Synergetic Engineering And Metabolic Regulation Of Clostridium Beijerinckii For Enhancing Xylose Metabolism And Butanol Biosynthesis

As a sustainable energy,bioenergy has attracted more attention than other renewable energy sources(such as wind energy and solar energy).The "14th Five-Year Plan for Biomass Energy Development" promulgated by my country pointed out that the focus of the new energy industry should be placed on the biomass energy industry.and fully develop and utilize biomass energy to improve the comprehensive utilization of biomass energy.As an emerging biomass energy,biobutanol is a new idea for alleviating the shortage of fossil energy and protecting the environment and green development.According to statistics,the demand for bioenergy has quadrupled over the past few decades.Meanwhile,experts predict that by 2060,bioenergy will account for more than 17% of global energy.However,the production of biobutanol by the traditional ABE fermentation(acetone-ethanol-butanol)method has problems such as high substrate cost and low yield and yield of butanol produced by solvent-producing Clostridium,making it impossible for large-scale production of biobutanol.Based on the absence of "carbon metabolism repression effect" in Clostridium beijerinckii,this study selected Clostridium Beijerinckii CC101 as the experimental strain.At the same time,considering that xylose is the five-carbon sugar with the highest content in lignocellulose hydrolyzate,C.beijerinckii CC101 has a lot of room for improvement in the ability to use xylose to produce butanol and to transport and metabolize xylose.Based on this,this study will overexpress,knock out or inhibit transcription of key genes in the xylose transport pathway,xylose metabolism pathway and butanol synthesis pathway through metabolic engineering technology and gene editing technology,and optimize the combination of genes to construct A strain that can efficiently utilize xylose to produce butanol is obtained.Under the enhanced xylose transport strategy,three genetically engineered bacteria were successfully constructed: CC101(thl-Xyl FGH),CC101(ptb-Xyl T)and CC101(thl-Xyl T).The recombinant strain CC101(ptb-Xyl T)was the best strain for xylose fermentation under this strategy,and the highest ABE yield was 0.41 g/g.When the xylose concentration increased to 55 g/L,the ABE yield increased to 14.6 g/L,an increase of 23.7% compared with the control group.The common feature of the three engineered bacteria was that when the xylose substrate concentration was gradually increased from 25 g/L to At 55 g/L,the xylose consumption,acetone production,butanol production and ABE production increased with the increase of xylose substrate concentration,while the yield of ABE decreased with the increase of xylose concentration.Under the strategy of strengthening xylose metabolism,two genetically engineered strains,CC101(Xyl A)and CC101(Xyl A/B),were successfully constructed.The consumption of xylose increased,and the yields and yields of butanol and ABE were different from those of the wild-type strains.improvement.The recombinant strain CC101(Xyl A/B)had the best xylose fermentation performance,and the highest ABE yield was 0.40 g/g.When the xylose concentration increased to 55 g/L,the ABE yieldincreased to 15.4 g/L,which was 30.5% higher than that of the control group;Under the enhanced butanol synthesis strategy,six genetically engineered bacteria with enhanced butanol synthesis were successfully constructed: CC101(Adh A),CC101(Adh B),CC101(Adh E),CC101(ΔADC),CC101(ΔADC-Adh E)and CC101(ΔADC-Adh E-Ctf A/B).The recombinant strain CC101(Adh B)had the best xylose fermentation performance,and the highest ABE yield was 0.40 g/g;when the xylose concentration increased to 55 g/L,the ABE yield increased to 14.9 g/L,an increase of26.3% compared with the control group At the same time,it was found that knocking out acetoacetate decarboxylase could almost completely block the synthesis of the by-product acetone,but it would lead to the growth inhibition of Clostridium beijerinckii and the reduction of solvent synthesis ability to varying degrees.Under the strategy of synergistically enhancing xylose metabolism and butanol synthesis,the yield and yield of ABE of the recombinant strain CC101(ptb-Xyl A/B-Adh B)were further enhanced,and the ABE yield of the recombinant strain remained at 0.39-0.40 g/ g,the ABE yield of the recombinant strain with a single enhancement strategy is more stable;the highest ABE yield can reach 16.5 g/L,which is twice as high as that of the wild strain!The research idea of this study is to carry out experiments from three strategies of strengthening xylose transport,strengthening xylose metabolism and strengthening butanol synthesis,and the thinking is relatively systematic and comprehensive.In this paper,the recombinant strain CC101(ptb-Xyl T-Xyl A/B-Adh B)was constructed by using ptb promoter and co overexpression of xylose isomerase(Xyl A),xylose kinase(Xyl B)and alcohol dehydrogenase(Adh B).It provides a new idea and valuable reference for the industrialized production of butanol.