CN115141816B

CN115141816B - Method for improving conversion of clostridium into butanol by using carbon source and application

Info

Publication number: CN115141816B
Application number: CN202210652505.0A
Authority: CN
Inventors: 张霏霏; 张志乾; 张侃; 吴奕瑞; 冼幸游; 王海梅; 邱�益; 刘月月
Original assignee: Guangzhou Qianxiang Biotechnology Co Ltd
Current assignee: Guangzhou Qianxiang Biotechnology Co Ltd
Priority date: 2022-06-09
Filing date: 2022-06-09
Publication date: 2023-08-08
Anticipated expiration: 2042-06-09
Also published as: CN115141816A

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a method for improving the conversion of butanol by clostridium using a carbon source and application thereof. According to the invention, a complete analysis system is established for exploring and analyzing related genes of the clostridium solvolicum strain WK in the carbon source metabolic process, and obtaining the key enzyme of regulating metabolism, namely UDP-glucose-hexose-1-phosphate uridyltransferase, based on the complete analysis system, the invention firstly discloses a new way for efficiently utilizing seaweed biomass rich in carbohydrates, such as red algae, green algae and the like by improving the content and/or activity of the UDP-glucose-hexose-1-phosphate uridyltransferase in clostridium, thereby improving the carbon source utilization rate of clostridium, promoting the generation of butanol in clostridium, promoting the generation of acetone in clostridium, promoting the growth of clostridium, and being beneficial to eliminating the influence of substrate feedback inhibition.

Description

Method for improving conversion of clostridium into butanol by using carbon source and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for improving the conversion of butanol by clostridium using a carbon source and application thereof.

Background

The industrial application of biological butanol has great potential due to the advantages of high calorific value, low volatility, low corrosiveness and the like of butanol, but the production cost required by the traditional acetone-butanol-ethanol (ABE) fermentation process of wild type solvolyte is relatively high, and the requirement of large-scale industrial production is not met.

As with other industrial microorganisms, the breeding of strains with industrial application value is always a long-term requirement for industrial production of biological butanol, and the microbial strains with the characteristics of high butanol tolerance, high butanol production proportion and the like or the transformation of the strains from acid production to solvent production without artificial regulation and control are capable of effectively improving the production efficiency and reducing the production cost, and meet the industrial production standard. On the other hand, during the whole fermentation process, most of the carbon source substrate can be used for self-growth and other metabolic pathways, which reduces the energy flowing to the synthesis of butanol, and therefore, the utilization efficiency of the carbon source substrate by the strain is increased, and the yield of butanol can be improved to a certain extent. In addition, most clostridium solvogenes can utilize a relatively broad substrate spectrum, but are generally weaker than glucose. By improving the utilization efficiency of other fermentable sugars such as galactose, the utilization of the biomass in red algae with high galactose content can be further expanded, so that the production cost can be effectively reduced, and the sustainability of industrial production can be increased. Ra et al found in "effect of galactose on rhodophyta ethanol fermentation suitability of yeast" (Bioprocess and Biosystems Engineering,2015, 38, 1715-1722) in the literature that strain Saccharomyces cerevisiae KCCM1129, which is not suitable for high concentration galactose, failed to completely consume galactose in the hydrolysate and consumed at a lower rate during ethanol production using rhodophyta biomass hydrolysate; and after the strain is completely suitable for high-concentration galactose, the utilization rate of the strain on a substrate and the overall bioconversion rate are greatly improved. Likewise, substrate utilization and consumption rates during clostridium fermentation should be taken into account while pursuing an increase in butanol yield.

In the process of fermenting biological butanol, all carbon source substrates are converted into pyruvic acid through a glycolysis pathway and then enter an ABE metabolic pathway, so that analysis of carbon source metabolic regulation is beneficial to regulating glycolysis efficiency, and further provides assistance for butanol synthesis. In addition to glucose, common substrates that can be fermented by clostridium include lactose, galactose, xylose, rhamnose, glycerol, and the like.

As a member of the third generation of algal biomass, red algae biomass such as agaropectins can be hydrolyzed by a simpler acid or enzyme process, so that a large amount of fermentable sugars can be released for subsequent microbial fermentation, wherein the galactose content can be more than 80%. However, in the actual fermentation process, the substrate selectivity of the strain is low, and the utilization rate of the wild strain Clostridium sp.wk to galactose is low, so that the problems of low product level, unsatisfactory conversion effect, increased cost, resource waste and the like caused by incomplete galactose consumption can occur when the seaweed hydrolysate with high galactose content is subsequently utilized.

Disclosure of Invention

It is an object of the first aspect of the present invention to provide the use of UDP-glucose-hexose-1-phosphate uridyltransferase.

The object of the second aspect of the present invention is to provide the use of biological materials related to UDP-glucose-hexose-1-phosphate uridyltransferase.

The object of the third aspect of the present invention is to provide the use of an agent for targeted up-regulation of the expression level of UDP-glucose-hexose-1-phosphate uridyltransferase and/or for enhancing UDP-glucose-hexose-1-phosphate uridyltransferase activity.

The fourth aspect of the present invention is directed to a method for constructing a recombinant bacterium.

The fifth aspect of the present invention is directed to a recombinant bacterium.

The object of the sixth aspect of the present invention is to provide the use of the recombinant bacterium of the fifth aspect of the present invention for the preparation of butanol and/or acetone.

The object of the seventh aspect of the present invention is to provide a process for preparing butanol and/or acetone.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention there is provided the use of UDP-glucose-hexose-1-phosphate uridyltransferase in at least one of (a 1) to (a 8);

(a1) The carbon source utilization rate of clostridium is improved;

(a2) Promote the generation of butanol in clostridium;

(a3) Promote the generation of acetone in clostridium;

(a4) Preparing a product that promotes the growth of clostridium;

(a5) Promoting the growth of clostridium;

(a6) Preparing a product for improving the carbon source utilization rate of clostridium;

(a7) Preparing a product that promotes the production of butanol in clostridium;

(a8) A product is prepared that promotes the production of acetone in clostridium.

Preferably, the clostridium comprises a solventogenic clostridium; further preferably, the Clostridium comprises Clostridium sp.wk.

Preferably, the carbon source comprises at least one of lactose, glucose, galactose, xylose, rhamnose; further preferably, the carbon source comprises galactose.

Preferably, the UDP-glucose-hexose-1-phosphate uridyltransferase is any one of c 1) to c 3);

c1 UDP-glucose-hexose-1-phosphate uridyltransferase with an amino acid sequence shown as SEQ ID NO. 6;

c2 UDP-glucose-hexose-1-phosphate uridyltransferase having the same function as SEQ ID No.6 by substitution and/or deletion and/or addition of one or more amino acids to SEQ ID No. 6;

c3 UDP-glucose-hexose-1-phosphate uridyltransferase having 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology with SEQ ID No.6 and having the same function as SEQ ID No. 6.

In a second aspect of the invention, there is provided the use of a biological material associated with UDP-glucose-hexose-1-phosphate uridyltransferase in at least one of (a 1) to (a 8);

(a1) The carbon source utilization rate of clostridium is improved;

(a2) Promote the generation of butanol in clostridium;

(a3) Promote the generation of acetone in clostridium;

(a4) Preparing a product that promotes the growth of clostridium;

(a5) Promoting the growth of clostridium;

(a8) Preparing a product that promotes the production of acetone in clostridium;

the biological material related to UDP-glucose-hexose-1-phosphate uridyltransferase comprises at least one of b 1) to b 12):

b1 A nucleic acid molecule encoding a UDP-glucose-hexose-1-phosphate uridyltransferase;

b2 An expression cassette comprising b 1) said nucleic acid molecule;

b3 A recombinant vector comprising the nucleic acid molecule of b 1);

b4 A recombinant vector comprising the expression cassette of b 2);

b5 A recombinant cell comprising the nucleic acid molecule of b 1);

b6 A recombinant cell comprising the expression cassette of b 2);

b7 A recombinant cell comprising the recombinant vector of b 3);

b8 A recombinant cell comprising the recombinant vector of b 4);

b9 A recombinant microorganism comprising the nucleic acid molecule of b 1);

b10 A recombinant microorganism comprising the expression cassette of b 2);

b11 A recombinant microorganism comprising the recombinant vector of b 3);

b12 A recombinant microorganism comprising the recombinant vector of b 4).

In a third aspect of the present invention, there is provided the use of an agent for targeted upregulation of the expression level of UDP-glucose-hexose-1-phosphate uridyltransferase and/or for enhancing UDP-glucose-hexose-1-phosphate uridyltransferase activity in at least one of (a 1) to (a 8);

(a1) The carbon source utilization rate of clostridium is improved;

(a2) Promote the generation of butanol in clostridium;

(a3) Promote the generation of acetone in clostridium;

(a4) Preparing a product that promotes the growth of clostridium;

(a5) Promoting the growth of clostridium;

In a fourth aspect of the present invention, there is provided a method for constructing a recombinant bacterium, comprising the steps of: increasing the content and/or activity of UDP-glucose-hexose-1-phosphate uridyltransferase in clostridium.

Preferably, the method for increasing the content and/or activity of UDP-glucose-hexose-1-phosphate uridyltransferase in Clostridium is to overexpress UDP-glucose-hexose-1-phosphate uridyltransferase in Clostridium.

Preferably, the method for increasing the content and/or activity of UDP-glucose-hexose-1-phosphate uridyltransferase in clostridium is to introduce a gene encoding UDP-glucose-hexose-1-phosphate uridyltransferase into clostridium.

Preferably, the coding gene of UDP-glucose-hexose-1-phosphate uridyltransferase is introduced into Clostridium by a recombinant vector; the recombinant vector is obtained by inserting the coding gene of the UDP-glucose-hexose-1-phosphate uridyltransferase into a multiple cloning site of an expression vector.

Preferably, the expression vector may be an expression vector commonly known in the art, for example: a pMTL80000 series clostridium specific expression vector comprising: pMTL83353, pMTL84422, pMTL85141, pMTL82151, pMTL83151, pMTL84151, pMTL85151, and the like.

Preferably, the expression vector comprises pMTL83353.

Preferably, the sequence of the coding gene of the UDP-glucose-hexose-1-phosphate uridyltransferase is any one of d 1) to d 3):

d1 As shown in SEQ ID NO. 7;

d2 A nucleotide sequence of SEQ ID NO.7 with the same function as SEQ ID NO.7 through substitution and/or deletion and/or addition of one or more nucleotides;

d3 A nucleotide sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology with SEQ ID NO.7 and having the same function as SEQ ID NO. 7.

Preferably, the recombinant vector is pMTL-galT, and the nucleotide sequence of the recombinant vector is shown in SEQ ID NO. 5.

In a fifth aspect of the present invention, there is provided a recombinant bacterium obtained by the construction method of the fourth aspect of the present invention.

In a sixth aspect, the invention provides the use of a recombinant bacterium according to the fifth aspect of the invention in at least one of e 1) to e 2);

e1 Butanol is prepared;

e2 Acetone is prepared.

In a seventh aspect of the invention, there is provided a method of inoculating the recombinant bacterium of the fifth aspect of the invention to a fermentation medium for fermentation; the method is used for at least one of e 1) to e 2):

e1 Butanol is prepared;

e2 Acetone is prepared.

Preferably, the fermentation medium is an anaerobic medium.

Preferably, the fermentation medium comprises a carbon source.

Preferably, the fermentation medium comprises: yeast extract, naHCO ₃ At least one of 2- (N-morpholinyl) ethane sulfonic acid, inorganic salt, trace elements and deoxidizing reducer.

Preferably, the inorganic salt comprises: naCl, mgCl ₂ 、KH ₂ PO ₄ 、NH ₄ Cl、KCl、CaCl ₂ At least one of them.

Preferably, the trace elements comprise: feCl ₂ 、CoCl ₂ 、MnCl ₂ 、ZnCl ₂ 、H ₃ BO ₃ 、Na ₂ MoO ₄ 、NiCl ₂ 、CuCl ₂ At least one of them.

Preferably, the oxygen-scavenging reducing agent comprises: dithiothreitol, L-cysteine, na ₂ At least one of S.

Preferably, the fermentation medium comprises: an anaerobic indicator.

Preferably, the anaerobic indicator comprises resazurin.

Preferably, the fermentation medium contains an antibiotic.

Preferably, the antibiotic comprises at least one of spectinomycin, penicillin, streptomycin, gentamicin; further comprising spectinomycin.

Preferably, the fermentation conditions are 25-35 ℃ and 100-200 rpm for 90-102 h.

The beneficial effects of the invention are as follows:

the invention is generalBy establishing a complete analysis system for exploring and analyzing related genes in the metabolic process of clostridium solvolicum strain WK on a carbon source and obtaining a key enzyme for regulating metabolism, namely UDP-glucose-hexose-1-uridine phosphate transferase, based on the complete analysis system, the method firstly discloses that the carbon source utilization rate of clostridium is improved, the production of butanol in clostridium is promoted, the production of acetone in clostridium is promoted and the growth of clostridium is promoted by improving the content and/or the activity of UDP-glucose-hexose-1-uridine phosphate transferase in clostridium, wherein the carbon source consumption is improved by 23.05%, the butanol yield is improved by 23.21%, and the acetone yield is improved by 40.22% relative to wild clostridium OD _600nm Improves the yield by 12.87 percent, is beneficial to eliminating the influence of substrate feedback inhibition (CCR), and provides a new way for efficiently utilizing seaweed biomass rich in carbohydrate, such as red seaweed, green seaweed and the like.

Drawings

FIG. 1 is a galactose metabolism pathway chart of a wild-type strain WK established in example 1 of the present invention.

FIG. 2 is a diagram showing a primer-verification analysis of a target gene involved in a metabolic pathway in example 1 of the present invention: wherein M represents Mraker; lanes 1-16 represent: aldose 1-epimerase (galM); a DNA recombinant repair protein gene (RecA); galactokinase (galK); phosphoglucomutase (pgm); DNA gyrase subunit B (gyrB); an adenylate kinase gene (adk); a DNA recombinant repair protein gene (RecA); UTP-glucose-1-phosphate uridyltransferase (galU-16); UDP-glucose-hexose-1-phosphate uridyltransferase (galT); a DNA recombinant repair protein gene (RecA); a DNA recombinant repair protein gene (RecA); a DNA recombinant repair protein gene (RecA); UTP-glucose-1-phosphate uridyltransferase (galU-4); UTP-glucose-1-phosphate uridyltransferase (galU-5); UDP-glucose 4-epimerase (galE-52); UDP-glucose 4-epimerase (galE-26).

FIG. 3 is a graph showing the difference in transcription level of different genes by real-time quantitative PCR analysis in example 1 of the present invention.

FIG. 4 is a map of the plasmid pMTL-galT constructed in example 2 of the invention.

FIG. 5 is a diagram showing PCR identification analysis of engineering strain WK:: galT in example 2 of the present invention: wherein M represents Mraker; lanes 1-5 represent: lanes 1-2 are bands amplified by using the primer 16s-F/16s-R with strains WK and WK:: galT genome as templates respectively; lanes 3-5 are bands amplified with Primer1-F/Primer1-R using the strain WK genome, strain WK:: galT genome, plasmid pMTL-galT as template.

FIG. 6 is a graph showing the growth curve, substrate consumption and product synthesis of the wild strain WK of example 3 of the present invention during fermentation with 40g/L galactose as a substrate.

FIG. 7 is a graph showing the growth curve, substrate consumption and product synthesis of the recombinant strain WK:: galT during fermentation with 40g/L galactose as substrate in example 3 of the present invention.

FIG. 8 is a graph showing comparison of biomass, substrate consumption and product synthesis during fermentation of recombinant strain WK in example 3 of the present invention, wherein p <0.05; * Represents p <0.01.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are commercially available ones unless otherwise specified.

1. Strain and carrier

(1) The shuttle plasmid pMTL83353 is a commercial product and is gratuitously given by the Shanzhi university synthetic biology and biochemical engineering laboratory and stored by Beijing state biosciences Limited company;

(2) Coli Top10 competent cells were purchased from Biotechnology (Shanghai) Inc.;

(3) The wild strain is Clostridium sp.wk (published in patent document CN112961799a, accession number is GDMCCNo: 61493), and is gratuitously given by the university of shan, synthetic biology and biochemical engineering laboratory, and is currently stored by Beijing state creature biotechnology limited company;

(4) The recombinant engineering strain is Clostridium sp.wk:: galT, which is a gene (galT) of over-expressed UDP-glucose-hexose-1-phosphate uridyltransferase based on wild strain WK, and is now stored by Beijing state biosciences.

2. Preparation of culture Medium

(1) Anaerobic Culture Medium (ACM), comprising per liter: yeast extract, 10g; naHCO (NaHCO) ₃ 2.52g;2- (N-morpholino) ethanesulfonic acid (MES), 2.132g;100 X10 mL of salt solution (containing NaCl,1.0 g/liter; mgCl) ₂ ·6H ₂ O，0.5g；KH ₂ PO ₄ ，0.2g；NH ₄ Cl，0.3g；KCl，0.3g；CaCl ₂ ·2H ₂ O,0.015 g); 1000 Xtrace element solution 1mL (FeCl per liter) ₂ ·4H ₂ O，1.5g；CoCl ₂ ·6H ₂ O，0.19g；MnCl ₂ ·4H ₂ O，0.1g；ZnCl ₂ ，0.07g；H ₃ BO ₃ ，0.006g；Na ₂ MoO ₄ ·2H ₂ O，0.036g；NiCl ₂ ·6H ₂ O，0.024g；CuCl ₂ ·2H ₂ O,0.002 g); 1mL of anaerobic indicator (1000 XResazurin) was added as a pure water preparation; finally adding deoxidizing reducer (dithiothreitol DTT,0.077g; L-cysteine L-Cys,0.0242g, 1000 XNa) ₂ S solution, 1 mL), pH was adjusted to 6.0, and N was continuously introduced into the medium ₂ Deoxidizing, packaging into serum bottles for anaerobic culture, sealing with butyl rubber plug and aluminum cap, and sterilizing at 121deg.C for 20min. The culture medium is used for activating and fermenting strains.

(2) Carbon source substrate mother liquor: the carbon source is glucose or galactose, which is prepared into 50% (w/v) mother liquor, and is packaged into serum bottle, and N is continuously introduced into the serum bottle ₂ Deoxidizing, and sterilizing at 115 ℃ for 15min for standby.

(3) LB medium, which contains per liter: naCl,10g; tryptone, 10g; yeast extract, 5g; the solid medium was prepared with pure water, and 1.5% (w/v) agar was added thereto, and the mixture was sterilized at 121℃for 20 minutes. The medium is used for the cultivation of E.coli strains containing the plasmid of interest.

(4) Electrotransport buffer (10% glycerol): 10mL of glycerol was dissolved in 90mL of ddH ₂ In O, N is continuously introduced ₂ Removing oxygen, sealing with butyl rubber plug and aluminum cap, sterilizing at 121deg.C for 20min, and preserving at 4deg.C.

(5) Clostridium enhancement medium (RCM) was prepared by adding 1.5% agar to the solid medium after 38g of RCM-containing medium powder (available from BD Co., U.S.A., cat# CNL17-B0061, lot # 9183138) was completely dissolved in pure water, and sterilizing at 121deg.C for 20min.

(6) Strain recovery medium, comprising per liter: yeast extract, 10g; naCl,5g; peptone, 16g; after complete dissolution, adding 1mL of anaerobic indicator, and uniformly stirring until the volume reaches 950mL; adding DTT 0.077g, L-Cys0.0242g and 1000 xNa in turn after constant volume ₂ S solution 1mL, and regulating pH to 6.0-7.0; continuously introducing N ₂ After the culture medium is completely deoxygenated, N is introduced into the culture medium ₂ Is divided into 100 or 20mL serum bottles according to the required amount, and is sterilized for 20min at 121 ℃ after being sealed by a butyl rubber plug and an aluminum cover. The culture medium is used for resuscitating and culturing target positive engineering strains.

(7) Anaerobic spectinomycin solution: spectinomycin powder 250mg/mL, dissolved in pure water, filtered through a 0.22 μm nylon membrane, and continuously introduced with N ₂ And (3) until oxygen is removed, sealing by using a butyl rubber plug and an aluminum cover, and preserving at 4 ℃.

(8) Proteinase K solution: 32mg of proteinase K was weighed accurately and dissolved in 80mL of 1 XTE buffer solution at pH 8.0 and stored at 4 ℃.

EXAMPLE 1 analysis of the galactose Metabolic pathway of wild type strain Clostridium sp.WK (WK)

1. Culturing and fermenting strain, extracting total RNA of sample and constructing cDNA library

Taking activated strain Clostridium sp.wk, accurately sucking 4% seed solution in an ultra-clean workbench, inoculating into 50mL anaerobic culture medium containing 30g/L glucose, and culturing at 37deg.C and 100rpm for 18 hr to obtain fermentation seed solution (OD) _600nm =0.8). Inoculating the seed solution to 150mL containing 30g/L galactose as base at an inoculum size of 4%In the anaerobic culture medium of the culture, 3mL of bacterial liquid is taken out every 2, 3 hours or 4 hours, and after centrifugation at 13000rpm for 10min at 4 ℃, bacterial cells are collected, and after re-suspension by 1mL of pre-cooled Trizol solution, the bacterial cells are rapidly placed at-80 ℃ for preservation. Anaerobic culture medium inoculated to 30g/L glucose is used as a control group, 3 parallel samples are arranged in the experimental group and the control group, and 3 samples at each sampling point are required to be mixed. In the whole fermentation process, fermentation index conditions such as biogas, residual sugar in fermentation liquid, pH, growth curve, product conditions in fermentation liquid and the like are detected in real time. With a slight modification with reference to the TRIzol Reagent instructions, total RNA from all samples was extracted with pre-chilled phenol and isopropanol, and immediately after RNA concentration detection, a cDNA library was constructed using Qiagen company QuantiTect Reverse Transcription Kit. Step optimization is carried out on the basis of instruction book of the reference kit, after gDNA is removed from the sample, reverse transcription is carried out for 25min at 42 ℃, then the reaction is stopped by heating for 3min at 95 ℃, and the obtained cDNA library is preserved at-20 ℃ for standby.

2. Analysis of galactose metabolism transcription level difference by real-time quantitative PCR technique

Based on genomic information of the strain Clostridium sp.WK and KEGG database analysis, the relevant genes of its galactose metabolic pathway were determined, and a metabolic pathway map was established including a main pathway composed of aldose 1-epimerase (galM), galactokinase (galK), UDP-glucose-hexose-1-phosphate uridyltransferase (galT), and a branch pathway composed of UDP-glucose 4-epimerase (galE-26/52), UTP-glucose-1-phosphate uridyltransferase (galU-4/5/16), phosphoglucomutase (pgm) (FIG. 1). By designing qPCR primers for detecting target genes, on the basis of verifying the amplification specificity of the primers (figure 2), a quantitative analysis system for different target genes is established by using an RT-qPCR technology, and a standard curve is established by using the logarithm of Ct value to the copy number of the target genes. qPCR analysis was performed using the cDNA obtained in step 1 as a template, and the reaction system was set to 20. Mu.L system containing 1. Mu.L of the DNA template and 0.4. Mu.L of each forward and reverse primer (primer sequences shown in Table 1, 2. Mu.M) and 10. Mu.L of each 2 XqPCR supermix; the reaction procedure is: pre-denaturation at 95℃for 2min, 30s at 95℃and Tm (annealing temperature adjusted by gene sequence) for 60s,45 cycles. Three parallel samples are arranged on each template, and a relative quantification method is used for the gene transcription amount analysis method, specifically:

delta Ct (gene of interest) =ct (gene of interest)/Ct (reference gene, recA);

relative transcript amount (%) =Δct (transcript amount when galactose is the substrate) - Δct (transcript amount when glucose is the substrate);

relative elevation (%) =relative transcription (%)/Δct (transcription when glucose is the substrate).

TABLE 1 primer sequences

The anaerobic culture medium of glucose is taken as a control group, through analyzing the expression quantity of related genes related to main and branch paths in the galactose metabolism process of a wild strain Clostridium sp.wk (figure 3), the monitoring result of the fermentation process shows that the expression quantity of galK and galT in the main path rapidly rises in the early fermentation period (2-21 h), wherein the expression quantity of galK reaches a peak value in 10h, the galactose consumption rate is the maximum (1.42 g/L/h), while the galT always maintains a high expression state in 2-36 h, and plays a role in global regulation in the galactose utilization process; in contrast, the majority of gene lifting by the branched pathway is not apparent. Therefore, it was confirmed that the galT gene encoding UDP-glucose-hexose-1-phosphate uridyltransferase (amino acid sequence shown as SEQ ID NO.6 and nucleic acid sequence shown as SEQ ID NO. 7) is the most critical gene regulating galactose metabolism of the strain WK.

EXAMPLE 2 construction of recombinant Strain for improving galactose metabolism WK:: galT

This example is based on the key gene galT in galactose metabolism process already determined in example 1, and a clostridium-based overexpression system is designed and constructed to increase the intracellular galT level of the strain to achieve the purpose of increasing the galactose substrate utilization rate. The method comprises the following steps: 1) Specific PCR primers were designed (galT-F: 5' -AGAGTCGACGTCATGATAAATATAA-3' (SEQ ID NO.3, underlined is the NdeI cleavage site), galT-R:5' -GCAGGCCTCGAGTTATAATATATT-3' (SEQ ID NO.4, underlined is the XmaI cleavage site)); 2) Amplifying target fragments from the genome of the strain WK by using high-fidelity enzyme, wherein the reaction system is set to be a 25 mu L system, and comprises 1 mu L of a DNA template (50-200 ng), 1 mu L, dNTP mu L of forward and reverse primers (10 mu M), 5 mu L of a5 Xhigh-fidelity enzyme reaction buffer solution and 0.5 mu L of high-fidelity polymerase; the reaction procedure is: pre-denaturation at 95℃for 5min, pre-denaturation at 95℃for 30s, pre-denaturation at 63℃for 30s, pre-denaturation at 72℃for 1.5min for 30 cycles, and extension at 72℃for 10min; 3) Detecting the amplified galT gene fragment through agarose gel electrophoresis, purifying and recovering the gene fragment by using a rapid purification kit, and measuring the concentration; 4) The galT and the clostridium-escherichia coli shuttle vector pMTL83353 were digested with restriction enzymes (NdeI, xmaI) respectively, and the digestion system was set to 50. Mu.L, which includes a target DNA fragment/pMTL 83353 (1. Mu.g), 5. Mu.L of 10 Xuniversal digestion buffer, and different restriction enzymes (NdeI, 20000U/mL; xmaI, 10000U/mL) were reacted at 37℃for 12 hours, and the digested products were recovered by agarose gel electrophoresis with a DNA recovery kit; 5) The digested gene fragment galT and linearization vector pMTL83353 are subjected to ligation reaction, the system is set to 10 mu L, wherein the ligation reaction comprises 0.03pmol of vector fragment, 0.3pmol of target DNA fragment, 1 mu L of T4 DNA ligase (350U/. Mu.L) and 1 mu L of 10 xT 4 DNA reaction buffer solution, the mixture is placed at 16 ℃ for reaction for 30min and then is transformed into an escherichia coli Top10 strain, positive clones are screened by using LB solid culture medium containing 250mg/mL spectinomycin resistance, and the galT gene overexpression plasmid is obtained through colony PCR and sequencing verification, the sequence of the galT gene overexpression plasmid is named pMTL-galT (the sequence of pMTL-galT is shown in SEQ ID NO. 5), and the map is shown in figure 4.

The plasmid pMTL-galT (5 μg) was introduced into the preactivated wild-type strain WK using an optimized clostridium electrotransformation system (electrotransformation process was completed with 10% glycerol as electrotransformation buffer, 2mm cuvette and 3.4ms shock time, voltage 1.5 kV). Coating the recovered bacterial liquid on an RCM agar plate containing 250mg/mL spectinomycin, culturing overnight for 48 hours to clone, selecting a colony to expand and culture in a strain recovery culture medium, and pretreating the bacterial liquid by using proteinase K after the bacterial liquid grows to a certain amount for bacterial liquid PCR, wherein the primers are respectively Primer1-F:5'-TAGTAGCCTGTGAAAT-3' (SE Q ID NO. 8); primer1-R:5'-TAGTATTGATCCTCCA-3' (SEQ ID NO. 9); 16s-F:5'-AGAG TTTGATCCTGGCTCAG-3' (SEQ ID NO. 28); 16s-R:5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 29) to verify whether the target plasmid was successfully introduced into Clostridium, the result of the verification is shown in FIG. 5, and the engineering strain WK:: galT was finally obtained.

Example 3 engineering strain WK Process monitoring of galT fermentation of biological butanol with galactose

The fermentation conditions of the engineering strain WK are compared with that of the wild strain WK, so that the effect of the engineering strain in promoting the galactose to be converted into the biological butanol is tested. Preparing an anaerobic culture medium, inoculating wild type strain WK and engineering strain WK obtained in example 2 according to the volume of the culture medium, wherein galT (the concentration of bacterial solution of the wild type strain WK and the engineering strain WK obtained in example 2 is the same), simultaneously adding 10g/L glucose, adding 250mg/mL spectinomycin into the culture medium of the engineering strain, carrying out anaerobic culture for 12h at 37 ℃ and 150rpm to obtain a seed solution, adding galactose into a new anaerobic culture medium to enable the initial carbon source concentration to be 40g/L, and inoculating the seed solutions of the strain WK, galT and WK into the anaerobic culture medium (45 mL) according to the inoculum size of 4% of the total volume of the culture medium, wherein the strain WK, galT and the seed solution of the strain WK are required to be additionally added with 1% of the spectinomycin solution (250 mg/mL), and carrying out constant-temperature fermentation for 96h at 30 ℃ and 150 rpm. In the whole fermentation process, exhaust sampling is carried out every 12 or 24 hours, and the growth condition of thalli, the galactose consumption condition and the fermentation product generation condition in the fermentation process are detected.

As shown in FIGS. 6 to 8, the wild-type strain WK consumed 25.55g/L galactose altogether, and the maximum biomass could reach OD _600nm Yields of acetone, ethanol and butanol were 2.76, 0.55 and 7.97g/L, respectively, =9.4, with overall ABE yield reaching 11.28g/L; under the same conditions, the galactose consumption of the strain WK is improved to 31.44g/L, which is 23.05% higher than that of the control group WK, the time period of the highest galactose consumption rate is mainly between 12 and 24 hours, the highest galactose consumption rate is 0.97g/L/h, and the maximum biomass in the fermentation process reaches OD _600nm =10.61, 12.87% improvement over WK control; at the end of fermentation, engineering strain WK is acetone, ethanol of galT,The total yield of butanol and ABE is 3.87, 0.80, 9.82 and 14.49g/L respectively, which are improved by 40.22%, 45.45%, 23.21% and 28.46% respectively compared with the strain WK. This result shows that the present example has successfully molecularly engineered the existing clostridium solvogenes WK and significantly improved its substrate utilization efficiency and biobutanol synthesis efficiency.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Guangzhou City dry phase Biotech Co., ltd

<120> method for improving conversion of butanol by clostridium using carbon source and application thereof

<130>

<160> 29

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> DNA

<213> artificial sequence

<400> 1

tcctccagtt gttgtttct 19

<210> 2

<211> 18

<212> DNA

<213> artificial sequence

<400> 2

ctcagttgcg gctttagt 18

<210> 3

<211> 25

<212> DNA

<213> artificial sequence

<400> 3

agagtcgacg tcatgataaa tataa 25

<210> 4

<211> 24

<212> DNA

<213> artificial sequence

<400> 4

gcaggcctcg agttataata tatt 24

<210> 5

<211> 6414

<212> DNA

<213> artificial sequence

<400> 5

cctgcaggat aaaaaaattg tagataaatt ttataaaata gttttatcta caattttttt 60

atcaggaaac agctatgacc gcggccgcgt gtagtagcct gtgaaataag taaggaaaaa 120

aaagaagtaa gtgttatata tgatgattat tttgtagatg tagataggat aatagaatcc 180

atagaaaata taggttatac agttatataa aaattacttt aaaaattaat aaaaacatgg 240

taaaatataa atcgtataaa gttgtgtaat ttttaaggag gtgtgttaca tatgatgata 300

aatataaatc atgaaataaa tagacttata aactttgcat tgaaaaaaaa tcttataaat 360

aatgatgata taatttactc taccaatatg attcttggag tattaaactt aaatgaattt 420

gaagttcgtg aagttgatga aactttagaa actccaactc ctatcttaga aaatatctta 480

gactacgcat gtgaaaataa tttaatagaa aatacagtga ctgaaagaga cttatttgat 540

actttaatta tgaactgcgt aatgccaaga ccttctgagg ttataaataa ctttaataat 600

ttatataata tctctccaac taaagctact gagtactatt atgatttaag catttcttct 660

aactatattc gaaaagatag aattgacaag aacataatct ggaaagcacc aacggaatat 720

ggagacttag atataacaat taatttatca aaaccagaaa aagatcctag ggatatagca 780

aaagctaaat taattaaatc tagttcatat cctaaatgtt tattatgtaa agaaaatgaa 840

ggattttatg gtcatataaa tcatcctgcc agacagactc atagaattat acctttggat 900

tttgataaac aaaaatattt cttgcagtat tccccttata cttattataa tgagcattgt 960

ataattctaa atagcgaaca tatacctatg aaaattaata aggatacttt taggaattta 1020

ttgattttta ctgatatact gcctcattac tttgctggat ctaacgctga cttgcctata 1080

gttggaggat caatactatc tcatgatcat tatcaaggtg gtcactacac atttgctatg 1140

gaaaaagctc caatagaaaa aaactactca gttaaaaatt atgaagatgt tgaaataggt 1200

agagtcaaat ggcctatgtc tgttgttagg ctttcaagta atgataaaga taaattatta 1260

gatttagctg atcatatttt aacttcatgg agaagttatt ctgatgaatc tgtaaatata 1320

ttaagtcata caatggatga accgcataat actattactc ctattgcaag aaaaaggaat 1380

aataagtacg aactagattt agtcttaagg aacaatagaa caagtactga gcaccctctt 1440

ggtattttcc atcctcatag tgaagttcat cacattaaaa aagaaaatat aggacttata 1500

gaagtcatgg gacttgcagt tcttcctgca agattaaaag aagaattaaa cattttaaag 1560

gaatacttaa ttaatagaaa agacaacatt ttagatgatg aaatggtggc aaaacattct 1620

gattggtata aatatcttct agaaaaacac tctaatatat ctcaagaaaa cgttgattct 1680

attttaaaag aggaagttgg atttaaattc ttagaagtat tatgtcactc cggagtattt 1740

aaaagaaata aagaaggatt tgctgccttt gataaattta taaatatatt ataacccggg 1800

gatcctctag agtcgacgtc acgcgtccat ggagatctcg aggcctgcag acatgcaagc 1860

ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 1920

aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 1980

gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctagca taaaaataag 2040

aagcctgcat ttgcaggctt cttattttta tggcgcgccg ccattatttt tttgaacaat 2100

tgacaattca tttcttattt tttattaagt gatagtcaaa aggcataaca gtgctgaata 2160

gaaagaaatt tacagaaaag aaaattatag aatttagtat gattaattat actcatttat 2220

gaatgtttaa ttgaatacaa aaaaaaatac ttgttatgta ttcaattacg ggttaaaata 2280

tagacaagtt gaaaaattta ataaaaaaat aagtcctcag ctcttatata ttaagctacc 2340

aacttagtat ataagccaaa acttaaatgt gctaccaaca catcaagccg ttagagaact 2400

ctatctatag caatatttca aatgtaccga catacaagag aaacattaac tatatatatt 2460

caatttatga gattatctta acagatataa atgtaaattg caataagtaa gatttagaag 2520

tttatagcct ttgtgtattg gaagcagtac gcaaaggctt ttttatttga taaaaattag 2580

aagtatattt attttttcat aattaattta tgaaaatgaa agggggtgag caaagtgaca 2640

gaggaaagca gtatcttatc aaataacaag gtattagcaa tatcattatt gactttagca 2700

gtaaacatta tgacttttat agtgcttgta gctaagtagt acgaaagggg gagctttaaa 2760

aagctccttg gaatacatag aattcataaa ttaatttatg aaaagaaggg cgtatatgaa 2820

aacttgtaaa aattgcaaag agtttattaa agatactgaa atatgcaaaa tacattcgtt 2880

gatgattcat gataaaacag tagcaaccta ttgcagtaaa tacaatgagt caagatgttt 2940

acataaaggg aaagtccaat gtattaattg ttcaaagatg aaccgatatg gatggtgtgc 3000

cataaaaatg agatgtttta cagaggaaga acagaaaaaa gaacgtacat gcattaaata 3060

ttatgcaagg agctttaaaa aagctcatgt aaagaagagt aaaaagaaaa aataatttat 3120

ttattaattt aatattgaga gtgccgacac agtatgcact aaaaaatata tctgtggtgt 3180

agtgagccga tacaaaagga tagtcactcg cattttcata atacatctta tgttatgatt 3240

atgtgtcggt gggacttcac gacgaaaacc cacaataaaa aaagagttcg gggtagggtt 3300

aagcatagtt gaggcaacta aacaatcaag ctaggatatg cagtagcaga ccgtaaggtc 3360

gttgtttagg tgtgttgtaa tacatacgct attaagatgt aaaaatacgg ataccaatga 3420

agggaaaagt ataatttttg gatgtagttt gtttgttcat ctatgggcaa actacgtcca 3480

aagccgtttc caaatctgct aaaaagtata tcctttctaa aatcaaagtc aagtatgaaa 3540

tcataaataa agtttaattt tgaagttatt atgatattat gtttttctat taaaataaat 3600

taagtatata gaatagttta ataatagtat atacttaatg tgataagtgt ctgacagtgt 3660

cacagaaagg atgattgtta tggattataa gcggccggcc caatgaatag gtttacactt 3720

actttagttt tatggaaatg aaagatcata tcatatataa tctagaataa aattaactaa 3780

aataattatt atctagataa aaaatttaga agccaatgaa atctataaat aaactaaatt 3840

aagtttattt aattaacaac tatggatata aaataggtac taatcaaaat agtgaggagg 3900

atatatttga atacatacga acaaattaat aaagtgaaaa aaatacttcg gaaacattta 3960

aaaaataacc ttattggtac ttacatgttt ggatcaggag ttgagagtgg actaaaacca 4020

aatagtgatc ttgacttttt agtcgtcgta tctgaaccat tgacagatca aagtaaagaa 4080

atacttatac aaaaaattag acctatttca aagaaaatag gagataaaag caacttacga 4140

tatattgaat taacaattat tattcagcaa gaaatggtac cgtggaatca tcctcccaaa 4200

caagaattta tttatggaga atggttacaa gagctttatg aacaaggata cattcctcag 4260

aaggaattaa attcagattt aaccataatg ctttaccaag caaaacgaaa aaataaaaga 4320

atatacggaa attatgactt agaggaatta ctacctgata ttccattttc tgatgtgaga 4380

agagccatta tggattcgtc agaggaatta atagataatt atcaggatga tgaaaccaac 4440

tctatattaa ctttatgccg tatgatttta actatggaca cgggtaaaat cataccaaaa 4500

gatattgcgg gaaatgcagt ggctgaatct tctccattag aacataggga gagaattttg 4560

ttagcagttc gtagttatct tggagagaat attgaatgga ctaatgaaaa tgtaaattta 4620

actataaact atttaaataa cagattaaaa aaattataaa aaaattgaaa aaatggtgga 4680

aacacttttt tcaatttttt tgttttatta tttaatattt gggaaatatt cattctaatt 4740

ggtaatcaga ttttagaagt ttaaactcct ttttgataat ctcatgacca aaatccctta 4800

acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 4860

agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 4920

ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 4980

cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa 5040

gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 5100

cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 5160

gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 5220

caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 5280

aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 5340

tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 5400

gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 5460

ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 5520

atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 5580

cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 5640

cagggccccc tgcttcgggg tcattatagc gattttttcg gtatatccat cctttttcgc 5700

acgatataca ggattttgcc aaagggttcg tgtagacttt ccttggtgta tccaacggcg 5760

tcagccgggc aggataggtg aagtaggccc acccgcgagc gggtgttcct tcttcactgt 5820

cccttattcg cacctggcgg tgctcaacgg gaatcctgct ctgcgaggct ggccggctac 5880

cgccggcgta acagatgagg gcaagcggat ggctgatgaa accaagccaa ccaggaaggg 5940

cagcccacct atcaaggtgt actgccttcc agacgaacga agagcgattg aggaaaaggc 6000

ggcggcggcc ggcatgagcc tgtcggccta cctgctggcc gtcggccagg gctacaaaat 6060

cacgggcgtc gtggactatg agcacgtccg cgagctggcc cgcatcaatg gcgacctggg 6120

ccgcctgggc ggcctgctga aactctggct caccgacgac ccgcgcacgg cgcggttcgg 6180

tgatgccacg atcctcgccc tgctggcgaa gatcgaagag aagcaggacg agcttggcaa 6240

ggtcatgatg ggcgtggtcc gcccgagggc agagccatga cttttttagc cgctaaaacg 6300

gccggggggt gcgcgtgatt gccaagcacg tccccatgcg ctccatcaag aagagcgact 6360

tcgcggagct ggtgaagtac atcaccgacg agcaaggcaa gaccgatcgg gccc 6414

<210> 6

<211> 499

<212> PRT

<213> Clostridium sp. WK

<400> 6

Met Ile Asn Ile Asn His Glu Ile Asn Arg Leu Ile Asn Phe Ala Leu

1 5 10 15

Lys Lys Asn Leu Ile Asn Asn Asp Asp Ile Ile Tyr Ser Thr Asn Met

20 25 30

Ile Leu Gly Val Leu Asn Leu Asn Glu Phe Glu Val Arg Glu Val Asp

35 40 45

Glu Thr Leu Glu Thr Pro Thr Pro Ile Leu Glu Asn Ile Leu Asp Tyr

50 55 60

Ala Cys Glu Asn Asn Leu Ile Glu Asn Thr Val Thr Glu Arg Asp Leu

65 70 75 80

Phe Asp Thr Leu Ile Met Asn Cys Val Met Pro Arg Pro Ser Glu Val

85 90 95

Ile Asn Asn Phe Asn Asn Leu Tyr Asn Ile Ser Pro Thr Lys Ala Thr

100 105 110

Glu Tyr Tyr Tyr Asp Leu Ser Ile Ser Ser Asn Tyr Ile Arg Lys Asp

115 120 125

Arg Ile Asp Lys Asn Ile Ile Trp Lys Ala Pro Thr Glu Tyr Gly Asp

130 135 140

Leu Asp Ile Thr Ile Asn Leu Ser Lys Pro Glu Lys Asp Pro Arg Asp

145 150 155 160

Ile Ala Lys Ala Lys Leu Ile Lys Ser Ser Ser Tyr Pro Lys Cys Leu

165 170 175

Leu Cys Lys Glu Asn Glu Gly Phe Tyr Gly His Ile Asn His Pro Ala

180 185 190

Arg Gln Thr His Arg Ile Ile Pro Leu Asp Phe Asp Lys Gln Lys Tyr

195 200 205

Phe Leu Gln Tyr Ser Pro Tyr Thr Tyr Tyr Asn Glu His Cys Ile Ile

210 215 220

Leu Asn Ser Glu His Ile Pro Met Lys Ile Asn Lys Asp Thr Phe Arg

225 230 235 240

Asn Leu Leu Ile Phe Thr Asp Ile Leu Pro His Tyr Phe Ala Gly Ser

245 250 255

Asn Ala Asp Leu Pro Ile Val Gly Gly Ser Ile Leu Ser His Asp His

260 265 270

Tyr Gln Gly Gly His Tyr Thr Phe Ala Met Glu Lys Ala Pro Ile Glu

275 280 285

Lys Asn Tyr Ser Val Lys Asn Tyr Glu Asp Val Glu Ile Gly Arg Val

290 295 300

Lys Trp Pro Met Ser Val Val Arg Leu Ser Ser Asn Asp Lys Asp Lys

305 310 315 320

Leu Leu Asp Leu Ala Asp His Ile Leu Thr Ser Trp Arg Ser Tyr Ser

325 330 335

Asp Glu Ser Val Asn Ile Leu Ser His Thr Met Asp Glu Pro His Asn

340 345 350

Thr Ile Thr Pro Ile Ala Arg Lys Arg Asn Asn Lys Tyr Glu Leu Asp

355 360 365

Leu Val Leu Arg Asn Asn Arg Thr Ser Thr Glu His Pro Leu Gly Ile

370 375 380

Phe His Pro His Ser Glu Val His His Ile Lys Lys Glu Asn Ile Gly

385 390 395 400

Leu Ile Glu Val Met Gly Leu Ala Val Leu Pro Ala Arg Leu Lys Glu

405 410 415

Glu Leu Asn Ile Leu Lys Glu Tyr Leu Ile Asn Arg Lys Asp Asn Ile

420 425 430

Leu Asp Asp Glu Met Val Ala Lys His Ser Asp Trp Tyr Lys Tyr Leu

435 440 445

Leu Glu Lys His Ser Asn Ile Ser Gln Glu Asn Val Asp Ser Ile Leu

450 455 460

Lys Glu Glu Val Gly Phe Lys Phe Leu Glu Val Leu Cys His Ser Gly

465 470 475 480

Val Phe Lys Arg Asn Lys Glu Gly Phe Ala Ala Phe Asp Lys Phe Ile

485 490 495

Asn Ile Leu

<210> 7

<211> 1500

<212> DNA

<213> Clostridium sp. WK

<400> 7

atgataaata taaatcatga aataaataga cttataaact ttgcattgaa aaaaaatctt 60

ataaataatg atgatataat ttactctacc aatatgattc ttggagtatt aaacttaaat 120

gaatttgaag ttcgtgaagt tgatgaaact ttagaaactc caactcctat cttagaaaat 180

atcttagact acgcatgtga aaataattta atagaaaata cagtgactga aagagactta 240

tttgatactt taattatgaa ctgcgtaatg ccaagacctt ctgaggttat aaataacttt 300

aataatttat ataatatctc tccaactaaa gctactgagt actattatga tttaagcatt 360

tcttctaact atattcgaaa agatagaatt gacaagaaca taatctggaa agcaccaacg 420

gaatatggag acttagatat aacaattaat ttatcaaaac cagaaaaaga tcctagggat 480

atagcaaaag ctaaattaat taaatctagt tcatatccta aatgtttatt atgtaaagaa 540

aatgaaggat tttatggtca tataaatcat cctgccagac agactcatag aattatacct 600

ttggattttg ataaacaaaa atatttcttg cagtattccc cttatactta ttataatgag 660

cattgtataa ttctaaatag cgaacatata cctatgaaaa ttaataagga tacttttagg 720

aatttattga tttttactga tatactgcct cattactttg ctggatctaa cgctgacttg 780

cctatagttg gaggatcaat actatctcat gatcattatc aaggtggtca ctacacattt 840

gctatggaaa aagctccaat agaaaaaaac tactcagtta aaaattatga agatgttgaa 900

ataggtagag tcaaatggcc tatgtctgtt gttaggcttt caagtaatga taaagataaa 960

ttattagatt tagctgatca tattttaact tcatggagaa gttattctga tgaatctgta 1020

aatatattaa gtcatacaat ggatgaaccg cataatacta ttactcctat tgcaagaaaa 1080

aggaataata agtacgaact agatttagtc ttaaggaaca atagaacaag tactgagcac 1140

cctcttggta ttttccatcc tcatagtgaa gttcatcaca ttaaaaaaga aaatatagga 1200

cttatagaag tcatgggact tgcagttctt cctgcaagat taaaagaaga attaaacatt 1260

ttaaaggaat acttaattaa tagaaaagac aacattttag atgatgaaat ggtggcaaaa 1320

cattctgatt ggtataaata tcttctagaa aaacactcta atatatctca agaaaacgtt 1380

gattctattt taaaagagga agttggattt aaattcttag aagtattatg tcactccgga 1440

gtatttaaaa gaaataaaga aggatttgct gcctttgata aatttataaa tatattataa 1500

<210> 8

<211> 16

<212> DNA

<213> artificial sequence

<400> 8

tagtagcctg tgaaat 16

<210> 9

<211> 16

<212> DNA

<213> artificial sequence

<400> 9

tagtattgat cctcca 16

<210> 10

<211> 18

<212> DNA

<213> artificial sequence

<400> 10

taccgatgga tgtgatag 18

<210> 11

<211> 17

<212> DNA

<213> artificial sequence

<400> 11

attccaggag tggttgt 17

<210> 12

<211> 20

<212> DNA

<213> artificial sequence

<400> 12

ctattgctat ggatggctac 20

<210> 13

<211> 18

<212> DNA

<213> artificial sequence

<400> 13

tcagctaagg cagtttca 18

<210> 14

<211> 19

<212> DNA

<213> artificial sequence

<400> 14

ctttgctgga tctaacgct 19

<210> 15

<211> 21

<212> DNA

<213> artificial sequence

<400> 15

aatgtgtagt gaccaccttg a 21

<210> 16

<211> 19

<212> DNA

<213> artificial sequence

<400> 16

tagacttagc agacgcaca 19

<210> 17

<211> 19

<212> DNA

<213> artificial sequence

<400> 17

agccaattcc attacctag 19

<210> 18

<211> 22

<212> DNA

<213> artificial sequence

<400> 18

gggcagaagt agtattagga ga 22

<210> 19

<211> 19

<212> DNA

<213> artificial sequence

<400> 19

aacagcatca acacctcgt 19

<210> 20

<211> 19

<212> DNA

<213> artificial sequence

<400> 20

tcaatgccag attcaacgg 19

<210> 21

<211> 20

<212> DNA

<213> artificial sequence

<400> 21

cagcaactaa agcccaacca 20

<210> 22

<211> 19

<212> DNA

<213> artificial sequence

<400> 22

atggcctcag ttccaatta 19

<210> 23

<211> 19

<212> DNA

<213> artificial sequence

<400> 23

accgaagaag caccaagta 19

<210> 24

<211> 20

<212> DNA

<213> artificial sequence

<400> 24

ccccttaacc ttagaaactc 20

<210> 25

<211> 18

<212> DNA

<213> artificial sequence

<400> 25

gcaggcagta gaaccaga 18

<210> 26

<211> 18

<212> DNA

<213> artificial sequence

<400> 26

actaaggctc ctattgac 18

<210> 27

<211> 17

<212> DNA

<213> artificial sequence

<400> 27

actttcatct ttggctc 17

<210> 28

<211> 20

<212> DNA

<213> artificial sequence

<400> 28

agagtttgat cctggctcag 20

<210> 29

<211> 22

<212> DNA

<213> artificial sequence

<400> 29

tacggctacc ttgttacgac tt 22

Claims

Use of udp-glucose-hexose-1-phosphate uridyltransferase as sole active ingredient in at least one of (a 1) to (a 8);

(a1) The carbon source utilization rate of clostridium is improved;

(a2) Promote the generation of butanol in clostridium;

(a3) Promote the generation of acetone in clostridium;

(a4) Preparing a product that promotes the growth of clostridium;

(a5) Promoting the growth of clostridium;

(a6) Preparing a product for improving the carbon source utilization rate of clostridium;

(a7) Preparing a product that promotes the production of butanol in clostridium;

(a8) Preparing a product that promotes the production of acetone in clostridium;

the Clostridium is Clostridium sp.wk, theClostridium sp, WK accession number is GDMCC No. 61493;

the carbon source is galactose;

the amino acid sequence of the UDP-glucose-hexose-1-phosphate uridyltransferase is shown as SEQ ID NO. 6.
2. Use of biological material related to UDP-glucose-hexose-1-phosphate uridyltransferase as sole active ingredient in at least one of (a 1) to (a 8);

(a1) The carbon source utilization rate of clostridium is improved;

(a2) Promote the generation of butanol in clostridium;

(a3) Promote the generation of acetone in clostridium;

(a4) Preparing a product that promotes the growth of clostridium;

(a5) Promoting the growth of clostridium;

(a6) Preparing a product for improving the carbon source utilization rate of clostridium;

(a7) Preparing a product that promotes the production of butanol in clostridium;

(a8) Preparing a product that promotes the production of acetone in clostridium;

the biological material related to UDP-glucose-hexose-1-phosphate uridyltransferase comprises at least one of b 1) to b 12):

b1 A nucleic acid molecule encoding a UDP-glucose-hexose-1-phosphate uridyltransferase;

b2 An expression cassette comprising b 1) said nucleic acid molecule;

b3 A recombinant vector comprising the nucleic acid molecule of b 1);

b4 A recombinant vector comprising the expression cassette of b 2);

b5 A recombinant cell comprising the nucleic acid molecule of b 1);

b6 A recombinant cell comprising the expression cassette of b 2);

b7 A recombinant cell comprising the recombinant vector of b 3);

b8 A recombinant cell comprising the recombinant vector of b 4);

b9 A recombinant microorganism comprising the nucleic acid molecule of b 1);

b10 A recombinant microorganism comprising the expression cassette of b 2);

b11 A recombinant microorganism comprising the recombinant vector of b 3);

b12 A recombinant microorganism comprising the recombinant vector of b 4);

the Clostridium is Clostridium sp.wk, theClostridium sp, WK accession number is GDMCC No. 61493;

the carbon source is galactose;

the amino acid sequence of the UDP-glucose-hexose-1-phosphate uridyltransferase is shown as SEQ ID NO. 6.
3. The construction method of the recombinant bacteria comprises the following steps: increasing the content of UDP-glucose-hexose-1-phosphate uridyltransferase in clostridium; the Clostridium is Clostridium sp.wk, theClostridium sp, WK accession number is GDMCC No. 61493;

the amino acid sequence of the UDP-glucose-hexose-1-phosphate uridyltransferase is shown as SEQ ID NO. 6;

among them, UDP-glucose-hexose-1-phosphate uridyltransferase is the only substance in the clostridium for increasing the content.
4. A method of construction according to claim 3, wherein: the method for increasing the content of UDP-glucose-hexose-1-phosphate uridyltransferase in clostridium is to overexpress the UDP-glucose-hexose-1-phosphate uridyltransferase in clostridium.
5. A method of construction according to claim 3, wherein:

the method for improving the content of UDP-glucose-hexose-1-phosphate uridyltransferase in clostridium is to introduce the coding gene of UDP-glucose-hexose-1-phosphate uridyltransferase into clostridium.
6. The construction method according to claim 5, wherein:

the coding gene of UDP-glucose-hexose-1-phosphate uridyltransferase is introduced into clostridium through a recombinant vector; the recombinant vector is obtained by inserting the coding gene of the UDP-glucose-hexose-1-phosphate uridyltransferase into a multiple cloning site of an expression vector.
7. A recombinant bacterium obtainable by the construction method of any one of claims 3 to 6.
8. Use of the recombinant bacterium of claim 7 in at least one of e 1) to e 2);

e1 Butanol is prepared;

e2 Acetone is prepared.
9. A method comprising inoculating the recombinant bacterium of claim 7 to a fermentation medium, and fermenting; the method is used for at least one of e 1) to e 2):

e1 Butanol is prepared;

e2 Acetone is prepared.