The Breeding Of High Biobutanol Producing Strains And The Mutagenic Effect About The Key Enzyme Genes Expression Analysis By Lignocellulose Fermentation

With the growing global fossil energy depletion and the rapid growth of energy demand,combined with increasing energy resources constraints and ecological environment problems outstanding constantly,guarantee energy security and development of green renewable energy has attracted great attention of national governments.Therefore,using renewable lignocellulose biomass to produce alternative fuel received widespread attention.Biobutanol as a kind of renewable clean energy,which can be used with gasoline blending in any proportion or even replace gasoline has also ushered in the new development opportunity.But,there are some challenges using lignocellulosic materials to transform biobutanol,such as low yield and low utilization rate of raw materials.In view of this,this study started with the breeding of strains,the strain Clostridium saccharobutylicum ATCC BAA-117 with a broad spectrum of carbohydrate fermentation ability was used as the original strain,the genome shuffling technology combined with evolutionary engineering was adopted to screen excellent varieties of Clostridium saccharobutylicum with high concentrations of poplar wood hydrolysate tolerance and high biobutanol production in small-scale fermentation of laboratory,and a high yield strain gsGD-1 was obtained,which can efficiently utilize poplar wood hydrolysate to produce biobutanol.The fermentation conditions were optimized for biobutanol production from poplar wood hydrolysates by Clostridium saccharobutylicum gsGD-1.Further,comparative RT-PCR analysis was conducted for key enzyme gene expression of butanol biosynthesis between the original strain and evolutionary strain(gsGD-1)using fluorescent quantitation RT-PCR.The specific research contents and the main achievements were described as following:1.Established the diversity of phenotype parent strain library.To construct the genetic diversity parent library,Clostridium sacchar-obutylicum ATCC BAA-117 cells were mutagenized with ultraviolet irradiation,streptomycin mutagenic treatment and butanol tolerance screenings,respectively.Nine mutant strains which can stable genetic and have positive traits were screened by UV mutagenesis,ribosome engineering breeding and butanol tolerance screenings,including six strains with high biobutanol yield,namely UV-1,UV-2,UV-3,RE-1,RE-2 and RE-3,were obtained by UV and ribosome engineering mutagenesis,the butanol production more than 20%higher than that of the original strain.Another three strains with high concentrations of butanol tolerance,namely HBT-1,HBT-2 and HBT-3,obtained by butanol tolerance screenings,the butanol tolerability was 25%stronger than the original strain.These nine mutant strains constituted the parental library used as the starting population for recursive protoplast fusion and genome shuffling.2.The optimum conditions of Clostridium saccharobutylicum protoplast preparation,regeneration and fusion were obtained.Through the single factor experiment,central composite design and response surface analysis:the optimal conditions for protoplast preparation and regeneration was:seed culture time 12 h,lysozyme concentration 7.11 mg/mL,enzymolysis time 1 h and temperature 37?,the product of protoplast preparation and regeneration rate reached 50.8%;The best conditions for protoplast fusion was:PEG4000 concentration of 30%,fusion temperature of 37?,the fusion time was 20 min and Ca2+ concentration was 45 mM,and fusion rate was up to 4.9×10-5.3.High biobutanol-yield fusion recombinants GS3-11 and GS3-255 were bred by genome shuffling.Nine strains of parent strain library were devoted to recursive protoplast fusion,after three rounds of effective genome shuffling,then the fusion by resistant plate filter and shaking flask fermentation screening,two stable genetic recombinants GS3-11 and GS3-255 with high-yield and high butanol resistant strains were obtained.In 3 L fermentation tank,biobutanol production of GS3-11 and GS3-255 were 12.51 g/L and 12.20 g/L,respectively,more than 60%higher than the original strain.4.An evolutionary strain gsGD-1 was selected with high concentration of poplar wood hydrolysates tolerance by evolution engineering.First,the generation time and selection pressure of GS3-11 and GS3-255 were determined under different concentration of poplar wood hydrolysate,and found that GS3-11 stress tolerance was superior to GS3-255.Further,continuous evolution with screening was carried out by strain GS3-11 under the conditions of constant concentration of poplar wood hydrolysate and gradient concentration of poplar wood hydrolysate.It was found that the efficiency of gradient selection for obtaining strains with higher concentrations of poplar wood hydrolysate tolerance was much better than that of immobilized concentration of poplar wood hydrolysate selection.In 250 mL shaking flask and 3 L fermentation tank,under the fermentation conditions of reducing sugar concentration was 60 g/L in poplar wood hydrolysate,the maximum biobutanol production was 10.21 g/L by gsGD-1,and maximum cell density OD value reached 2.05,bioutanol production increased by 196%compared with the starting strain GS3-11(3.45 g/L).5.The fermentation medium and fermentation conditions were optimized for biobutanol production from poplar wood hydrolysates by gsGD-1.The Plackett-Burman design,central composite design and response surface methodology were adopted to optimize the fermentation medium and fermentation conditions.The obtained optimal culture medium and fermentation conditions were:reducing sugar 52.79g/L,ammonium sulfate 3.0 g/L,yeast extract 2.54 g/L,CaCO3 5.43 g/L,KH2PO4 0.75 g/L,K2HPO40.81g/L,MgSO4·7H2O 0.2g/L,FeS04·7H2O 0.3 g/L,MnSO40.01 g/L,initial pH7.0,inoculum size 9.7%,loading volume 76.4%and fermentation temperature 36.5?.Under this optimal condition,gsGD-1 showed maximum biobutanol production of 15.64g/L,led to 24.4%increase in biobutanol production from 12.57 g/L(un-optimized)to 15.64 g/L,increased by 120%compared with the original strain ATCC BAA-117(7.12 g/L).6.The key enzyme genes differential expression of butanol biosynthesis pathway in gsGD-1 and original strain was quantitated by fluorescence quantitative RT-PCR.The housekeeping 16s rRNA was selected as internal control gene,using real-time fluorescence quantitative PCR and 2-??Ct method to analyze the key enzyme genes relative expression between the original strain Clostridium saccharobutylicum ATCC BAA-117 and evolved strain gsGD-1 in the mid-acidogenesis and the mid-solventogenesis phase of butanol biosynthetic pathway.The following was the 2-??Ct relative quantitative analysis results.(1)Compared with the original strain,the acidogenic genes ack and buk in the mid-acidogenesis phase had a decreased transcription in gsGD-1,ack and buk reduced their expression to nearly 65%;while in the mid-solventogenesis phase,the transcript level of ack and buk in gsGD-1 was turned to higher levels,was about 1.5 times that of the original strain.(2)Compared with the original strain,in the mid-acidogenesis phase,the solventogenic genes adhE,ctfAB and bdhB had an increased transcription except that aadc expression had no obvious difference in gsGD-1,expression of adhE,ctfAB and bdhB achieved 2.67,1.8,and 1.6 times,respectively;while in the mid-solventogenesis phase,the transcript level of adhE,aadc,ctfAB and bdhB was still maintained at higher levels(2.51,2.82,10.5,and 1.34 times).(3)Compared with the original strain,butanol tolerance related genes gldA had a decreased transcription in gsGD-1,reduced its expression to nearly 40%of original strain in the mid-acidogenesis phase,and the transcript level of gldA was still maintained at lower level(0.6 times)in the mid-solventogenesis phase.The expression of hsp90in gsGD-1had a slightly higher than the original strain in the mid-acidogenesis phase,while in the mid-solventogenesis phase,the transcript level of hsp90 had an obviously increasment,expression of hsp90 achieved 1.5 times of original strain.Visibly,the biobutanol production of gsGD-1 was higher than the original strain,it was closely associated with the up-regulated genes of the solventogenic genes and differential expression of butanol tolerance related genes(down-regulated expression of gldA and up-regulated expression of hsp90).