阅读说明：本技术 利用木糖生产丁醇的工程菌株及其构建方法和应用 (Engineering strain for producing butanol by using xylose and construction method and application thereof ) 是由 赵春华 张延平 李寅 珍保罗西努瓦 张天瑞 董红军 于 2019-04-15 设计创作，主要内容包括：本发明公开了利用木糖生产丁醇的工程菌株及其构建方法和应用。本发明提供了一种构建重组菌的方法,包括如下步骤：提高生产丁醇的出发菌的基因组中xylE、xylFGH、xylA、xylB、rpe、tktA、rpiA、talB的表达和/或活性,且抑制所述出发菌中的glk基因表达得到的菌。本发明的实验证明了本发明构建的重组菌可以利用木糖为碳源生产丁醇,将其发酵生物质,可以提高生物质的利用率,实现有效利用廉价生物质(多为木质纤维素)或其水解液。(The invention discloses an engineering strain for producing butanol by using xylose and a construction method and application thereof. The invention provides a method for constructing a recombinant bacterium, which comprises the following steps: the expression and/or activity of xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB in the genome of the producing bacteria for producing butanol is improved, and the obtained bacteria can inhibit the expression of glk genes in the producing bacteria. Experiments prove that the recombinant strain constructed by the invention can utilize xylose as a carbon source to produce butanol, ferment the butanol with the xylose to improve the utilization rate of biomass, and realize effective utilization of cheap biomass (mostly lignocellulose) or hydrolysate thereof.)

1. A method for constructing a recombinant bacterium comprises the following steps: improving the expression and/or activity of at least one of genes xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB related to xylose transport and metabolism in the producing bacteria for producing butanol, and inhibiting the bacteria obtained by the expression of glk genes in the producing bacteria.

2. The method of claim 1, wherein:

the improvement of the expression and/or activity of at least one of genes xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB related to the transportation and metabolism of xylose in the butanol-producing bacteria is to improve the expression and/or activity of genes xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB related to the transportation and metabolism of xylose in the butanol-producing bacteria integrated on the genome.

3. The method of claim 2, wherein:

the improvement of the expression and/or activity of genes xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB related to xylose transport and metabolism in the butanol-producing outbreak is to increase the copy number of xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB in the genome of the outbreak.

4. The method of claim 3, wherein:

the copy number of xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB in the genome of the outbreak is increased, wherein the homologous fragment containing the xylE gene and the promoter thereof is replaced by paaI gene in the outbreak, the homologous fragment containing the xylFGH gene and the promoter thereof is replaced by ykgF gene in the outbreak, the homologous fragment containing the xylA, xylB gene and the promoter thereof is replaced by lldD gene in the outbreak, the homologous fragment containing the fusion gene formed by rpe and tktA and the promoter thereof is replaced by crr gene in the outbreak, and the homologous fragment containing the fusion formed by rpiA and talB and the promoter thereof are replaced by ptsG gene in the outbreak;

or, the inhibition of the expression of the glk gene in the outbreak is a knock-out or inhibition of the glk gene in the outbreak.

5. The method of claim 4, wherein:

the promoters of all the genes are constitutive promoters;

or the constitutive promoter is specifically a minipTac promoter, and the nucleotide sequence of the constitutive promoter is a sequence 12 in the sequence table.

6. The method according to claim 4 or 5, wherein:

the knockout or the replacement is carried out by adopting a mode of genome site-directed editing and/or homologous recombination;

or the genome site-directed editing is specifically ZFN editing, TALEN editing or CRISPR/Cas9 editing.

7. The method according to any one of claims 1 to 6, wherein: the outbreak bacteria for producing the butanol are escherichia coli which uses glucose as a carbon source to produce the butanol.

8. A recombinant bacterium produced by the method according to any one of claims 1 to 7.

9. The recombinant bacterium according to claim 8, wherein: the preservation number of the recombinant strain is CGMCC No. 17141.

10. Use of the recombinant bacterium of claim 8 or 9 for the production of butanol using xylose as a carbon source;

or, a microbial agent comprising the recombinant bacterium of claim 8 or 9 and Escherichia coli for producing butanol using glucose as a carbon source;

or, the use of the microbial inoculum in the production of butanol by using xylose and glucose;

or the application of the microbial inoculum in producing butanol by fermenting biomass or hydrolysate thereof;

or, a method for producing butanol, comprising the steps of: fermenting the recombinant bacteria or the microbial inoculum by taking xylose as a carbon source;

or, a method for producing butanol, comprising the steps of: fermenting the microbial inoculum by taking xylose and glucose as carbon sources;

or, a method for producing butanol, comprising the steps of: and fermenting the microbial inoculum by using biomass or hydrolysate thereof as a carbon source.

Technical Field

The invention relates to the technical field of biology, in particular to an engineering strain for producing butanol by using xylose and a construction method and application thereof.

Background

With the forward development of global economy, a sustainable green economy model has become a consensus of human society. Industries that rely on non-renewable resources such as petroleum are gradually shifting to green industries that rely on biomass. Cell factories are important ways of producing chemicals or fuels from biomass. At present, alcohols such as ethanol and acids such as succinic acid are produced in cell factories.

Butanol is a more potent fuel than ethanol. It has high heat value and strong hydrophobicity, can be mutually dissolved with gasoline in any proportion, and is an excellent biofuel capable of replacing gasoline. In addition, butanol is also an important chemical product and raw material, can be directly used as an organic solvent, is a precursor for synthesizing various ester compounds, and is widely applied to various plastic and rubber products.

Naturally occurring in nature is the Clostridium acetobutylicum (Clostridium acetobutylicum) naturally producing butanol, a strain capable of converting starch into acetone, butanol and ethanol. Since clostridium acetobutylicum is a gram-positive bacterium which strictly grows anaerobically, the genetic operation system is complex, and the clostridium acetobutylicum is not beneficial to the research and industrial production in laboratories. Therefore, in recent years researchers have turned their attention to the model microorganism Escherichia coli (E.coli). Coli have been studied for producing butanol using glucose as a substrate, but no strain capable of efficiently utilizing xylose has been reported. In order to produce butanol by efficiently using inexpensive biomass (mostly lignocellulose) or hydrolysate thereof, it is necessary to construct a strain capable of efficiently producing butanol by using xylose, particularly a strain capable of efficiently using xylose even in the presence of a plurality of sugars.

Disclosure of Invention

In order to construct a strain capable of efficiently utilizing xylose to produce butanol, the invention provides the following technical scheme:

one purpose of the invention is to provide a method for constructing a recombinant bacterium, which comprises the following steps: improvement ofXylose transport and metabolism-related gene xylE (xylose: Hydrogen ion cotransporter Gene, xylose: H) in butanol-producing outbreak bacteria⁺symportor), xylFGH (xylose transporter gene, xylose ABC transporter), xylA (xylose isomerase gene, xylB (xylulokinase gene, xylulokinase), rpe (ribulose phosphate epimerase gene, ribulose-phosphate epimerase), tktA (transketolase gene, transketolase), rpiA (5-phosphoribose isomerase gene, ribose-5-phosphate isomerose), talB (aldolase gene, transaldolase), and inhibiting the expression of glk gene (hexokinase gene, glucokinase).

In the above method, in one embodiment of the present invention, the increasing the expression and/or activity of at least one of genes xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA, talB related to xylose transport and metabolism in the butanol-producing bacteria is to increase the expression and/or activity of genes xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA, and talB related to xylose transport and metabolism in the butanol-producing bacteria integrated on the genome.

In the above method, the increasing of the expression and/or activity of the genes xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB related to xylose transport and metabolism in the outgrowth bacteria for producing butanol is to increase the copy number of xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB in the genome of the outgrowth bacteria.

In the method, the copy number of xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB in the genome of the haired bacteria is increased, wherein a homologous fragment containing the xylE gene and a promoter thereof is replaced by a paaI gene in the haired bacteria, a homologous fragment containing the xylFGH gene and a promoter thereof is replaced by a ykgF gene in the haired bacteria, a homologous fragment containing a fusion gene formed by xylA and xylB and a promoter thereof is replaced by an lldD gene in the haired bacteria, a fusion gene formed by rpe and tktA and a homologous fragment containing a promoter thereof are replaced by a crr gene in the haired bacteria, and a homologous fragment containing a fusion gene formed by rpiA and talB and a promoter thereof are replaced by a ptsG gene in the haired bacteria;

or, the inhibition of the expression of the glk gene in the outbreak is a knock-out or inhibition of the glk gene in the outbreak.

The knockout or the replacement is carried out by adopting a mode of genome site-directed editing and/or homologous recombination;

or the genome site-directed editing is specifically ZFN editing, TALEN editing or CRISPR/Cas9 editing.

In the method, the nucleotide sequence of the homologous fragment containing the xylE gene and the promoter thereof is a sequence 15 in a sequence table;

the nucleotide sequence of the homologous fragment containing the xylFGH gene and the promoter thereof is a sequence 18 in a sequence table;

the nucleotide sequence of the fusion gene formed by xylA and xylB and the homologous fragment of the promoter thereof is a sequence 21 in a sequence table;

the nucleotide sequence of the homologous fragment containing the fusion gene formed by rpe and tktA and the promoter thereof is a sequence 24 in a sequence table;

the nucleotide sequence of the homologous fragment containing the fusion gene formed by the rpiA and the talB and the promoter thereof is a sequence 27 in a sequence table.

In the above method, the promoters of the genes are all constitutive promoters in the embodiment of the present invention; the constitutive promoter is specifically a miniPtac promoter in the embodiment of the invention, and the nucleotide sequence of the constitutive promoter is a sequence 12 in a sequence table.

In the method, the expression of the glk gene in the outbreak is knocked out or suppressed.

In the method, the knockout or the replacement is carried out by adopting a genome site-directed editing and/or homologous recombination mode;

or the genome site-directed editing is specifically ZFN editing, TALEN editing or CRISPR/Cas9 editing.

The above substitution is carried out by introducing a plasmid expressing sgRNA of the gene to be substituted, a homologous recombination fragment, and a pCas plasmid into the host cell.

The plasmid expressing sgRNA of the replaced gene is specifically as follows:

a pTargetF-ptsG plasmid which contains a coding gene of ptsG sgRNA and expresses ptsG sgRNA (sequence 9), wherein the target sequence of the ptsG sgRNA is sequence 10;

a pTargetF-paaI plasmid which contains a coding gene of paaI sgRNA and expresses paaI sgRNA (sequence 13), wherein the target sequence of the paaI sgRNA is sequence 14; a plasmid expressing the sgRNA of the replaced gene;

a pTargetF-ykgF plasmid containing a gene encoding ykgF sgRNA, expressing ykgF sgRNA (SEQ ID NO: 16) whose target sequence is SEQ ID NO: 17;

a pTargetF-lldD plasmid which contains a coding gene of lldD sgRNA and expresses the lldD sgRNA (sequence 19), and the target sequence of the lldD sgRNA is sequence 20;

pTargetF-crr plasmid, which contains coding gene of crr sgRNA and expresses crr sgRNA (sequence 22), wherein the target sequence of the crr sgRNA is sequence 23;

pTargetF- Δ ptsG plasmid containing a gene encoding ptsG sgRNA, which expresses ptsG sgRNA (SEQ ID NO: 25) whose target sequence is SEQ ID NO: 26.

The glk gene in the above knockout or suppression of the gene glk in the starting bacteria is introduced into the starting bacteria as a plasmid expressing sgRNA of the replaced gene glk, a glk homologous recombination fragment, and a pCas plasmid.

And (3) a plasmid pTargetF-glk for expressing the sgRNA of the replaced gene glk, wherein the plasmid contains a coding gene of the glk sgRNA, and the target sequence of the glk sgRNA is sequence 29.

The nucleotide sequence of the glk homologous recombination fragment is sequence 30.

In the method, the fermentation bacteria for producing butanol is escherichia coli which uses glucose as a carbon source to produce butanol, and in the embodiment of the invention, the escherichia coli which uses glucose as a carbon source to produce butanol is specifically escherichia coli EB243CGMCCNo.12191.

The recombinant bacteria prepared by the method are also within the protection scope of the invention.

The preservation number of the recombinant strain is CGMCC No. 17141.

The application of the recombinant bacterium in the production of butanol by taking xylose as a carbon source is also within the protection scope of the invention;

the invention also provides a microbial inoculum which comprises the recombinant strain and escherichia coli (in the embodiment of the invention, specifically escherichia coli EB243CGMCC No.12191) for producing butanol by taking glucose as a carbon source;

or, the application of the microbial inoculum in the production of butanol by utilizing xylose and glucose is also the protection scope of the invention;

or, the application of the microbial inoculum in the production of butanol by fermenting biomass or hydrolysate thereof is also within the protection scope of the invention;

alternatively, the present invention also provides a method for producing butanol, comprising the steps of: fermenting the recombinant bacteria or the microbial inoculum by taking xylose as a carbon source;

alternatively, the present invention also provides a method for producing butanol, comprising the steps of: fermenting the microbial inoculum by taking xylose and glucose as carbon sources;

alternatively, the present invention also provides a method for producing butanol, comprising the steps of: and fermenting the microbial inoculum by using biomass or hydrolysate thereof as a carbon source.

Escherichia coli EB243TAM-X has been deposited in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of Beijing, West Lu No.1 of the sunward region, Ministry of microbiology, Japan, postal code 100101) in 2019, at 1 month and 8 days, and has a collection number of CGMCC No.17141, and is classified and named as Escherichia coli.

Experiments prove that the strain EB243TAM-X is obtained by introducing various genes including xylE, xylFGH, xylA, xylB, rpe, tktA, rpiA and talB containing constitutive promoters into a starter for producing butanol and knocking out glk genes in the starter. The strain can utilize xylose as a carbon source to produce butanol, ferment the butanol to biomass, improve the utilization rate of the biomass, and realize effective utilization of cheap biomass (mostly lignocellulose) or hydrolysate thereof.

Drawings

FIG. 1 shows xylose metabolism pathways in E.coli.

FIG. 2 is a graph showing the comparison of xylose utilization by fermentation using strains EB 243. delta. ptsG, EB243T, EB243TA and EB243 TAM.

FIG. 3 is a diagram showing the process of fermenting mixed sugars (glucose + xylose) by strain EB243 TAM.

FIG. 4 is a diagram showing the process of fermenting mixed sugars by strain EB243 TAM-X.

FIG. 5 is a graph of butanol yield of mixed sugars fermented by strains EB243 and EB243 TAM-X.

FIG. 6 is a graph showing the process of inoculating different proportions of EB243 and EB243TAM-X strains into small tubes for fermentation of mixed sugar and the yield of butanol.

FIG. 7 is a diagram showing the fermentation process of mixed sugar in a fermentation tank after strains EB243 and EB243TAM-X are inoculated in different proportions.

FIG. 8 is a graph showing the fermentation process of mixed sugars in a fermentor after inoculation of strains EB243 and EB243TAM-X at a ratio of 2:1 (OD 600 value).

FIG. 9 is a diagram of the process of fermentation of mixed sugars in separate fermenters for strains EB243 and EB243 TAM-X.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following formula of M9 culture medium is: 17.1g/l Na₂HPO₄·12H₂O、3.0g/l KH₂PO₄、0.5g/lNaCl、1.0g/l NH₄Cl, 0.5mg/l vitamin B1, 20g/l C₅H₁₀O₅、2mM MgSO₄·7H₂O、0.1mM CaCl₂And water, and glucose, xylose or mixed sugar can be added into the culture medium as carbon source for fermentation.

The glucose-containing M9 medium was obtained by adding glucose at a certain concentration to M9 medium as a carbon source.

The xylose-containing M9 medium is obtained by adding xylose with a certain concentration as a carbon source to M9 medium.

The M9 culture medium containing glucose and xylose is obtained by adding xylose with a certain concentration and glucose with a certain concentration as carbon sources (the mass ratio of glucose to xylose is 1:1) into M9 culture medium.

The starting bacterium Escherichia coli EB243 has been deposited in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Lu 1 of the national institute of sciences, Japan, and postal code 100101) at 2016, 3, 9, 3, and 9 days, with the preservation number of CGMCC No.12191, and is classified and named as Escherichia coli.

The host bacterium Escherichia coli EB243 is EB216, which knocks out pykA gene on the genome and improves the expression and/or activity of fdh gene on the genome; then domesticating the target bacterium A in an M9 culture medium with an insufficient nitrogen source to obtain a target bacterium B; inhibiting the expression and/or activity of genes yieP, stpA, yqeG and yagM on the target bacterium B genome, and improving the expression and/or activity of the ter gene and the crt gene in the target bacterium B to obtain the recombinant bacterium.

The host bacterium Escherichia coli EB243 is a strain which utilizes glucose as a carbon source to produce butanol through fermentation.