The Regulation Mechanism Of Substrate Utilization In Thermoanaerobacterium Aotearoense SCUT27 And Its Application

Due to the increasingly prominent issues of energy,resources and environment,the conversion of biomass into biofuels and high value-added products by microorganisms has become a research hotspot owing to its economic,environmental and renewable advantages.China is a large agricultural country with an abundant reserve of lignocellulose.Large amouts of glucose,xylose and cellobiose could be obtained from the partial hydrolysis of biomass.With completely hydrolysis of lignocellulose,glucose and xylose can be obtained(the mass ratio is about 2:1).The economics of green bio-manufacturing depends on whether the biocatalyst can efficiently co-utilize cellobiose,glucose and xylose produced from the hydrolysis of lignocellulose.Thermoanaerobacterium aotearoense SCUT27(SCUT27)screened by our lab could co-utilize glucose and xylose,xylose and cellobiose but cannot co-utilize glucose and cellobiose.Though SCUT27 can co-utilize xylose and glucose,the utilization rate of xylose was significantly lower than that of glucose.In the early stage,our team performed the RNA-seq to study the transcript profile changes of SCUT27 in different carbon sources at different growth stages.Based on the transcriptome data,key genes or regulatory factors were mined and the corresponding metabolic engineerings were performed to strengthen the ability of the substrate utilization and ethanol production in SCUT27.SCUT27 cannot co-utilize glucose and cellobiose,and the mechanism of the carbon catabolite repressor was CcpA-independent.After mining the genome of SCUT27,four gene clusters annotated as cellobiose related genes were found and named as Cel1,Cel2,Cel3 and Cel4,the co-transcription of which was studied by RT-PCR.Results showed that Cel2 that contained PTS transporter and glycoside hydrolase,was the only intact operon for cellobiose utilization.After analyzing the promoter with the software,it was found that the promoter of Cel2 was inducible and activated by the cooperation of ?54 and CelRcel2.To make Cel2 constituitive expression,the promoter along with celRcel2 was replaced with the promoter of adhE and SCUT27/?celRcel2/padhE was obtained and able to co-utilize glucose and cellobose.Redox sensing protein(Rex)can regulate the expression of target genes by sensing the changes of NADH/NAD+ ratio in cells and is regarded as the global transcriptional regulator.In the genome of SCUT27,the gene with locus V518₂238 is annotated as redox sensing protein(Rex).With rex knockedout,the maximum OD600 of SCUT27/?rex increases by 95.36%-103.16%,the yield of ethanol improves by 63.16%-88.23%and the yields of acetic acid and lactic acid decrease by 44.44%-60.98%,respectively,along with sugar consumption rate increased by 74.19%-130.56%,when compared with that of SCUT27 under glucose,xylose or glucose/xylose mixture.Results above indicated that with rex deletion,the distribution of carbon flow in the mutant had been changed by metabolic synthesis disturbances and a new and stable redox balance had been remodelled,resulting in the improved substrate utilization and enhanced ethanol production.To further enhance the ethanol yield,SCUT27/?ldh/?rex was obtained with the increase of ethanol titer and yield from about 4.56 g/L and 0.32 g/g to 7.45 g/L and 0.38 g/g,respectively.Compared with SCUT27/?ldh,SCUT27/?ldh?rex had the stronger resistance to the toxicity of lignocellulose hydrolysates,especially under the hydrolysis of wheat straw,with the ethanol concentration increased by 37.08%and the ethanol production rate enhanced by 36.67%.Arginine repressor(ArgR)is a regulatory protein related to arginine metabolism and also regarded as the global regulator.In the genome of SCUT27,three genes are annotated as ArgRs.Results of gene knockout and fermentation tests showed that only argR1864 deletion had a significant effect on carbon metabolism of SCUT27 with the glucose utilization rate increased by 38.46%and the xylose utilization rate improved by?50.00%.The metabolic flux of SCUT27 and SCUT27/?argR1864 under glucose or xylose was analyzed,and results showed that the carbon metabolic flux of SCUT27/?argR1864 had changed,and more carbon source flowed to the strains growth(the mole yield of biomass improved by?31.82%),ethanol production(the mole yield of ethanol improved by?82.95%)and acetic acid formation(the mole yield of acetic acid improved by?40.00%),suggesting that SCUT27/?argR1864 not only had improved co-utilization ability of glucose and xylose but also had improved ethanol production.Fermentation results under the lignocellulose hydrolysate pretreated with dilute acid showed that the xylose utilization rate was enhanced by 25.00%-62.96%and the glucose utilization rate was improved by 21.43%-118.75%,along with the ethanol concentration and yield significantly increased by 113.86%-760.00%and 88.89%-400.00%,respectively,indicating that knocking out argR1864 is an effective strategy to engineer SCUT27 for ethanol production from acid pretreated lignocellulose hydrolysate.Transcriptome analysis of SCUT27/?argR1864 under xylose was used for the in-depth exploration of the physiological functions of ArgR1864 in SCUT27.Results showed that argR1864 is the negative regulator not only for genes related to arginine metabolism,but also for genes related to heat shock proteins.Results of spot assays experiments,intracellular reactive oxygen species(ROS)measurement and lignocellulose-derived inhibitors tolerance experiments proved that SCUT27/?argR1864 had the stronger ability to resist external environmental pressures.Enzymatically pretreated lignocellulose hydrolysates were used for fermentation test.Compared with SCUT27,the sugar consumption rate of SCUT27/?argR1864 had improved by 52.88%-84.62%,along with ethanol concentration enhanced by 117.55%-186.96%,which is consistent with the fermentation phenotype of SCUT27/?argR1864 under lignocellulose hydrolysate pretreated with dilute acid,suggesting that the fermentation performance of SCUT27/?argR1864 is very stable under various lignocellulose hydrolysates.In summary,the mechanism of carbon catabolite repressor between glucose and cellobiose in SCUT27 was CcpA-independent,and the promoter of Cel2 was activated by the cooperation of ?54 and CelRcel2.SCUT27/?celRcel2/padhE was successfully achieved by promoter replacement with the ability to co-utilize glucose and cellobiose.Rex and ArgR1864 are the transcriptional regulators of SCUT27,both of which can cause metabolic synthesis disturbances in SCUT27 with gene knockedout.A new and stable metabolic flow distribution was established in mutants with the ability to promote substrate utilization and ethanol production.