Regulation Of Histidine Kinases On Sporulation And Butanol Metabolism Of Clostridium Acetobutylicum

Butanol,an important industrial chemical and a promising biofuel,has been produced in acetone-butanol-ethanol(ABE)fermentation by solventogenic clostridia,mainly Clostridium acetobutylicum.The metabolism of C.acetobutylicum is typically characterized by a biphasic fermentation pattern with acidogenesis and solventogenesis.During acidogenesis,organic acid accumulation is associated with the drop of pH and unfavorable condition for cell growth,which causes the metabolism shift to butanol formation or trigger sporulation to increase survival ability in hostile environments.Naturally,sporulation is accompanying with solventogenesis,and eventually halts all metabolic activities associated with butanol biosynthesis,which limits its use in butanol production.Spo0A,a global transcriptional regulator in C.acetobutylicum,participates in regulation of various physiological functions including cell growth,butanol metabolism and sporulation in its phosphorylation state.Five histidine kinases including Cac3319,Cac0323,Cac0903,Cac2730 and Cac0437 were responsible for phosphorylating/dephosphorylating Spo0A.It is presumed that the Spo0A-P level regulated by histidine kinases engineering could determine cell fates between solventogenesis and sporulation for survival.Firstly,we analyzed the genetic background,sporulation and fermentation performance of C.acetobutylicum ATCC 55025.Comparative genomics analysis of ATCC 55025 and ATCC 824 revealed that they had high sequence identity and synteny.ATCC 55025 completed the formation of forespore in stage Ⅲ of sporulation cycle,but no mature spore formed.The cell morphology,metabolism and cell autolysis of ATCC 55025 were all similar with ATCC 824.Therefore,these two strains have similar genetic backgrounds,physiological and metabolic characteristics.C.acetobutylicum ATCC 55025 is the most suitable option to investigate the promoting or suppressing effect of HKs on sporulation and cell stability as it only forms forespore and can produce butanol in a continuous process.Secondly,the regulatory function of histidine kinases on cellular metabolism was investigated by constructing mutant strains with different histidine kinase expression levels.Except for cac2730,altering the expressions of other four histidine kinases had different effects on organic acid and butanol metabolisms,and cac3319 and cac0437 played the predominant role.The cac3319 knockout strain produced 49.1%more butanol(16.4 g/L)and 18.6%less acid(5.7 g/L)while the overexpression strain produced 44.5%less butanol(6.1 g/L)and 83%more acid(12.8 g/L)compared to ATCC 55025(11.0 g/L butanol and 7.0 g/L acid).All three cac0437 mutants produced less butanol(1.9-6.5 g/L)and more acid(11.4-14.9 g/L),suggesting that altering cac0437 expression was detrimental to acid reassimilation.Knocking out both cac3319 and cac0323 could further improve butanol titer to 18.2 g/L.Thirdly,we analyzed the cell morphologies and sporulation of cac3319KO,cac3319OS and cac0437KO,whose metabolites were significantly changed.cac3319 knockout displayed only the vegetative cell form throughout the fermentation without granulose accumulation,forespore formation or any cell autolysis in the decline phase,suggesting the cell did not initiated sporulation.On the contrary,overexpressing cac3319 in ATCC 55025 resulted in more granulose accumulation and obvious formation of spore cortex and spore coat in the solventogenic phase,which developed into mature spores in the decline phase.For cac0437 knockout,cells with a discernible phase-dark region at one pole of the cell were observed in the solventogenic phase.However,neither granulose accumulation nor septum was found in these cells,suggesting that sporulation initiated,but no further forespore development.The histidine kinase engineered strains without sporulation/degeneration showed enhanced cell stability and were stable for long-term culture.In high-cell-density fermentation,the cac3319/cac0323KO was able to produce up to 20.6 g/L with butanol yield and productivity of 0.23 g/g and 0.61 g/L/h,respectively.In repeated batch fermentation,the average butanol titer and productivity were up to 20.1 g/L and 0.46 g/L/h,which were 55.8%and 117.4%higher than those obtained in the parental strain.Enhancement of cell stability by histidine kinases engineering allowed for continuous biosynthesis of butanol during prolonged fermentation and improved the stability of the butanol production process.Finally,the transcriptional profiles of genes involved in sporulation and solventogenesis were analyzed with the RNA-Seq analysis to guide subsequent metabolic engineering work.For cac3319 knockout,all genes involved in cell division,sporulation and cell autolysis were differently down-regulated.The sol operon responsible for acid re-assimilation and solvent formation up-regulated 4-fold in the solventogenic phase,which directly contributed to enhanced butanol production.Considering that the acetone and hydrogen biosynthesis genes adc and hydA were both significantly down-regulated,knocking out these two genes might decrease by-product formation and promoting butanol biosynthesis.The acetone was decreased to 0.1 g/L and the butanol ratio was improved from 63.0%to 87.6%by knocking adc.The strain with hydA knockout produced 31.2%and 31.9%less hydrogen and acetone,respectively,and selectively promoted butanol production.RNA-Seq showed that knocking out cac3319 weakened the butanol synthesis pathway by adhE2,and overexpression of adhE2 in cac3319/cac0323 knockout strain further improved butanol production to 19.7 g/L.In this study,we demonstrated that engineering histidine kinases in C.acetobutylicum could regulate sporulation and butanol metabolism to improve cell stability and solventogenesis in ABE fermentation,enabling stable and energy-efficient butanol production.