Preliminary Research Of The Regulation And Enhancement Of Hexose And Pentose Utilization In Thermoanaerobacterium Aotearoense SCUT27

With the depletion of traditional fossil energy and the sharply decrease of the stock of oil,it is vital to find or develop a renewable clean energy sources.Nowadays,along with the goal of energy security,and therefore environmental protection,overall renewable clean energy sources,such as solar energy,wind energy and biomass energy,are envisaged.Lignocellulose and hemicellulose are the most abundant renewable resources on the earth,and has received considerable attention as a sustainable feedstock that can replace diminishing fossil fuels for the production of ethanol,methane and hydrogen.Thermoanaerobacteria has a strong ability of hydrolyzing cellulose,high pentose conversion rate,and broad carbon resource utilization spectrum.T.aotearoense SCUT27,a Gram-positive thermophilic anaerobic bacterium,not only metabolizes a variety of hexoses,pentoses and disaccharides but also simultaneously ferments xylose,glucose,fructose and mannose into ethanol.This feature is of particular industrial interest because the pentose D-xylose is the primary ingredient of the hemicellulose fraction of lignocellulosic biomass.However,carbon catabolite repression（CCR）between arabinose and glucose still existed and the utilization rate of xylose only accounted for 42.67%that of glucose.Thus,it is meaningful to study the mechanism of CCR and find out the genes related to xylose and glucose metabolism of T.aotearoense SCUT27In this study,SCUT27/△ccpA had been constructed to study the role of CcpA in the suppression of arabinose and hexose catabolism.The fermentation results showed that the phenomenon of catabolism suppression between arabinose and glucose or fructose was alleviated,with OD600 of the mutant decreased by 27.34%,and total sugar consumption decreased by 35.07%.In order to verify the functions of the co-repressor proteins HPr and Crh,the hpr knockout vector was constructed successfully,but the positive clones were failed to be obtained,indicating that HPr is very critical for the survival of the bacteria and could not be completely knocked out.After crh knockout,the mutant SCUT27/△crh neither relieved the suppression of catabolic metabolites between arabinose and glucose or fructose,nor changed the growth of cells and the ability of sugar consumption.The mutant SCUT27/△hprlhprS46A could relieve the suppression of catabolic metabolites between arabinose and glucose or fructoseIn order to improve the co-utilization of glucose and xylose in T.aotearoense SCUT27,the transcriptome data with xylose or glucose as the sole carbon source was mined to reveal genes related to xylose and glucose metabolism of SCUT27.Omics data indicate that gene clusters V518₁054-1059 maybe were xylose metabolism related genes,including xylose isomerase（xylA）,xylulose kinase（xylB）,ABC transporter（xylT）and three possible regulatory factors genes rok（V518₁060）,lacl（V518₁062）and mtlR（V518₂192）containing PRDs domain.The fermentation data of SCUT27/△mtlR showed that MtlR is a positive regulator of xylose metabolism.With mtlR deletion,the xylose utilization of the mutant decreased by 67.56%,along with the transcription levels of xylA and xylB decreased by-39.67%,and that of ABC transporter gene dropped by 86.93%.With mtlR overexpression,the transcription levels of the genes xylA and xylB increased by about 44.75%,and the ability of xylose utilization increased by 25.71%With further analysis of the transcriptome,four transporters that may be involved in glucose transport including an ABC transporter and three PTS transport systems had been explored.Mutants with the deficiency of predicted glucose transporter showed no significant changes on glucose utilization,while fructose utilization was ceased by knocking out genes related to PTS（V518₀375-0376）or the DeoR family transcriptional activator gene（V518₀378）.Subsequently,the engineering strain SCUT27/△hpr/hprH15D was obtained by replacing the histidine at position 15 of the HPr protein with aspartate,the glucose consumption decreased by 18.02%,which did not cease the glucose consumption,indicating that glucose is mainly transported through non-PTS pathways.In order to improve the PTS pathway of glucose metabolism,the HPr protein of SCUT27 was replaced by HPr from Clostridium acetobutylicum ATCC824.The sugar utilization capacity of the mutant increased by 63.15%.Under the mixed culture of xylose and glucose,glucose utilization increased by 68.18%,xylose utilization increased by 46.15%In summary,this study had verified that the carbon catabolite repression（CCR）between arabinose and glucose or fructose in T.aotearoense SCUT27 is CcpA-dependent CCR,and the co-repressor protein in the regulation process is HPr rather Crh.Our results showed that the xylose metabolic pathway was activated by the transcription factor MtlR in T.aotearoense SCUT27.It also showed that fructose was mainly transported through PTS.However,glucose is mainly transported through non-PTS pathways.By incorporating the HPr of Clostridium acetobutylicum ATCC824 into T.aotearoense SCUT27,the co-utilization ability and rate of glucose and xylose were improved,which provided new ideas for improving the sugar utilization rate of anaerobic thermophilic bacteria and enhancing the co-utilization ability of xylose and hexose.