RNA-Seq And Whole-Genome Sequencing Revealed Ethanol Tolerance Mechanism Of Thermoanaerobacterium Aotearoense

As the increasingly serious problems of fossil energy depletion and environmental pollution,exploiting the clean renewable energy resources has become very urgent.The development and utilization of biomass as the raw material to produce ethanol will become tendency in energy industry.Cellulose and hemicellulose as the main component of plant biomass are the most abundant and renewable resource on earth.Thermophilic anaerobic bacteria as a desirable biological catalyst to produce ethanol from cellulosic biomass have attracted great interest over the past years.However,it's generally low ethanol tolerance,limited the industrial application.Meanwhile,relevant research about ethanol tolerance mechanism of Thermoanaerobacterium was still comparatively rare.In this paper,Whole-genome sequencing,RNA-seq and following metabolic engineering were applied to reveal the ethanol tolerance mechanism of Thermoanaerobacterium aotearoense ?ldh which lactate dehydrogenase was deleted to improve the yield of ethanol.After being maintained for 410 generations in increasing ethanol adaptive culture,a mutant strain T.aotearoense ET7 that grew under 6% ethanol?v/v?was isolated,and it's ethanol tolerance was significantly improved by 100%.The genome DNA of ET7 was extracted for Whole-genome sequencing.After comparative analysis with the wild type strain T.aotearoense SCUT27,113 SNP mutation sites and 20 InDel sites come to light.Among that,111 mutant sites distributed in 100 genes were detected in CDS region,the general mutation rate were 3.73%.83 amino acid and 5 multiple mutant genes were found by sequence analysis.When T.aotearoense ?ldh was grown to exponential phase,0%,2%,3% ethanol was added to the medium,and cells were harvested after 10 min,45 min,120 min incubation in different ethanol concentration respectively to prepare RNA-seq samples.According to transcriptome analysis,the number of differentially expressed genes?DEGs?was decreased with the extension of ethanol stress time,but the fold change increased.And by contrast to 2%,more DESs and bigger fold change were found under 3% ethanol.By contrast to the blank samples,1214,1021,669 DEGs in 10 min,45 min,120 min was detected respectively at the concentration of 2% and 3% ethanol?v/v?.Finally,a comprehensive analysis of all samples at the three time points was conducted to discover 1516 DEGs related to the reponse to ethanol stress.The analysis of heatmap and expression cluster pattern showed that 50 DEGs showed strong response to ethanol.Including 13 significant down-regulated genes,15 significant up-regulated genes,5 DEGs essential for cell growth were over-expressed significantly,17 low expression level genes but positively correlated to the concentrations of ethanol stress.Further venn diagram analysis showed 76 DEGs involved in the response of ethonal stress,combine with 50 DEGs obtained by cluster analysis 22 key genes which showed strong connection to ethanol stress were found.Finally,8 key genes related to ethanol tolerance were picked up for metabolic engineering of T.aotearoense ?ldh.Five engineered strains were obtained by gene overexpression or knock out according to the result of transcriptome analysis.With 20 g/L glucose as carbon source,the concentration and yield of ethanol from engineered strain ?ldh/adhE were 7.27 g/L and 0.45 g/g which increased by 18.8% and 12.5%,respectively.The maximum cell density,acetic acid concentration and yield of pflA gene knock out strain fell rapidly,but its ethanol concentration and yield increased significantly,reached up to 6.49 g/L and 0.50 g/g,which increased by 6.1% and 25%,respectively.The yield was close to the theoretical yield.Cell density of engineered strain ?ldh/?APS was increased by 24%,and the acetic acid concentration increased significantly reaching 2.73 g/L.With 20 g/L glucose as carbon source,3.5%,4.0% and 4.5% ethanol was added to the medium respectively when the engineered strains reached exponential phase(OD₆₀₀=1).The ability of engineered strains to ethanol tolerance were arranged as ?ldh/SH > ?ldh/?APS > ?ldh/adhE > ?ldh.Specially,the maximum cell density and maintain time of engineered strain ?ldh/SH under high ethanol concentration significantly higher than other strains.When ethanol was added before cell inoculation,all the engineered strains showed the same ethanol tolerance to ?ldh.The probable reason is that the SH and adhE genes required synergistic combination of other genes expressed in stationary phases.