The Research On Ethanol Metabolism Of Thermoanaerobacterium Calidifontis Rx1

The production of bioethanol from lignocellulose is a hot research topic in the field of bioethanol.Thermophilic anaerobic bacteria have been considered to potential fermentation strains because of their capacity to degrade polysaccharides such as cellulose and/or hemicellulose.Thermoanaerobacterium calidifontis RxI is a grampositive bacterium isolated from Baoshan Hot Springs in Yunnan province,China.Acetate kinase gene of Rxl was knocked out to construct the mutant T.calidifontis Rxl△ack.In this study,both of strains were used to analyze the difficulty of the construction of the gene knockout system and the effect on Rxl transcriptome of acetate kinase disruption.At the same time,ferment conditions optimization for bioethanol production and the mechanism was studied.The result obtained were as follows:1.Transcript analysis was performed on T.calidifontis Rxl and T.calidifontis Rxl△ack.Gene expression analysis showed that 2283 genes were expressed in both strains,205 genes were expressed exclusively in wild-type strain,and 73 genes were expressed in mutant strains.A total of 324 genes were differentially expressed,with up-regulated expression of 104(32.1%),down-regulated expression of 220(67.9%).GO analysis shows that most of the genes differentially expressed were biological processes,particularly in organic substrate catabolic process.It indicating that disruption of ack has great impact on the metabolic process.KEGG analysis showed that 209 differentially expressed genes are.involved in 42 different metabolic or signaling pathways.Among them,40(19.1%)genes belonged to Metabolic pathway,15(7.2%)genes belonged to Biosynthesis of secondary metabolites,15(7.2%)genes belong to Biosynthesis of antibiotics,13(6.2%)genes are involved in Microbial metabolism in diverse environments.At the same time,the loss of acetate kinase resulted in the downregulation of xylose transporter,xylose isomerase and xylulokinase.In addation,inactivation of acetate kinase led to the accumulation of acetyl phosphate,which makes the expression of PTA down regulated.2.From the feed fermentation of Rx1,it was found that the substrate comsumption was stopped or slowed after the strain reached the maximum cell density,which may be due to the continuous decrease in the pH.Thus,the effect of pH control on Rxl fermentation was subsequently studied.The pH value of the wild type uncontrolled decreased 4.33 at 24 h,and the mutant decreased to 4.79 at 32 h.Rxl and Rxl Δack could not utilized entire organic substrate in uncontrolled pH condition but fully utilized in controlled pH=6.0 or 7.0 condition.However,controlled condition could reduce the utilized rate of substrate and.result in the decrease in growth rate of wild type strain.In the case of metabolites,the control of pH=6.0 or 7.0 reduced the lactate production of T.calidifontis Rxl and T.calidifontis Rxl Δack,and the carbon would flow to ethanol and ethanol become the main end-product of 3.865 g/L.3.The pH metabolic regulation mechanism of Rxl and its mutant was studied by fluorescence quantitative PCR and enzyme activity.Compared with the uncontrolled condition,the expression of lactate hydrogenase and pyruvate kinase gene ldh and pk were significantly increased by more than twice of wild type Rxl under the pH controlled at 7.0,and the acetate kinase gene ack was significantly down-regulated.The expression of acetaldehyde/alcohol dehydrogenase gene AdhE was not significantly different.The expression levels of pk,ldh and ack genes in the mutant strain Rxl Δack were both tripled and the expression of AdhE was not significantly different.Under the uncontrolled condition,the activity of pyruvate kinase PK of wild type Rxl was increased by 88%,while the activities of ALDH and ADH were decreased by 39%and 51%respectively,Lactate dehydrogenase LDH(pH=5.3)activity did not change significantly.The activity of PK,LDH and ALDH in the mutant strain Rxl Δack increased by 18%,37%and 31%respectively,and the activity of ADH did not change significantly.As in the previous reports,the mutant still retained part of the activity of acetate kinase,pH control did not have any effect on the activity of the mutant acetate kinase.In addition,LDH had higher activity at lower pH.Under the condition of pH=7.3,LDH had only 50%activity compare with pH=5.3.This result can explain the reason for the drastic decrease in lactate production under controlled pH in the fermentation experiment.4.DNA restriction endonuclease activity was detected in cell-extracts of T.calidifontis Rxl using plasmid or linear DNA isolated from E.coli.It was preliminary confirmed that restriction endonuclease was type 2 restriction endonuclease DpnII by protein sequence alignment.Methylation of plasmid or linear DNA by methyltransferase could resist the degradation from endonuclease.Plasmid hosts such as E.coli JM109 and E.coli BL21,were associated with their own methylation enzymes,can be methylated and effectively resistant to degradation.Different transformation method was carried out for Rxl gene knock-out,however,no transformant had been obtained.We speculated that the main reason was the low transformation efficiency or the plasmid could not replicate in Rx l cells.