Characterization Of Thermoanaerobacterium Calidifontis Rx1 With Disrupted Ack Gene On Metatolic Pastways

From rich lignocellulosic feedstock,the second-generation of bioethanol has became a major research focus to researchers.However,two problems:the high cost and lack of fermenting strains for pentose were the main obstacles to the current cellulosic ethanol fermentation process.Thermophilic anaerobic bacterium can metabolize almost all of lignocellulosic sugars to produce ethanol.They were one of the candidate microorganisms for ethanol fermentation.For lactic acid and acetic acid metabolic pastway competition of carbon and redox with ethanol,knockout the genes of lactate dehydrogenase and phosphate acetyltransferase are the major metabolic engineering strategys to increase ethanol.But the ethanol yeld of these muant types is still lower than that of Saccharomyces cerevisiae.Maybe we should find out the physiological functions and metabolic regulation mechanism of ldh and pta in thermophilic anaerobic bacterium primarily to provide a theory guidance to the konchout strategy.In this study,we knockouted the gene of acetate kinase(ack)in the acetate pastway of T.calidiforntis Rx1 preserved in the laboratory.Analysis the activity of the key enzymes in the carbon center metabolism and their expression were to study the impacts on the metabolic regulation of glycolysis and ethanol fermentation pastway.And the analysis the link between NADH/NAD +,ATP/ADP ratio and regulation of metabolism within the mutant and wild cell were done to explore the mechanism of metabolic regulation of ack gene.The results obtained were as follows:Firstly,based on acetyl phosphate acyltransferase gene(pta)and acetate kinase gene(ack)sequences,upstream and downstream of the homologous fragments were designed,and inserted into pBluescript ? SK(-)plasmid with a kanamycin resistance gene to rebuilded the recombinant vector pBSkpa.The recombinant vector pBSkpa was transformed into T.calidiforntis Rxl by electricity,and four strains of mutant type were screened preliminaryly.The mutant strains were verified by PCR,sequencing and metabolite analysis.One of the fastest growth rate of strains was named ?ack and used to explore the impacts of ack on metabolic pastways.Continuous cultivating ?ack for 30 generations,PCR results confirmed that the kanamycin resistance gene was still inserted in the genome,and its growth,the yeld of ethanol and acetic acid were also relatively stable.?ack is an ideal mutant strain.The lag phase of ?ack was longer than wild stain with glucose and xylose as substrate.The wild strain was 4%ethanol tolerance,and ?ack increased to 6%.Then glucose fermentation,cellobiose fermentation,xylose fermentation,acid hydrolyzate of corncob of ?ack mutant and the wild strain were performed respectively to produce ethanol and lactate.The results indicate that the acetate of ?ack mutant is much lower as compared with the wild.Dray cell weight of the mutant is always lower than that of the wild under four conditions.However,the yield of ethanol or lactate is more than the wild.When ?ack mutant used cellobiose to produce ethanol,the yield is 3.60 g/L higher than another three substrates.At the same time,it could be exist approximative 40 mM acetate in the hydrolysate,so the output of lactate and ethanol of the wild are more than that with xylose fermentation.Key enzymes of T.calidiforntis Rxl central carbon metabolism pathway include two rate-limiting enzymes glucokinase(GK),pyruvate kinase(PK)in the glycolytic pathway and lactate dehydrogenase(LDH),phosphate acetyltransferase(PTA),acetate kinase(ACK),aldehyde dehydrogenase(ALDH),alcohol dehydrogenase(ADH),pyruvate ferredoxin oxidoreductase(POR)in the fermentation metabolic pathway.Real time fluorescence quantitative PCR showed that the expression of ?ack key enzyme genes were lower than the wild strain,expect pta and adh.Enzymatic activity analysis was results that PK and POR of ?ack was higher than the wild,but the other enzymes were lower,expecially PTA reduced significantly.NADH,NAD+,ATP,ADP concentration were measured by HPLC to analyze the changes for NADH/NAD+ and ATP/ADP in mutant strain with disrupted ack dependent ATP.The results displayed that ATP/ADP ratio was almost equal in the exponential growth phase of the two type cells,and ATP/ADP ratio in the exponential growth phase was higher than that in stationary phases of wild-type strain,but mutant strain was just the opposite;for intracellular redox levels NADH/NAD+,NADH/NAD+ ratio in stationary phases was consistent with that in the exponential growth phase of wild-type strain,while that of the mutant strain was higher than in the exponential growth phase.