Studies On Carbon Catabolite Repression And Dynamic Cell Responses Under Hyperosmotic Stress Of Thermoanaerobacterium Sp.

Thermoanaerobes has gained great attention from biofuel researchers over the past decade years,motivated by their broader substrate spectra,simpler metabolites and higher product yields.Thermoanaerobacterium aotearoense LA1002 is capable of producing lactic acid and hydrogen by directly fermenting various single carbon sources,such as glucose,xylose,cellobiose and glucan,with the yield of lactic acid is very close to the theoretical value?1.0 g/g of glucose?,which has the potential to apply in industry.This study aims to investigate and promote efficiency of carbon metabolism mechanism in T.aotearoense LA1002.Carbon catabolite repression?CCR?is widely observed in bacteria,which is one kind of adaptive carbon regulation mechanisms.Although the physiological outcome is similar,the global mechanisms underlying CCR are completely different in diverse bacteria.The co-culture study of mixed sugars showed that the utilization of cellobiose,galactose and lactose were strongly inhibited by 2-deoxyglucose and glucose in LA1002,which meant obvious CCR existed in this bacterium.Thus,the CCR related genes,ptsH and hprK were confirmed to exist in T.aotearoense LA1002 through gene cloning and protein characterization in this study.The highly purified Histidine-containing Protein?HPr?could be specifically phosphorylated at Serine 46 by HPr kinase/phosphatase?HPrK/P?with no need to add fructose-1,6-bisphosphate?FBP?or glucose-6-phosphate?Glc-6-P?in the reaction mixture.The specific protein-interaction of catabolite control protein A?CcpA?and phosphorylated HPr was proved via affinity chromatography and the equilibrium binding constant?K_D?was determined as 2.22±0.36 nM by surface plasmon resonance?SPR?analysis.The research above suggested that the CCR of T.aotearoense LA1002 belongs to that kind of Firmicutes,and was helpful to further study of relief CCR and co-utilization of mixed sugars.On the other hand,substrate repression would lead to a reduction in cell growth,fermentation rate and production capacity in industrial microbial fermentation process.To relieve the inhibition of high concentration substrate?>50 g/L sugars?to the engineered lactate-production strain T.aotearoense LA1002,the adaptive evolution to high sugar medium was employed to gain a stable,rapid carbon metabolism mutated strain,named LA1002-G40.When supplied with 100 g/L carbon source?90 g/L glucose and 10 g/L fructose?in serum bottle,there is no obvious lag phase existed in LA1002-G40 after inoculation,and OD₆₀₀ reached about 1.3 at 24 h,with no growth observed in the original LA1002 under the same condition.Moreover,fed by mixed bakery waste?MBW?as substrates?75 g/L glucose and 16 g/L fructose?,the fermentation could finish in 48 h,and the titer and yield of lactic acid were 68.03±0.77 g/L and 0.79±0.03 g/g,respectively,with a good stability between batches.Transcriptome analysis showed that there were 239 differential expression genes,and6 metabolic pathways enriched by at least 5 altered genes.Intracellular solute pools analysis confirmed the hypothesis from transcriptome analysis.Under high concentration substrate,the glucosyl donor UDP-Glucose of LA1002-G40 was increased by 1.88-fold.As important compatible solutes,glutamate and glycine in LA1002-G40 were improved by 2.90-and1.42-fold,respectively,as well as the related genes about DNA repair,compared with the original strain LA1002.This study provided a good example to Thermoanaerobes for microorganism industrialization,and indicated directions to rational engineering of strain for the relief of inhibition of high sugar medium.