Genetically engineered bacteria for preparing ethanol from lignocellulosic sugar hydrolysis and its application

Abstract

The present invention belongs to the field of microbial technology, and relates to a genetically engineered bacterium for preparing ethanol based on hydrolyzed sugar from wood fiber and its application. The bacterium is a high xylose utilization and inhibitor tolerance engineered bacterium. The engineered bacterium is based on Saccharomyces cerevisiae S288C as a chassis strain, and co-expresses heterologous xylose reductase-xylitol dehydrogenase pathway-related genes, xylose transport-related genes, xylose isomerase-related genes and xylose isomerase genes, and over-expresses endogenous xylulose kinase-related genes, non-oxidative pentose phosphate pathway-related genes, reduced glutathione system genes, superoxide dismutase expression system-related genes, NADPH / NADP + The genes and transcription factors related to the cofactor circulation system were knocked out, the genes related to the competitive metabolic pathway were knocked out, and the bacteria were domesticated with sugar sources and inhibitors as pressure; the engineered bacteria can efficiently metabolize xylose and produce high-concentration ethanol in non-detoxified lignocellulose hydrolysate with high inhibitor concentrations and without any nutrient salt addition.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to genetically engineered bacteria for preparing ethanol based on hydrolyzing sugar from wood fiber and applications thereof. Background Art

[0002] Second-generation bioethanol production utilizes lignocellulose as a feedstock, converting it into ethanol through bioconversion technology. Lignocellulose is produced from plant fiber resources such as discarded agricultural straw, forestry sawdust, and branches. These waste resources are plentiful, readily available, and do not compete with food resources, making them more environmentally friendly and sustainable. Through effective pretreatment technologies and bioethanol production and utilization, this technology will significantly contribute to reducing fossil energy consumption, improving energy security, and promoting the development of a low-carbon economy.

[0003] Selecting the right bacterial strain is crucial for the production of second-generation bioethanol. Saccharomyces cerevisiae, with its clear genetic background, mature genetic manipulation, and good environmental adaptability, has become a key cell factory for the second-generation bioethanol industry. However, using Saccharomyces cerevisiae to produce second-generation bioethanol still faces several bottlenecks. For one thing, xylose, the second-most abundant sugar in lignocellulose, is difficult for Saccharomyces cerevisiae to metabolize. Currently, two main strategies for introducing heterologous pathways are being used to engineer Saccharomyces cerevisiae strains to efficiently utilize xylose for bioethanol production: heterologous introduction of the xylose isomerase pathway and heterologous expression of the xylose reductase-xylitol dehydrogenase pathway. Furthermore, the presence of inhibitory chemicals in lignocellulosic hydrolysates, such as weak acids, furans, and phenolic compounds, is a major obstacle to the production of second-generation bioethanol. Existing research has proven difficult to construct ethanol fermentation strains that simultaneously demonstrate high xylose utilization and, in particular, high inhibitor tolerance. This severely limits their application in producing high-concentration second-generation bioethanol from lignocellulosic hydrolysates containing high inhibitor concentrations.

[0004] Therefore, there is an urgent need to research and develop an engineered Saccharomyces cerevisiae strain with high xylose utilization and high inhibitor tolerance. Summary of the Invention

[0005] The purpose of the present invention is to provide a genetically engineered bacterium for preparing ethanol based on lignocellulosic hydrolyzed sugars based on the problems existing in the prior art. The genetically engineered bacterium has high xylose utilization and high inhibitor tolerance. The genetically engineered bacterium is used to prepare ethanol from lignocellulosic hydrolyzed sugars, has high xylose assimilation efficiency, high robustness, high ethanol conversion rate, and is easy to industrialize.

[0006] To this end, the present invention provides a genetically engineered bacterium for preparing ethanol based on the hydrolysis of wood fiber sugars, which is a recombinant Saccharomyces cerevisiae that co-expresses heterologous xylose reductase-xylitol dehydrogenase pathway-related genes, xylose transport-related genes, and xylose isomerase-related genes.

[0007] In some embodiments of the present invention, the xylose reductase-xylitol dehydrogenase pathway-related genes include genes encoding xylose reductase from Pichia stipitis XYL1 or a codon-optimized gene encoding xylose reductase from Pichia stipitis XYL1 , a gene encoding the xylose reductase mutant R276H from Pichia stipitis mXYL1 or a codon-optimized gene encoding the xylose reductase mutant R276H from Pichia stipitis mXYL1 and a gene encoding xylitol dehydrogenase from Pichia stipitis XYL2 or a codon-optimized gene encoding xylitol dehydrogenase from Pichia stipitis XYL2 One or more of the .

[0008] In some embodiments of the present invention, the xylose transport-related gene includes a gene encoding a high-affinity xylose transporter from Pichia stipitis XUT4 and / or genes XUT6 , or a codon-optimized gene encoding a high-affinity xylose transporter from Pichia stipitis XUT4 and / or genes XUT6 .

[0009] In some embodiments of the present invention, the xylose isomerase-related gene includes a gene encoding xylose isomerase from Clostridium plant fermentans. xylA or a codon-optimized gene encoding xylose isomerase from Clostridium plantarum xylA .

[0010] According to some embodiments of the present invention, the genetically engineered bacteria also overexpress endogenous xylulose kinase-related genes, non-oxidative pentose phosphate pathway-related genes, reduced glutathione system genes, superoxide dismutase expression system-related genes, NADPH / NADP + One or more genes and transcription factors related to the cofactor circulation system.

[0011] In some embodiments of the present invention, the xylulokinase-related gene includes a gene encoding xylulokinase XKS1 .

[0012] In some embodiments of the present invention, the non-oxidative pentose phosphate pathway-related genes include genes encoding transketolase TKL1 , Gene encoding glucose-7-phosphate:D-glyceraldehyde-3-phosphate transaldolase TAL1 , the gene encoding 5-phospho-ribose isomerase RKI1and the gene encoding ribulose 3-phosphate epimerase RPE1 One or more of the .

[0013] In some embodiments of the present invention, the reduced glutathione system-related genes include genes encoding glutathione-disulfide reductase GLR1 , genes encoding glutamate-cysteine ​​ligase GSH1 , genes encoding glutathione synthetase GSH2 and the gene encoding superoxide dismutase PRX1 One or more of the .

[0014] In some embodiments of the present invention, the superoxide dismutase expression system-related genes include genes encoding superoxide dismutase SOD1 , genes encoding peroxidase A CTA1 and the gene encoding catalase T CTT1 One or more of the .

[0015] In some embodiments of the invention, the NADPH / NADP + Cofactor recycling system-related genes include the gene encoding 6-phosphate glucose-1-dehydrogenase ZWF1 , gene encoding 6-phosphogluconate dehydrogenase GND1 and / or genes GND2 , genes encoding 2-glycerol dehydrogenase GCY1 , the gene encoding isocitrate dehydrogenase IDP1 and / or genes IDP2 and / or genes IDP3 One or more of the .

[0016] In some embodiments of the present invention, the transcription factor comprises a DNA-binding transcription factor YAP1 , drug response transcription factors PDR3 , stress response transcription activator MSN2 and stress response transcription activator MSN4 One or more of the .

[0017] According to some embodiments of the present invention, the genetically engineered bacteria have also undergone chassis modification, which includes knocking out genes related to the competitive metabolic pathway of the chassis microorganisms.

[0018] In some embodiments of the present invention, the competitive metabolic pathway-related genes of the chassis microorganisms include genes encoding aldose reductase GRE3 , genes encoding alkaline phosphatase PHO13 , Gene encoding 3-phosphate-glycerol dehydrogenase GPD1 and the gene encoding potassium-activated aldehyde dehydrogenase ALD4 One or more of the .

[0019] According to some embodiments of the present invention, the genetically engineered bacteria have also undergone sugar source adaptation and / or inhibitor pressure adaptation domestication.

[0020] In some embodiments of the present invention, the sugar source includes xylose or xylose and glucose.

[0021] In the present invention, the inhibitors include one or more of phenolic monomers, lignin oligomers, organic acids and furan compounds.

[0022] In some embodiments of the present invention, the phenolic monomer includes one or more of a syringylphenol monomer, a guaiacylphenol monomer, and a p-hydroxyphenylphenol monomer.

[0023] In some embodiments of the present invention, the lignin oligomers include: one or more of: syringyl lignin oligomers, guaiac lignin oligomers, p-coumaric acid lignin oligomers, and syringyl-guaiac p-coumaric acid mixed lignin oligomers.

[0024] In some embodiments of the present invention, the organic acid includes one or more of formic acid, oxalic acid, acetic acid and butyric acid.

[0025] In some embodiments of the present invention, the furanic compound includes furfural and / or 5-hydroxymethylfurfural.

[0026] The present invention also provides the use of the genetically engineered bacteria in the preparation of ethanol based on the hydrolysis of sugar raw materials from wood fibers.

[0027] In some embodiments of the present invention, the application includes inoculating genetically engineered bacteria into a fermentation medium, and fermenting and culturing non-detoxified wood fiber hydrolyzed sugar raw materials as substrates to produce ethanol.

[0028] In the present invention, the lignocellulosic sugar raw material is a non-detoxified lignocellulosic enzymatic hydrolysate; preferably, the non-detoxified lignocellulosic enzymatic hydrolysate has a solid content of 20%-40% w / v.

[0029] The inventors used Saccharomyces cerevisiae S288C as a base microbial cell and heterologously introduced genes encoding xylose reductase-xylitol dehydrogenase, xylose isomerase, and xylose transport. Genes related to the pentose phosphate pathway, reduced glutathione system, and superoxide dismutase system were also enhanced. Regulatory transcriptional activators were optimized, and genes related to competing metabolic pathways were weakened. This resulted in the modification of the Saccharomyces cerevisiae base microbial cell by laboratory adaptive evolution using sugar sources (e.g., C5 / C6) and inhibitors as stressors. Through combined optimization of metabolic engineering and laboratory adaptive evolution strategies, the engineered strain YLE702 was constructed and obtained, demonstrating high xylose utilization and high inhibitor tolerance. Compared to other xylose-engineered strains, the engineered strain YLE702 possesses the ability to efficiently metabolize xylose and produce ethanol from lignocellulosic hydrolysate. This strain can efficiently metabolize xylose and produce high-concentration ethanol in non-detoxified lignocellulosic hydrolysate containing high inhibitor concentrations and without the addition of any nutrients, making it readily adaptable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the present invention easier to understand, the present invention will be described in detail below with reference to the accompanying drawings.

[0031] Figure 1 The diagram shows the metabolic pathway for producing ethanol from lignocellulose using the genetically engineered bacteria constructed in the present invention.

[0032] Figure 2 The fermentation performance of the strains after acclimation and screening and the strains before screening in producing ethanol in 40 g / L xylose synthetic medium, glucose and xylose mixed sugar medium and undetoxified steam explosion corn straw hydrolysate is shown; where A is the growth rate (OD) of the strains after acclimation and screening and the strains before screening in 40 g / L xylose synthetic medium. 600) changes with fermentation time; B is the graph showing the changes in xylose concentration in 40 g / L xylose synthetic medium of the strain after acclimation and screening and the strain before screening; C is the graph showing the changes in xylose concentration in 40 g / L xylose synthetic medium of the strain after acclimation and screening and the strain before screening g / L xylose synthetic medium; D is a graph showing changes in glucose concentration over fermentation time in a mixed sugar medium of glucose and xylose for the strains that have been acclimated and screened and the strains before screening; E is a graph showing changes in xylose concentration over fermentation time in a mixed sugar medium of glucose and xylose for the strains that have been acclimated and screened and the strains before screening; F is a graph showing ethanol production in a mixed sugar medium of glucose and xylose for the strains that have been acclimated and screened and the strains before screening; G is a graph showing changes in glucose concentration over fermentation time in a non-detoxified steam-exploded corn straw hydrolysate for the strains that have been acclimated and screened and the strains before screening; H is a graph showing changes in xylose concentration over fermentation time in a non-detoxified steam-exploded corn straw hydrolysate for the strains that have been acclimated and screened and the strains before screening; I is a graph showing ethanol production in a non-detoxified steam-exploded corn straw hydrolysate for the strains that have been acclimated and screened and the strains before screening.

[0033] Figure 3 The ethanol fermentation performance of the engineered strain overexpressing key genes of the glutathione system and the superoxide dismutase system using undetoxified steam-exploded corn straw hydrolysate is shown; wherein, A is a graph showing the change of glucose concentration in the undetoxified steam-exploded corn straw hydrolysate of the engineered strain overexpressing key genes of the glutathione system and the superoxide dismutase system with fermentation time; B is a graph showing the change of xylose concentration in the undetoxified steam-exploded corn straw hydrolysate of the engineered strain overexpressing key genes of the glutathione system and the superoxide dismutase system with fermentation time; C is a graph showing the ethanol yield in the undetoxified steam-exploded corn straw hydrolysate of the engineered strain overexpressing key genes of the glutathione system and the superoxide dismutase system; and D is a comparative graph showing the ethanol yield and yield obtained by fermentation of different strains.

[0034] Figure 4 The ethanol fermentation performance of the engineered strain overexpressing other genes of the glutathione system and the superoxide dismutase system using the undetoxified steam-exploded corn straw hydrolysate is shown; wherein, A is a graph showing the change of glucose concentration in the undetoxified steam-exploded corn straw hydrolysate of the engineered strain overexpressing other genes of the glutathione system and the superoxide dismutase system with fermentation time; B is a graph showing the change of xylose concentration in the undetoxified steam-exploded corn straw hydrolysate of the engineered strain overexpressing other genes of the glutathione system and the superoxide dismutase system with fermentation time; C is a graph showing the ethanol yield in the undetoxified steam-exploded corn straw hydrolysate of the engineered strain overexpressing other genes of the glutathione system and the superoxide dismutase system; and D is a comparative graph showing the ethanol yield and yield obtained by fermentation of different strains.

[0035] Figure 5 Overexpression of NADPH / NADP+ Ethanol fermentation performance of undetoxified steam-exploded corn straw hydrolysate using engineered strains expressing key genes of the cofactor recycling system; A is overexpressed NADPH / NADP + Glucose concentration changes with fermentation time in the undetoxified steam-exploded corn straw hydrolysate of engineered strains expressing key genes of the cofactor circulation system; B is the overexpression of NADPH / NADP + The xylose concentration in the undetoxified steam-exploded corn straw hydrolysate of the engineered strain expressing key genes of the cofactor circulation system changes with fermentation time; C is the overexpression of NADPH / NADP + Figure 4. Ethanol production in undetoxified steam-exploded corn straw hydrolysate by engineered strains expressing key genes in the cofactor circulation system. Figure D is a comparison of ethanol production and yield obtained by fermentation with different strains.

[0036] Figure 6 The ethanol fermentation performance of the engineered strain overexpressing the transcription activator using the undetoxified steam-exploded corn straw hydrolysate is shown; wherein, A is a graph showing the change of glucose concentration in the undetoxified steam-exploded corn straw hydrolysate of the engineered strain overexpressing the transcription activator with fermentation time; B is a graph showing the change of xylose concentration in the undetoxified steam-exploded corn straw hydrolysate of the engineered strain overexpressing the transcription activator with fermentation time; C is a graph showing the ethanol yield of the engineered strain overexpressing the transcription activator in the undetoxified steam-exploded corn straw hydrolysate; and D is a comparison of the ethanol yield and yield obtained by fermentation of different strains.

[0037] Figure 7 The results of applying engineered bacteria with high xylose utilization and high inhibitor tolerance in batch fed-batch enzymatic hydrolysis and batch fermentation to produce ethanol are shown; wherein A is the batch fed-batch enzymatic hydrolysis of steam-exploded pulp; B is a graph of glucose, xylose, and ethanol concentrations in ethanol fermentation of the selected engineered bacteria in a 5-liter fermentor.

[0038] Figure 8 The figures show the performance comparison results of ethanol fermentation in undetoxified steam-exploded corn straw hydrolysate using three improved Saccharomyces cerevisiae strains (YLE533, YLE608 and YLE702); wherein, A is a graph showing the changes in glucose concentration in undetoxified steam-exploded corn straw hydrolysate of the three improved Saccharomyces cerevisiae strains over fermentation time; B is a graph showing the changes in xylose concentration in undetoxified steam-exploded corn straw hydrolysate of the three improved Saccharomyces cerevisiae strains over fermentation time; and C is a graph showing the ethanol yield of the three improved Saccharomyces cerevisiae strains in undetoxified steam-exploded corn straw hydrolysate. DETAILED DESCRIPTION

[0039] To facilitate understanding of the present invention, the present invention will be described in further detail below with reference to specific embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to be limiting. The examples provided below are intended to serve as a guide for those skilled in the art to make further improvements and are not intended to limit the present invention in any way.

[0040] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0041] I. Terminology

[0042] The term "chassis microorganism" in the present invention is also called "chassis microbial cell" or "chassis strain", which refers to the use of microbial cells as a platform and the placement of them into a functionalized biological system so that the cells can have the functions required by humans and be used for biosynthesis.

[0043] The term "genetically engineered bacteria" as used herein refers to strains, such as Saccharomyces cerevisiae, that have been engineered to produce a desired product by introducing a target gene into a host organism (i.e., a host cell or chassis microorganism) for expression, or by modifying the chassis microorganism to enhance pathways or knock out or weaken genes associated with competing metabolic pathways. The core technology of genetic engineering is DNA recombination; therefore, genetically engineered bacteria are also referred to as recombinant microorganisms in this invention.

[0044] The terms "laboratory adaptive evolution" and "domestication" are used interchangeably in the present invention.

[0045] The term "overexpression" in the present invention refers to the selection of a strong promoter to integrate the gene expression cassette into a specific site of the Saccharomyces cerevisiae chromosome. For example, the strong promoters used in the present invention are shown in Table 2.

[0046] The terms "include", "comprising", "having", "containing", etc. used in the present invention are all open terms, which mean including but not limited to.

[0047] II. Implementation Plan

[0048] As mentioned above, existing second-generation bioethanol production has disadvantages such as low xylose conversion rate during the production process and high toxicity of inhibitors to the strain, making it difficult to achieve industrialization. Moreover, it is difficult to simultaneously construct ethanol fermentation strains with high xylose utilization, especially high inhibitor tolerance, in existing research. This has become a barrier that technicians in this field have always wanted to solve but have not been able to successfully solve, seriously limiting its application in producing high-concentration second-generation bioethanol from lignocellulose hydrolysate with high inhibitor concentrations.

[0049] In order to solve the above problems, the present inventors have conducted a lot of research on the transformation of Saccharomyces cerevisiae. Saccharomyces cerevisiae ) strain S288C was used as the chassis microbial cell. By heterologously introducing genes related to the xylose reductase-xylitol dehydrogenase pathway, xylose isomerase pathway, and xylose transport, the pentose phosphate pathway, reduced glutathione system, and superoxide dismutase system were strengthened, the regulatory transcription activators were optimized, and genes related to competitive metabolic pathways were weakened to transform the chassis microorganism of Saccharomyces cerevisiae. Then, laboratory adaptive evolution was carried out using sugar sources (for example, C5 / C6) and inhibitors as pressure. Based on the combined optimization of metabolic engineering (xylose assimilation pathway, reduced glutathione system genes, superoxide dismutase expression system genes, NADPH / NADP+ cofactor cycle system genes and transcription factors, etc.) and laboratory adaptive evolution strategies, a genetically engineered bacterium with high xylose utilization and high inhibitor tolerance was constructed and obtained.

[0050] Therefore, the present invention provides a genetically engineered bacterium for preparing ethanol based on hydrolyzing sugar from wood fiber, which is a genetically engineered bacterium with high xylose utilization and high inhibitor tolerance.

[0051] According to the embodiment of the first aspect of the present invention, the genetically engineered bacteria for preparing ethanol based on hydrolysis of wood fiber sugars are recombinant cerevisiae bacteria that co-express heterologous xylose reductase-xylitol dehydrogenase pathway-related genes, xylose transport-related genes, and xylose isomerase-related genes, thereby constructing and strengthening the xylose assimilation pathway.

[0052] In some embodiments of the present invention, the xylose reductase-xylitol dehydrogenase pathway-related genes include genes derived from Pichia stipitis ( Scheffersomyces stipitis ) encoding xylose reductase XYL1 (its sequence is shown in SEQ ID No. 1, GeneBank ID: XM_001385144.1) or a codon-optimized gene encoding xylose reductase from Pichia stipitis XYL1 (whose sequence is shown in SEQ ID No. 2), a gene encoding a xylose reductase mutant R276H from Pichia stipitis mXYL1or a codon-optimized gene encoding the xylose reductase mutant R276H from Pichia stipitis mXYL1 (whose sequence is shown in SEQ ID No. 3) and a gene encoding xylitol dehydrogenase from Pichia stipitis XYL2 (its sequence is shown in SEQ ID No. 4, GeneBank ID: XM_001386945.1) or a codon-optimized gene encoding xylitol dehydrogenase from Pichia stipitis XYL2 (whose sequence is shown in SEQ ID No. 5).

[0053] It should be understood by those skilled in the art that the codon-optimized gene encoding the xylose reductase mutant R276H from Pichia stipitis is mXYL1 (The sequence is shown in SEQ ID No. 3) is a codon-optimized gene encoding xylose reductase from Pichia stipitis XYL1 Mutated (Watanabe, S., Abu Saleh, A., et al. “Ethanol production from xylose by recombinant Saccharomycescerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis”. Microbiology, (2007)153(9), 3044–3054.).

[0054] In some embodiments of the present invention, the xylose transport-related gene includes a gene encoding a high-affinity xylose transporter from Pichia stipitis. XUT4 (whose sequence is shown in SEQ ID No. 6, GeneBank ID: XM_001386678.1), and / or a gene encoding a high-affinity xylose transporter from Pichia stipitis XUT6 (whose sequence is shown in SEQ ID No. 7, GeneBank ID: XM_001386552.1) or a codon-optimized gene encoding a high-affinity xylose transporter from Pichia stipitis XUT4 (whose sequence is shown in SEQ ID No. 8), and / or a codon-optimized gene encoding a high-affinity xylose transporter from Pichia stipitis XUT6(Its sequence is shown in SEQ ID No. 9).

[0055] In some embodiments of the present invention, the xylose isomerase-related genes include those derived from Clostridium plantarum ( Lachnoclostridium phytofermentans ) encoding xylose isomerase xylA Or the codon-optimized gene encoding xylose isomerase from Clostridium plant fermentans xylA (Its sequence is shown in SEQ ID No. 10).

[0056] It should be understood by those skilled in the art that the codon-optimized gene encoding xylose isomerase from Clostridium plant fermentans is xylA (The sequence is shown in SEQ ID No. 10, K. Wang, C. Su, H. Bi, et al. “The transition from 2G to 3G-feedstocks enabled efficient production of fuels and chemicals”. Green Energy & Environment 1 (2023) 65-74.).

[0057] According to the embodiment of the second aspect of the present invention, the genetically engineered bacteria also overexpresses endogenous xylulose kinase-related genes, non-oxidative pentose phosphate pathway-related genes, reduced glutathione system-related genes, superoxide dismutase expression system-related genes, NADPH / NADP + One or more genes and transcription factors related to the cofactor circulation system strengthen the pentose phosphate pathway, the reduced glutathione system, the xylulokinase system and the superoxide dismutase system, optimize the regulation of transcription activators, and thereby enhance the tolerance of Saccharomyces cerevisiae in the presence of inhibitors.

[0058] In some embodiments of the present invention, the endogenous xylulose kinase-related gene includes an endogenous gene encoding xylulose kinase XKS1 (The sequence is shown in SEQ ID No. 11, GeneBank ID: NM_001181323.3).

[0059] In some embodiments of the present invention, the endogenous non-oxidative pentose phosphate pathway-related genes include endogenous genes encoding transketolase (Transketolase) TKL1(its sequence is shown in SEQ ID No. 12, GeneBank ID: NM_001184171.1), the gene encoding glucose-7-phosphate: D-glyceraldehyde-3-phosphate transaldolase TAL1 (Sequence No. 13, GeneBank ID: NM_001182243.1), gene encoding 5-phosphate-ribose isomerase RKI1 (its sequence is shown in SEQ ID No. 14, GeneBank ID: NM_001183514.1) and the gene encoding ribulose-phosphate 3-epimerase RPE1 (whose sequence is shown in SEQ ID No. 15, GeneBank ID: NM_001181554.1).

[0060] In some embodiments of the present invention, the endogenous reduced glutathione system-related genes include endogenous genes encoding glutathione-disulfide reductase (Glutathione reductase) GLR1 (its sequence is shown in SEQ ID No. 16, GeneBank ID: NM_001183905.1), the gene encoding glutamate-cysteine ​​ligase GSH1 (its sequence is shown in SEQ ID No. 17, GeneBank ID: NM_001181534.1), the gene encoding glutathione synthetase GSH2 (its sequence is shown as SEQ ID No. 18, GeneBank ID: NM_001183303.1) and the gene encoding superoxide dismutase (Peroxiredoxin) PRX1 (whose sequence is shown in SEQ ID No. 19, GeneBank ID: NM_001178304.1).

[0061] In some embodiments of the present invention, the endogenous superoxide dismutase expression system-related genes include endogenous genes encoding superoxide dismutase (Superoxide dismutase) SOD1(its sequence is shown in SEQ ID No. 20, GeneBank ID: NM_001181762.1), the gene encoding peroxisomal catalase A CTA1 (its sequence is shown in SEQ ID No. 21, GeneBank ID: NM_001180564.1) and the gene encoding catalase T CTT1 (whose sequence is shown in SEQ ID No. 22, GeneBank ID: NM_001181217.1).

[0062] In some embodiments of the invention, the endogenous NADPH / NADP + Cofactor recycling system-related genes include the endogenous gene encoding glucose-6-phosphate 1-dehydrogenase ZWF1 (its sequence is shown in SEQ ID No. 23, GeneBank ID: NM_001183079.1), the gene encoding 6-phosphogluconate dehydrogenase GND1 (6-phosphogluconate dehydrogenase, decarboxylating 1) (whose sequence is shown in SEQ ID No. 24, GeneBank ID: NM_001179314.3) and / or a gene encoding 6-phosphogluconate dehydrogenase GND2 (6-phosphogluconate dehydrogenase, decarboxylating 2) (its sequence is shown as SEQ ID No. 25, GeneBank ID: NM_001181385.3), the gene encoding 2-glycerol dehydrogenase (Glycerol 2-dehydrogenase) GCY1 (its sequence is shown in SEQ ID No. 26, GeneBank ID: NM_001183539.1), the gene encoding isocitrate dehydrogenase IDP1 (whose sequence is shown in SEQ ID No. 27, GeneBank ID: NM_001180125.1) and / or a gene encoding isocitrate dehydrogenase IDP2 (whose sequence is shown in SEQ ID No. 28, GeneBank ID: NM_001182061.1) and / or a gene encoding isocitrate dehydrogenase IDP3(whose sequence is shown in SEQ ID No. 29, GeneBank ID: NM_001182848.3).

[0063] In some embodiments of the present invention, the endogenous transcription factor includes an endogenous DNA-binding transcription factor (AP-1-like transcription factor) YAP1 (its sequence is shown in SEQ ID No. 30, GeneBank ID: NM_001182362.1), drug response transcription factor PDR3 Drug-responsive transcription factor (SEQ ID No. 31, GeneBank ID: NM_001178245.1), stress-responsive transcriptional activator MSN2 (its sequence is shown in SEQ ID No. 32, GeneBank ID: NM_001182534.1) and stress response transcription activator MSN4 (whose sequence is shown in SEQ ID No. 33, GeneBank ID: NM_001179628.1).

[0064] According to the implementation of the third aspect of the present invention, the genetically engineered bacteria have also undergone chassis modification, which includes knocking out genes related to the competitive metabolic pathway of the chassis microorganisms and weakening genes related to the competitive metabolic pathways to perform chassis microbial modification on brewer's yeast.

[0065] In some embodiments of the present invention, the competitive metabolic pathway-related genes of the chassis microorganisms include genes encoding aldose reductase (NADPH-dependent aldose reductase) GRE3 (its sequence is shown in SEQ ID No. 34, GeneBank ID: NM_001179234.1), the gene encoding alkaline phosphatase (4-nitrophenylphosphatase) PHO13 (Sequence No. 35, GeneBank ID: NM_001180296.1), gene encoding glycerol-3-phosphate dehydrogenase GPD1(whose sequence is shown in SEQ ID No. 36, GeneBank ID: NM_001180081.1) and a gene encoding potassium-activated aldehyde dehydrogenase ALD4 (Potassium-activatedaldehyde dehydrogenase) (whose sequence is shown in SEQ ID No. 37, GeneBank ID: NM_001183794.1).

[0066] According to an embodiment of the fourth aspect of the present invention, the genetically engineered bacteria have also undergone sugar source adaptive acclimation (laboratory adaptive evolution) and / or inhibitor pressure adaptive acclimation (laboratory adaptive evolution).

[0067] According to some embodiments of the present invention, the laboratory adaptive evolution includes laboratory adaptive evolution of sugar sources and laboratory adaptive evolution with multiple inhibitors as pressure.

[0068] In some embodiments of the present invention, the sugar source includes xylose (pentose), or xylose (pentose) and glucose (hexose).

[0069] In the present invention, the inhibitors include one or more of phenolic monomers, lignin oligomers, organic acids and furan compounds.

[0070] In some embodiments of the present invention, the phenolic monomer includes one or more of a syringylphenol monomer, a guaiacylphenol monomer, and a p-hydroxyphenylphenol monomer.

[0071] In some embodiments of the present invention, the lignin oligomers include one or more of syringyl lignin oligomers, guaiac lignin oligomers, p-coumaric acid lignin oligomers, and syringyl-guaiac p-coumaric acid mixed lignin oligomers.

[0072] In some embodiments of the present invention, the organic acid includes one or more of formic acid, oxalic acid, acetic acid and butyric acid.

[0073] In some embodiments of the present invention, the furanic compound includes furfural and / or 5-hydroxymethylfurfural.

[0074] In the present invention, the cerevisiae yeast strain S288C (deposit number: ATCC 26108; American Type Culture Collection, Biowind) is used as a host cell (chassis strain), and the xylose assimilation pathway is constructed and strengthened by heterologous introduction and co-expression of heterologous xylose reductase-xylitol dehydrogenase pathway-related genes, xylose transport-related genes, and xylose isomerase-related genes; by overexpressing endogenous xylulose kinase-related genes, non-oxidative pentose phosphate pathway-related genes, reduced glutathione system genes, superoxide dismutase expression system-related genes, NADPH / NADP + The cofactor circulation system-related genes strengthen the pentose phosphate pathway, reduced glutathione system, xylulose kinase system and superoxide dismutase system, optimize the regulation of transcriptional activators, and thus enhance the tolerance of Saccharomyces cerevisiae in the presence of inhibitors; by weakening the genes related to the competitive metabolic pathway, the chassis microorganism of Saccharomyces cerevisiae is modified; and then the laboratory adaptive evolution (domestication) is carried out with sugar sources (for example, C5 / C6) and inhibitors (for example, phenolic monomers, lignin oligomers, organic acids and furan compounds) as pressure to construct and obtain a recombinant Saccharomyces cerevisiae YLE702 with high xylose utilization and high inhibitor tolerance, and the metabolic pathway of ethanol production based on lignocellulose using this genetically engineered bacterium is as follows Figure 1 As shown, the genetically engineered bacteria can achieve C5 / C6 co-utilization and has significantly improved tolerance to inhibitors. It has the characteristics of high xylose utilization and high inhibitor tolerance, can efficiently metabolize xylose and produce ethanol in lignocellulose hydrolysate, and is easy to use for industrial production.

[0075] The present invention also provides the use of the genetically engineered bacteria of the present invention in preparing ethanol based on the hydrolysis of sugars from wood fibers, which can be understood as a method for preparing ethanol based on the hydrolysis of sugars from wood fibers using the genetically engineered bacteria of the present invention.

[0076] According to the present invention, the application includes inoculating genetically engineered bacteria into a fermentation medium, and fermenting and culturing lignocellulosic hydrolyzed sugar raw materials as substrates to produce ethanol.

[0077] In the present invention, the lignocellulosic hydrolyzed sugar raw material is an undetoxified lignocellulosic hydrolyzed liquid; the undetoxified lignocellulosic hydrolyzed liquid contains undetoxified lignocellulosic hydrolyzed sugars. The detoxification process removes toxic inhibitors from the lignocellulosic hydrolyzed liquid to reduce the fermentation burden of the strain. Research results show that the genetically engineered strain provided by the present invention can still normally perform ethanol fermentation in undetoxified lignocellulosic hydrolyzed liquid. Moreover, compared with the detoxified process, the undetoxified process has many advantages, such as simplicity and convenience, low sugar loss rate, and reduced risk of bacterial contamination.

[0078] Preferably, the solid content (mass to volume ratio, g / mL) of the non-detoxified wood fiber hydrolysate is 20%-40% (w / v), more preferably 20%, 30% or 40% (w / v).

[0079] After testing, the undetoxified wood fiber hydrolyzate contained wood fiber hydrolyzed sugars, including 70.50 g / L glucose and 24.70 g / L xylose, or 112.23 g / L glucose and 44.31 g / L xylose, 141.32 g / L glucose and 70.24 g / L xylose.

[0080] There is no particular limitation on the fermentation method in the present invention. For example, continuous culture, fed-batch culture or batch culture may be used.

[0081] The research results show that the strain provided by the present invention can produce cellulosic ethanol at a concentration of up to 92.61 g / L (ethanol yield of 0.438 g / g) in non-detoxified steam explosion hydrolysate containing high inhibitor concentrations without the addition of any nutrient salts, and the residual xylose content is very low, which is much higher than the level recorded in existing literature. Example

[0082] The present invention is described in detail below by way of specific examples. The examples are provided for the purpose of illustrating the present invention only and are not intended to limit the scope of the invention. The experimental methods described below are all routine laboratory methods unless otherwise specified. The experimental materials described below are all commercially available unless otherwise specified.

[0083] The culture medium formulations used in the following examples are as follows:

[0084] The culture medium of the present invention is formulated as follows:

[0085] 1. Liquid culture medium

[0086] YPD medium (10 g / L Yeast Extract, 20 g / L Peptone, 20 g / L glucose) was sterilized at 116°C for 25 min and used for pre-culture of Saccharomyces cerevisiae and chassis microorganisms.

[0087] YPX medium (10 g / L Yeast Extract, 20 g / L Peptone, 20 g / L Bleomycin, used for seed liquid culture of Saccharomyces cerevisiae before fermentation. Xylose) was sterilized at 116°C for 25 min and 200 μg / mL G418 or 200 μg / mL Xylose were added as needed.

[0088] 2. Solid culture medium

[0089] LB / Amp medium (10 g / L Peptone, 5 g / L Yeast Extract, 10 g / L NaCl) was sterilized at 116°C for 25 min and 100 μg / mL ampicillin was added for culturing Escherichia coli.

[0090] YPD / G418 / Zeocin solid medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 20 g / L agar) was sterilized at 116°C for 25 min, cooled to 60°C, and supplemented with 200 μg / mL G418 or 200 μg / mL zeocin. This medium was used to culture Saccharomyces cerevisiae. Depending on the experimental situation, two antibiotics were added to select for yeast transformants. Example 1

[0091] The relevant primers for gene expression and plasmid construction in this example are shown in Table 1.

[0092] Table 1 Primers related to gene expression and plasmid construction

[0093]

[0094] 1. Acquisition of xylose assimilation pathway genes

[0095] Gene encoding xylose reductase XYL1 , gene encoding xylose reductase mutant R276H mXYL1 , genes encoding xylitol dehydrogenase XYL2 , genes encoding xylulose kinase XKS1 , encoding a high-affinity xylose transporter XUT4 and XUT6 and the gene encoding xylose isomerase xylA Codon optimization and full gene synthesis were performed. Gene encoding transketolase TKL1 , Gene encoding glucose-7-phosphate:D-glyceraldehyde-3-phosphate transaldolase TAL1 , the gene encoding 5-phospho-ribose isomerase RKI1 and encoding ribulose 3-phosphate epimerase RPE1Obtained by amplification on the genomic DNA template of Saccharomyces cerevisiae. Amplification was performed using the primers required for gene amplification in Table 1 to obtain fragments of the expected size. The amplification system was configured using PrimSTARMAX DNA polymerase (TAKARA), and the amplification system was: 2×PrimSTAR MAX DNApolymerase 25 μL, 2 μL each of F / R end primers, 1 μL of genomic DNA / plasmid template, and ddH2O was added to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98°C for 3 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 30 minutes (34 cycles); extension at 72°C for 10 minutes (1 cycle). The target gene fragment and the corresponding vector were then digested with the corresponding restriction endonuclease, and the digested fragments were recovered by gel cutting. Among them TEF2p-XYL1-TPIt 、 TEF1p-mXYL1 (R276H)-PGIt 、 PGK1p-XYL2-CYC1t 、 HXT7p-XKS1-HXT7t The corresponding vector is pRS425, PGK1p - XUT4- CYC1t and PGK1p - XUT6-CYC1t The corresponding vector is pSP-GM1, PGK1p - xylA-CYC1t The corresponding vector is pIYC04, TDH1p-RPE1-PGI1t 、 PGK1p-TAL1-Hxt7t 、 TEF1p-RKI1-tCYC1 、 TDH3p-TKL1-GPDt The corresponding vector is pRS416. The target gene and the corresponding vector fragment were then ligated using T4 DNA ligase or Gibson enzyme and chemically transformed into Escherichia coli trans10 (competent cells, Beijing Quanshijin Biotechnology Co., Ltd.).

[0096] 2. Acquisition of reduced glutathione system genes

[0097] Gene encoding glutathione-disulfide reductase GLR1 , genes encoding glutamate-cysteine ​​ligase GSH1 , genes encoding glutathione synthetase GSH2 and the gene encoding superoxide dismutase PRX1 The target gene fragment and the corresponding vector were then digested with the corresponding restriction endonucleases, and the digested fragments were recovered by gel excision. PGK1p-GSH1-CYC1t 、 TEF1p-GLR1-ADH1t 、 PGK1p-GSH2-CYC1tThe target gene and the corresponding vector fragment were ligated using Gibson enzyme and then transformed into Escherichia coli trans10 by chemical transformation.

[0098] 3. Acquisition of superoxide dismutase system genes

[0099] Gene encoding superoxide dismutase SOD1 , genes encoding peroxidase A CTA1 and the gene encoding catalase T CTT1 The target gene fragment and the corresponding vector were then digested with the corresponding restriction endonucleases, and the digested fragments were recovered by gel excision. PGK1p-CTT1-CYC1t 、 PGK1p-CTA1-CYC1t and TDH1p-SOD1- HXT7t The target gene and the corresponding vector fragment were ligated using Gibson enzyme and then transformed into Escherichia coli trans10 by chemical transformation.

[0100] 4. NADPH / NADP + Acquisition of genes for the cofactor recycling system

[0101] Gene encoding 6-phosphoglucose-1-dehydrogenase ZWF1 , gene encoding 6-phosphogluconate dehydrogenase GND1 and GND2 , genes encoding 2-glycerol dehydrogenase GCY1 , the gene encoding isocitrate dehydrogenase IDP1 、 IDP2 and IDP3 The target gene fragment and the corresponding vector were then digested with the corresponding restriction endonucleases, and the digested fragments were recovered by gel excision. TDH3p - ZWF1 - HXT7t 、 PGI1p - GND1 - PGI1t 、 PGI1p - GND2 - PGI1t 、 PGI1p - GCY1 - PGI1t 、 PGI1p - IDP1 - PGI1t 、 PGI1p - IDP2 - PGI1t and PGI1p - IDP3 - PGI1t The target gene and the corresponding vector fragment were ligated using Gibson enzyme and transformed into Escherichia coli trans10 by chemical transformation.

[0102] 5. Acquisition of transcription activator genes

[0103] DNA-binding transcription factors YAP1 , drug response transcription factors PDR3 , stress response transcription activator MSN2 and MSN4 The target gene fragment and the corresponding vector were then digested with the corresponding restriction endonucleases, and the digested fragments were recovered by gel excision. PGK1p-YAP1-CYC1t 、 PGK1p-PDR3-CYC1t 、 PGK1p-MSN2-CYC1t 、 PGK1p-MSN4- CYC1t The target gene and the corresponding vector fragment were ligated using Gibson enzyme and transformed into Escherichia coli trans10 by chemical transformation.

[0104] Example 2: Construction of engineered bacteria

[0105] 1. Preparation of competent yeast

[0106] A single colony of the starting strain was cultured in YPD medium at 30°C and 200 rpm overnight, and inoculated into 50 mL of YPD medium at a final cell turbidity of OD600nm=0.3 and cultured at 30°C to OD600nm=1.2-1.6; the bacterial solution was centrifuged at 3000 rpm for 3 min at 4°C and collected into a 50 mL sterile centrifuge tube; the supernatant was discarded and the tube was washed once with 20 mL of sterile water; the supernatant was discarded again and the tube was washed once with 20 mL of 1 mol / L sorbitol; the Saccharomyces cerevisiae cells were re-pipetted and mixed in 16 mL of 1 mol / L sorbitol, 2 mL of 10×TE and 2 mL of 10×LiOAc were added and mixed thoroughly, and kept at 30°C for 30 min; 200 μL of 1 mol / L DTT stock solution was added to the mixture and kept on a shaker for 15 min; and the mixture was centrifuged at 3000 rpm for 3 min at 4°C. min to collect Saccharomyces cerevisiae cells; discard the supernatant and wash twice with 20 mL of 1 mol / L sorbitol; after centrifugation, remove as much liquid as possible and resuspend the yeast cells in 200 μL of 1 mol / L sorbitol, and aliquot to obtain competent cells.

[0107] The recombinant strain described below is constructed by simultaneously incorporating an expression element for the Cas9 protein, a recombinant plasmid expressing a guide RNA (using the G418 sulfate antibiotic as a selection marker, as shown in Table 2), and a linearized homologous recombination fragment. The recombinant plasmid expressing the guide RNA recognizes and binds to the specific PAM region at the corresponding site, activating and directing the Cas9 protein on the recombinant plasmid to cleave the double-stranded DNA at the corresponding site. The expression element containing the homologous region or the homologous recombination fragment is then integrated into the corresponding site in the strain's chromosomal DNA through homologous recombination repair.

[0108] 2. Construction of recombinant plasmid containing gRNA and linearized Donor

[0109] Construction of pLacZ-cas9 plasmid: pLacZ vector was used as template, which carries pTEF1- iCas9-tADH1, the Cas9 protein expression element. Primers were used to amplify the pSNR52-gRNA1-gRNA scaffold-tSNR52-G418-pSNR52-gRNA2-gRNA scaffold-tSNR52 fragment (this fragment includes gRNA expression cassettes 1 and 2 and the G418 expression cassette; the specific gRNA sequences are shown in Table 2). The vector and fragments were ligated using a Golden-Gate reaction and transformed into trans10 competent cells. The plasmid was extracted to obtain the gRNA-containing recombinant plasmid. The Golden-Gate reaction system consisted of 1.6 μL of BsaI, 0.4 μL of T4 DNA ligase, and 2 μL of T4 buffer. ddH2O was added to bring the total volume to 20 μL. Amplification conditions were as follows: 37°C for 10 minutes (1 cycle); 37°C for 10 minutes, 16°C for 5 minutes (16 cycles); 16°C for 30 minutes (1 cycle); 37°C for 30 minutes (1 cycle); and 80°C for 5 minutes (1 cycle).

[0110] Linearized Donor Construction: Using the constructed plasmid as a template, amplify the linearized donor using the primers listed in Table 1 using PrimSTAR MAX DNA polymerase. The upper and lower homology arms of the linearized donor are both 500 bp. The resulting PCR product, the linearized donor, is then concentrated to at least 15,000 ng using alcohol precipitation for gene knockout / integration preparation.

[0111] Table 2 Related plasmids for gene expression

[0112]

[0113] 3. Construction of genetically engineered bacteria

[0114] Chromosomal Integration of Genetically Engineered Bacteria: The construction of genetically engineered strains such as YL10 and YL13 involved chromosomal integration of genes. Competent cells were prepared by culturing the starting strain overnight in YPD liquid medium. The corresponding pLacZ-cas9 recombinant plasmid containing the gRNA and the linearized donor were then added. The cells were then pipetted until thoroughly mixed and electroporated at 1.5 kV in an electroporator. 1 mL of 1 M sorbitol was immediately added to the electroporated cells. The electroporated cells in the cuvette were then placed in a 50 mL centrifuge tube containing 6 mL of culture medium (sorbitol:YPD = 1:1) and allowed to recover at 30°C on a shaker for 5 hours. Finally, the medium was changed to YPD / G418 medium and cultured for 24 hours. 100 μL of the mixture was then plated onto a YPD / G418 plate. Colonies that emerged from the plates were rapidly extracted and amplified using the primers listed in Table 3. The correct colonies were identified as the corresponding strains. The obtained strain was then cultured in YPD medium for several generations to lose the pLacZ-cas9 recombinant plasmid containing gRNA.

[0115] Gene Knockout in Genetically Engineered Bacteria: The construction of genetically engineered strains YL11 and YL12 both involved gene knockout. Competent cells were prepared by culturing the starting strain overnight in YPD liquid medium. The corresponding pLacZ-cas9 recombinant plasmid containing the gRNA and the linearized donor were added. The cells were then pipetted until thoroughly mixed and electroporated at 1.5 kV in an electroporator. 1 mL of 1 M sorbitol was immediately added to the electroporated cells. The electroporated cells in the cuvette were then placed in a 50 mL centrifuge tube containing 6 mL of culture medium (sorbitol:YPD = 1:1) and allowed to recover in a shaker at 30°C for 5 h. Finally, one side was washed with sterile water, and 100 μL of the mixture was plated onto a YPD / G418 plate. Colonies that emerged from the plates were rapidly extracted and amplified using primers. The correct colonies were identified as the strains listed in Table 3. The resulting strains were then cultured in YPD medium for several passages to eliminate the pLacZ-cas9 recombinant plasmid containing the gRNA.

[0116] Plasmid transformation of genetically engineered bacteria: The construction of genetically engineered strains such as YLE601 and YLE602 involved plasmid transformation. Competent cells were prepared by culturing the starting strain overnight in YPD liquid medium. The corresponding recombinant plasmid containing the gene expression cassette was then added. The cells were then pipetted until thoroughly mixed. Electroporation was performed at 1.5 kV in an electroporator. 1 mL of 1 M sorbitol was immediately added to the electroporated cells. The electroporated cells in the cuvette were then placed in a 50 mL centrifuge tube containing 6 mL of culture medium (sorbitol:YPD = 1:1) and allowed to recover at 30°C on a shaker for 2 hours. Finally, one side was washed with sterile water. 100 μL of the mixture was plated onto a YPD / G418 plate. Colonies representing the corresponding strains listed in Table 3 were obtained.

[0117] Table 3 Chassis bacteria and constructed engineered strains

[0118]

[0119] Example 3: Laboratory Adaptive Evolution of Engineered Bacteria

[0120] Laboratory adaptive evolution using sugar as a source: The engineered strain seed solution was inoculated into a medium containing 20 mL YPX20 (20 g / L xylose) to ensure an initial OD 600 1. After the strain grows to the middle and late logarithmic growth phase in YPX20 medium, the initial OD 600 The inoculum size was 1 and inoculated into 20 mL of YPX20 (20 g / L xylose) medium. After about 85 days of circulation, the engineered strain with high xylose utilization was screened. The growth of the strain in the xylose synthesis medium was evaluated from the OD 600 The xylose consumption rate increased significantly from 4.31 to 10.14.

[0121] Laboratory adaptive evolution was performed using a synthetic medium containing corn straw steam explosion enzymatic hydrolysate inhibitors: the engineered strains that had undergone the first round of laboratory adaptive evolution were inoculated again into a medium containing 20 mL corn straw steam explosion enzymatic hydrolysate inhibitors to ensure that the initial OD 600 After about 24 h of growth and fermentation, the initial OD 600 The inoculum size was 1 and the culture medium containing 20 mL of corn straw steam explosion hydrolysate inhibitor was used. After about 180 days of circulation, the average xylose consumption rate of the yeast strain in the presence of the inhibitor increased from the initial 0.17 g / L / h to 0.44 g / L / h.

[0122] After domestication, three engineered strains, YLE531, YLE532, and YLE533, were obtained through screening (Table 3). The fermentation performance of these three strains was compared with that of strain YLE43 ( Figure 2 ). The engineered strain was picked from a single colony on a plate and cultured in YPX medium as a seed liquid, and then inoculated into a 20 mL working volume of fermentation medium at a 10% v / v inoculation rate for fermentation analysis. Glucose, xylose, and ethanol in the fermentation process were detected by HPLC. HPLC determination conditions: This high performance liquid chromatography was equipped with an Aminex HPX-87H column (7.8×300 mm) and a differential detector was used for detection at 50°C, with a mobile phase flow rate of 0.6 mL / min and a column temperature of 65°C. Fermentation in a synthetic medium containing 40 g / L xylose showed that YLE533 had the best xylose fermentation performance, with an OD of 0.05 after 72 h of fermentation. 600maxThe ethanol yield of YLE533 was 10.55 (compared to 8.48 for YLE43), and the ethanol production concentration was 12.29 g / L (compared to 11.15 g / L for YLE43). After 72 h of fermentation in YPDX medium containing 70 g / L glucose and 30 g / L xylose as sugar sources, YLE533 exhibited the best fermentation performance, achieving a residual xylose concentration of only 2.15 g / L and an ethanol concentration of 40.60 g / L. The fermentation performance of the evolved strains was further verified in a steam-exploded hydrolysate of undetoxified corn stover containing 20% ​​(w / v) solids (without exogenous nutrient addition). YLE533 exhibited the best fermentation performance, achieving an ethanol concentration of 38.98 g / L in the steam-exploded hydrolysate, with a residual xylose content of only 4.27 g / L. The ethanol yield of YLE533 was 0.408 g / g (the sum of the glucose and xylose in the lignocellulosic hydrolysate).

[0123] Example 4: Application of glutathione system-enhanced and superoxide dismutase system-enhanced engineered bacteria in the production of ethanol from wood fiber sugar hydrolysis

[0124] Furfural, phenols and other inhibitors in corn straw steam explosion hydrolysate can severely damage mitochondria and vacuoles, leading to ROS accumulation in Saccharomyces cerevisiae cells and affecting normal cell growth and metabolism. In order to reduce ROS in cells, the glutathione (GSH) system, as an antioxidant system, can oxidize GSH into glutathione disulfide (GSSG) and simultaneously convert excess hydrogen peroxide in the cell (reduced to O by superoxide dismutase SOD1) into glutathione disulfide. 2- Production) is reduced to H2O. In order to increase the reduced GSH in the cell and reduce ROS, genes related to the GSH system and superoxide dismutase system were overexpressed. The engineered strains YLE601-YLE611 were fermented in 30% (w / v) (mass volume percentage) solid content of non-detoxified corn straw steam explosion hydrolysate (no exogenous nutrients added). The fermentation results showed that overexpression of GSH system-related genes significantly enhanced the tolerance of Saccharomyces cerevisiae in corn straw steam explosion hydrolysate, and strain YLE608 showed the best ethanol fermentation performance, with residual xylose of 0.89 g / L, ethanol concentration of 63.77 g / L, and ethanol yield of 0.444 g / g (the sum of the mass of glucose and xylose in the lignocellulose hydrolysate) ( Figure 3 and Figure 4 ).

[0125] Example 5: Application of Transcription Activator Enhanced Engineering Bacteria in the Production of Ethanol from Lignocellulosic Sugar Hydrolysis

[0126] Under the influence of enzymatic hydrolysis, Saccharomyces cerevisiae is prone to stress response. The control of gene expression under stress conditions is mediated by multiple transcription factors, which are the main regulatory factors for responding to oxidative stress. In order to enhance the robustness of the engineered bacteria, multiple related transcription activators were overexpressed. Among them, transcription factors Yap1p (regulates the endoplasmic reticulum folding protein response and participates in oxidative stress) and Msn4p (positive transcriptional regulator, stress response transcriptional activator) showed the best yeast ethanol fermentation performance in the steam-exploded enzymatic hydrolysate of non-detoxified corn straw with a solid content of 25% (w / v) (mass volume percentage), with ethanol concentrations of 59.53 g / L and 59.64 g / L ( Figure 5 ).

[0127] Example 6: NADPH / NADP + Application of engineered bacteria with enhanced cofactor recycling system in ethanol production from lignocellulosic sugar hydrolysis

[0128] To reduce ROS and improve tolerance in Saccharomyces cerevisiae, NADPH / NADP was enhanced + The cofactor circulation system is also crucial. The engineered strains YLE701-YLE708 were fermented in a 25% (w / v) solid content steam-exploded enzymatic hydrolyzate of non-detoxified corn straw without adding any exogenous nutrients. The fermentation results are shown in Figure 2. Figure 6 As shown, NADPH / NADP was enhanced in the steam explosion enzymatic hydrolysate. + The engineered strain with a cofactor recycling system significantly enhanced ethanol fermentation. After 96 hours of fermentation, the ethanol titer of engineered strain YLE702 increased by 2.44% compared to YL608 and by 9.43% compared to YLE533. The final ethanol concentration of engineered strain YLE702 was 65.11 g / L, with an ethanol yield of 0.454 g / g (the sum of glucose and xylose in the lignocellulosic hydrolysate).

[0129] Example 7: Application of high xylose utilization and high inhibitor tolerance engineered bacteria in the production of ethanol from wood fiber sugar hydrolysis

[0130] Based on the above glutathione system, superoxide dismutase system, transcription activator and NADPH / NADP +The combined strengthening of the cofactor circulation system screened out the optimal engineered strain YLE702. In order to obtain a higher concentration of bioethanol in the undetoxified corn straw steam-exploded hydrolysate, a batch fed saccharification process was adopted, which can maintain good homogenization and mass transfer and achieve a higher concentration of monosaccharides. Batch fed saccharification and batch fermentation were carried out in a 5L fermenter. When the final solid content in the corn straw steam-exploded hydrolysate was 30% (w / v), after 96 hours of batch fed hydrolysis, the glucose content of the corn straw steam-exploded hydrolysate reached 112.23 g / L and the xylose content reached 44.31 g / L ( Figure 7 A). Ethanol fermentation was performed with the engineered strain YLE702, which has high xylose utilization and high inhibitor tolerance, at a 10% inoculum without any exogenous nutrients. After 96 h of fermentation, the final ethanol concentration reached 70.52 g / L, the ethanol productivity reached 0.59 g / L / h, and the ethanol yield was 0.450 g / g (the sum of the glucose and xylose in the lignocellulose hydrolysate). Figure 7 B).

[0131] After 96 hours of batch hydrolysis at a solid content of 40% (w / v), the glucose content of the corn straw steam-exploded hydrolysate reached 141.32 g / L and the xylose content reached 70.24 g / L. The engineered strains YLE533, YLE608, and YLE702, which were good at xylose utilization and inhibitor tolerance at various stages of transformation, were used to perform bioethanol fermentation on the undetoxified corn straw steam-exploded hydrolysate without adding any exogenous nutrients for 120 hours. Figure 8 As shown in Table 4, the final engineered strain YLE702 showed the best ethanol fermentation performance, with an ethanol concentration of 92.61 g / L and an ethanol yield of 0.438 g / g (the sum of the mass of glucose and xylose in the lignocellulose hydrolysate) (Table 4). The results showed that after laboratory adaptive evolution and the activation of the glutathione system, superoxide dismutase system, transcription activator and NADPH / NADP + The ethanol production and yield of the strain with enhanced cofactor circulation system were greatly improved in corn straw steam explosion enzymatic hydrolysate.

[0132] Table 4 Bioethanol production and yield of different engineered bacteria

[0133]

[0134] It should be noted that the embodiments described above are only preferred embodiments of the present invention and are used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A genetically engineered bacterium for preparing ethanol based on hydrolyzed sugars from wood fiber, which is a recombinant Saccharomyces cerevisiae that co-expresses heterologous xylose reductase-xylitol dehydrogenase pathway-related genes, xylose transport-related genes, and xylose isomerase-related genes; The xylose reductase-xylitol dehydrogenase pathway-related gene is a codon-optimized gene encoding xylose reductase from Pichia stipitis XYL , a codon-optimized gene encoding a xylose reductase mutant R276H from Pichia stipitis mxDV and a codon-optimized gene encoding xylitol dehydrogenase from Pichia stipitis XYL2 ; The xylose transport-related gene is a codon-optimized gene encoding a high-affinity xylose transporter from Pichia stipitis. XUT4 and genes XUT6 ; The xylose isomerase-related gene is a codon-optimized gene encoding xylose isomerase from plant fermentation Clostridium. xyA ; The genetically engineered bacteria also overexpressed endogenous xylulose kinase-related genes, non-oxidative pentose phosphate pathway-related genes, reduced glutathione system genes, NADPH / NADP + Genes related to the cofactor recycling system; The xylulokinase-related gene is a gene encoding xylulokinase XKS1 ; The non-oxidative pentose phosphate pathway-related gene is a gene encoding transketolase TKL1 , Gene encoding glucose-7-phosphate:D-glyceraldehyde-3-phosphate transaldolase TAL1 , Gene encoding 5-phospho-ribose isomerase RKI1 and the gene encoding ribulose 3-phosphate isomerase RPE1 ; The reduced glutathione system-related gene is a gene encoding glutathione-disulfide reductase GLR1 and the gene encoding glutamate-cysteine ​​ligase GSH1 ; The NADPH / NADP + The gene related to the cofactor circulation system is the gene encoding 6-phosphogluconate dehydrogenase GND1 ; The genetically engineered bacteria have also undergone chassis modification, which is the knockout of genes related to the competitive metabolic pathway of the chassis microorganisms; the genes related to the competitive metabolic pathway of the chassis microorganisms are genes encoding aldose reductase GRE3 , gene encoding alkaline phosphatase PHO13, Gene encoding 3-phosphoglycerate dehydrogenase GPD1 and the gene encoding potassium-activated aldehyde dehydrogenase ALD4 ; The genetically engineered bacteria have also undergone sugar source adaptation and inhibitor pressure adaptation domestication; The sugar source is xylose; The inhibitors are phenolic monomers, lignin oligomers, organic acids and furan compounds; The phenolic monomers are syringylphenol monomers, guaiacylphenol monomers and p-hydroxyphenylphenol monomers; The lignin oligomers are syringa-type lignin oligomers, guaiac-type lignin oligomers, p-coumaric acid-type lignin oligomers, and syringa-guaiac-p-coumaric acid mixed lignin oligomers; The organic acids are formic acid, oxalic acid, acetic acid and butyric acid; The furanic compounds are furfural and 5-hydroxymethylfurfural; The host cell of the recombinant Saccharomyces cerevisiae is the Saccharomyces cerevisiae strain S288C with a deposit number of ATCC 26108.

2. The use of the genetically engineered bacteria as claimed in claim 1 in the preparation of ethanol based on lignocellulosic hydrolyzed sugar raw materials; the use comprises inoculating the genetically engineered bacteria into a fermentation medium, and fermenting and culturing the lignocellulosic hydrolyzed sugar raw materials as substrates to prepare ethanol.

3. The use according to claim 2, characterized in that: The wood fiber hydrolyzed sugar raw material is a wood fiber enzymatic hydrolyzate without any nutrient salt added and detoxified; the solid content of the wood fiber enzymatic hydrolyzate without any nutrient salt added and detoxified is 20%-40% w / v.

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