upstream homology arm and xylB downstream homology arm;

PCR reaction system and response procedures the same.

Amplified production carries out DNA gel electrophoretic analysis, cuts the nucleic acid fragment of glue recovery with correct target stripe.

2. the induction of Red recombination system and the preparation of competent cell

By transforming the intestinal bacteria HK101 having pKD46, in 30 DEG C of overnight incubation; Next day is seeded to 100mLLB substratum (ampicillin concentration is for 50mg/mL) with 1:50, and 30 DEG C are cultured to OD ₆₀₀when=0.25, add L-arabinose to 5mmol/L, induction 1h (OD ₆₀₀can not more than 0.6), Exo, Bet and Gam tri-albumen on pKD46 are given full expression to.Precooling 10min, 4000r/min on ice, 4 DEG C of centrifugal 10min, abandon substratum; With precooling 10% glycerine centrifuge washing 3 times, concentrated 100 times of competent cells becoming 1mL, often pipe 100 μ L.

3.-4. utilize the method identical with " in the knocking out of (1) ptsG gene 3.-4. ", obtain and knocked out xylA, xylB and the clone of chlorampenicol resistant disappearance, adopt primer Del-xylA-P1 and Del-xylB-P2 to carry out identifying (see Fig. 5).PCR reaction system and response procedures the same.To identify that the clone designation that correct ptsG has been knocked is HK301.

(3) the knocking out of ptsF gene

With W3110 genome for template, utilize primer Del-ptsF-up-P1 and Del-ptsF-up-P2 to increase, obtain the upstream homology arm of ptsF gene:

Del-ptsF-up-P1：5’-TGAATAAATTCACCGTGCTGTGC-3’；

Del-ptsF-up-P2：

5’-GACGTGTAATGCTGCAATCTTAAAAGGTGTGTTACAGGGCAGAAA-3’；

PCR reaction system and response procedures the same.

Amplified production carries out DNA gel electrophoretic analysis, cuts the nucleic acid fragment of glue recovery with correct target stripe.

With plasmid pKD3 for template, utilize primer ptsF-FRT-P1 and ptsF-FRT-P2 to increase, obtain Cm gene:

ptsF-FRT-P1：

5’-GCCCTGTAACACACCTTTTAAGATTGCAGCATTACACGTCTTGA-3’；

ptsF-FRT-P2：

5’-CTGACAGCAGGAGAGGCATAGCAATTTTCAGTGACACAGGAACA-3’；

PCR reaction system and response procedures the same.

Amplified production carries out DNA gel electrophoretic analysis, cuts the nucleic acid fragment of glue recovery with correct target stripe.

With W3110 genome for template, utilize primer Del-ptsF-do-P1 and Del-ptsF-do-P2 to increase, obtain the downstream homology arm of ptsF gene:

Del-ptsF-do-P1：

5’-CCTGTGTCACTGAAAATTGCTATGCCTCTCCTGCTGTCAGTTAAAA-3’；

Del-ptsF-do-P2：5’-CGTGGCCAAAGTATTAAAAGACCTG-3’；

2. the induction of Red recombination system and the preparation of competent cell

By transforming the intestinal bacteria HK301 having pKD46, in 30 DEG C of overnight incubation; Next day is seeded to 100mLLB substratum (ampicillin concentration is for 50mg/mL) with 1:50, and 30 DEG C are cultured to OD ₆₀₀when=0.25, add L-arabinose to 5mmol/L, induction 1h (OD ₆₀₀can not more than 0.6), Exo, Bet and Gam tri-albumen on pKD46 are given full expression to.Precooling 10min, 4000r/min on ice, 4 DEG C of centrifugal 10min, abandon substratum; With precooling 10% glycerine centrifuge washing 3 times, concentrated 100 times of competent cells becoming 1mL, often pipe 100 μ L.

3.-4. utilize the method identical with " in the knocking out of (1) ptsG gene 3.-4. ", obtain and knocked out xylA, xylB and the clone of chlorampenicol resistant disappearance, adopt primer Del-ptsF-up-P1 and Del-ptsF-do-P2 to carry out identifying (see Fig. 5).PCR reaction system and response procedures the same.To identify that the clone designation that correct ptsG has been knocked is HK401.

3, the structure of genetic engineering bacterium

By recombinant expression vector pTrc99a-rbs-xr6600 electric transformation of E. coli competent cell W3110, HK101, HK301, HK401 respectively, the bacterium colony that picking ampicillin plate grows, after extracting plasmid, order-checking, the correct positive colony of checking is named engineering bacteria HK009, HK102, HK302, HK402 respectively.

4, the detection of expression of each engineering bacteria

Engineering bacteria HK009, HK102, HK302, HK402 are inoculated in by 2% the improvement M9 substratum spent the night and (in 1L substratum, contain 4 ~ 6g Na ₂hPO ₄, 2 ~ 5g KH ₂pO ₄, 1 ~ 2g NH ₄cl, 1 ~ 5g NaCl, 1 ~ 5mMMgSO ₄, 1 ~ 5mM CaCl ₂, 2 ~ 10g/L yeast extract paste) in, cultivate at 30 DEG C.Investigate the fermentation character of each engineering bacteria, investigate result as shown in Fig. 6 a, 6b, 6c, 6d.

From Fig. 6 a, the Host Strains of engineering bacteria HK009 is wild-type E.coli K-12W3110, and it shows obvious glucose effect during the fermentation;

From Fig. 6 b, knock out the engineering bacteria HK102 of glucose phosphotransferase, although utilize the speed of xylose and glucose all also slow, can utilize xylose and glucose simultaneously;

From Fig. 6 c, knocked out the engineering bacteria HK302 of xylose isomerase and xylulokinase further, it is all significantly improved to the wear rate of wood sugar and the throughput rate of Xylitol;

From Fig. 6 d, knock out the engineering bacteria HK402 of fructose phosphate transferring enzyme further, it is all further improved to the wear rate of wood sugar and the throughput rate of Xylitol, and the wear rate of wood sugar is up to 0.598g/L/h, and the throughput rate of Xylitol is up to 0.61g/L/h.

Embodiment 2 utilizing works bacterium HK402 produces the example of Xylitol

1, engineering bacteria utilizes corn cob hemicellulose hydrolysate to produce Xylitol

In order to verify that can the engineering bacteria of structure utilize half fiber hydrolysis to produce Xylitol, carry out shake flask fermentation (250ml, liquid amount is 50ml) with engineering bacteria HK402.

(1) engineering bacteria HK402 is inoculated in the seed culture medium spent the night by 2%, at 30 DEG C, cultivate 8h, obtain seed liquor;

The formula of seed culture medium and fermention medium is: in 1L substratum, containing 4 ~ 6g Na ₂hPO ₄, 2 ~ 5g KH ₂pO ₄, 1 ~ 2g NH ₄cl, 1 ~ 5g NaCl, 1 ~ 5mM MgSO ₄, 1 ~ 5mM CaCl ₂, the yeast extract paste of 2 ~ 10g/L.

(2) being carried out by hemicellulose hydrolysate Rotary Evaporators being concentrated into Xylose Content is 20%-50%, to add in sterilized fermention medium after the hemicellulose hydrolysate sterilizing after concentrated, the whole content of wood sugar in fermention medium is made to be 2%, adding glucose to its final concentration is again 1%, obtains fermentation culture;

(3) seed liquor is seeded in fermentation culture by 2%, at 30 DEG C, is cultured to OD ₆₀₀during for 0.6-1.0, add the IPTG of 0.05 ~ 0.5mM, abduction delivering at 30 DEG C.

Investigate the fermentation character of engineering bacteria HK402 in corn cob hemicellulose hydrolysate, see Fig. 7 a.

From Fig. 7 a, engineering bacteria HK402 can effectively utilize hemicellulose hydrolysate to produce Xylitol, and Growth of Cells is not suppressed.

2, recombinant bacterial strain utilizes pure sugar batch fermentation

(1) engineering bacteria HK402 is inoculated in the seed culture medium spent the night by 2%, at 30 DEG C, cultivate 8h, obtain seed liquor;

The formula of seed culture medium and fermention medium is: in 1L substratum, containing 4 ~ 6g Na ₂hPO ₄, 2 ~ 5g KH ₂pO ₄, 1 ~ 2g NH ₄cl, 1 ~ 5g NaCl, 1 ~ 5mM MgSO ₄, 1 ~ 5mM CaCl ₂, 10 ~ 20g/L peptone, the yeast extract paste of 2 ~ 8g/L.

(2) seed liquor is seeded to by 10% is equipped with in the 5L fermentor tank of 2L fermention medium, at 30 DEG C, be cultured to OD ₆₀₀when being 5 ~ 15, add in fermented liquid wood sugar to final concentration be 100g/L, add glucose to final concentration be 50g/L, add the IPTG of 0.05 ~ 0.5mM, abduction delivering at 30 DEG C simultaneously.

Prepare muscovado solution (glucose, wood sugar, pectinose, the each 10g/L of Xylitol), adopt Dionex UltiMate3000 high performance liquid phase system, Corona Charged Aerosol detector, Aminex HPX-87C (7.8mm × 300mm) sugared post, moving phase is pure water (0.8mL min ^-1, 76 DEG C) and obtain typical curve (see Fig. 8); When abduction delivering starts, adopting uses the same method detects the content of each essential substance in fermented liquid, and detected result is shown in Fig. 9.

Investigate the fermentation character of engineering bacteria HK402 in pure sugar-fermenting liquid, see Fig. 7 b.

From Fig. 7 b, engineering bacteria HK402 can Bioconversion of D-xylose To Produce Xylitol fast, and in fermented liquid, Xylitol ultimate density can reach 118g/L, and production efficiency is 1.74g/L, can obtain higher Xylose reductase enzyme work simultaneously, be up to 3.86 ten thousand U/L fermented liquids.

3, recombinant bacterial strain utilizes pure sugared fed-batch fermentation

(1)-(2) same to part 2 " engineering bacteria utilizes pure sugar batch fermentation " (ferment in 15L fermentor tank carry out), 80g/L and 40g/L is respectively to adding xylose and glucose during 37h in addition to final concentration at abduction delivering, investigate the fermentation character of engineering bacteria HK402, see Fig. 7 c.

From Fig. 7 c, through the fermentation of 110 hours, all sugar can consume by engineering bacteria HK402, and in fermented liquid, Xylitol ultimate density reaches 172.4g/L.

4, engineering bacteria utilizes hemicellulose hydrolysate batch fermentation

(1)-(2) same to part 2 " engineering bacteria utilizes pure sugar batch fermentation ", but add glucose and the hemicellulose hydrolysate containing 43% wood sugar when step (2) abduction delivering starts, when abduction delivering is started, in fermented liquid, wood sugar final concentration is 143.79g/L, and glucose final concentration is 80g/L.

Investigate the fermentation character of engineering bacteria HK402, see Fig. 7 d.

From Fig. 7 d, in 5L fermentor tank, engineering bacteria HK402 effectively can transform the xylose production Xylitol in hemicellulose hydrolysate equally, Xylitol concentration reaches 150g/L, production efficiency reaches 1.4g/L/h, and this utilizes Bacterial Transformation hemicellulose hydrolysate to produce the maximum concentration of Xylitol and most high efficiency at present.

Claims (10)

1. a genetic engineering bacterium, comprise intestinal bacteria and proceed to colibacillary expression vector, it is characterized in that, the promotor downstream of described expression vector is inserted with Xylose reductase gene, and the base sequence of described Xylose reductase gene is as shown in SEQ ID No.4.

2. genetic engineering bacterium as claimed in claim 1, it is characterized in that, described promotor is Trc promotor, and 5 ' end band of described Xylose reductase gene has ribosome bind site, and the base sequence of described ribosome bind site is:

5’-AGAAGGAGATATACAT-3’。

3. genetic engineering bacterium as claimed in claim 1, it is characterized in that, described expression vector is pTrc99a.

4. the genetic engineering bacterium as described in as arbitrary in claims 1 to 3, it is characterized in that, in described intestinal bacteria, glucose phosphotransferase gene is suppressed or knock out.

5. the genetic engineering bacterium as described in as arbitrary in claims 1 to 3, is characterized in that, in described intestinal bacteria, in xylose isomerase gene and xylulokinase gene, at least one is suppressed or knock out.

6. the genetic engineering bacterium as described in as arbitrary in claims 1 to 3, it is characterized in that, in described intestinal bacteria, fructose phosphate transferase gene is suppressed or knock out.

7. the construction process of genetic engineering bacterium as described in as arbitrary in claim 1 ~ 6, is characterized in that, comprise the following steps:

(1) glucose phosphotransferase gene knockout carrier, xylose isomerase gene knockout carrier, xylulokinase gene knockout carrier, fructose phosphate transferase gene knockout carrier is built respectively, and get at least one transformation of E. coli competent cell in four kinds of knockout carriers, obtain the intestinal bacteria having knocked out at least one in glucose phosphotransferase gene, xylose isomerase gene, xylulokinase gene and fructose phosphate transferase gene;

(2) Xylose reductase gene of 5 ' end band ribosome bind site is connected to the Trc promotor downstream of pTrc99a, obtains expression vector;

(3) by the intestinal bacteria that described expression vector step of converting (1) obtains, described genetic engineering bacterium is obtained.

8. genetic engineering bacterium as described in as arbitrary in claim 1 ~ 6 is producing the application in Xylitol.

9. apply as claimed in claim 8, it is characterized in that, comprising:

Described genetic engineering bacterium is seeded in nutrient solution and is cultured to OD ₆₀₀be 0.6 ~ 1.0, add inductor and carry out abduction delivering, after completing, from nutrient solution, separation and purification obtains Xylitol.

10. apply as claimed in claim 9, it is characterized in that, abduction delivering 40 ~ 120h at 26 ~ 30 DEG C.