Zn²⁺-mediated Regulation And Underlying Mechanism For ABE Fermentation By Clostridium Acetobutylicum

Biobutanol,as one of the promising alternatives for fossil fuels,is subjected to several obstacles from the viewpoint of industrialization.On the othe hand,Zn²⁺ is not only an essential micronutrient,but also a key cofactor due to its pleiotropic roles in global levels for microbial metabolism.Therefore,investigation of Zn²⁺-mediated responses on cellular metabolism of Clostridium acetobutylicum is of great importance for improving butanol production through acetone-butanol-ethanol?ABE?fermentation.The impact of Zn²⁺ on ABE fermentation by C.acetobutylicum was investigated from the viewpoint of bioprocess engineering,and significant improvement on butanol production was observed when 1 mg/L ZnSO4*7H2O was supplemented into the fermentation medium,which might be due to the pleiotropic role of Zn²⁺ in cell growth,sugar utilization,acids re-assimilation,and ABE biosynthesis,as well as enhanced tolerance of the cells to formate,acetate,butyrate and butanol.As a result,butanol productivity of 0.32 g/L/h was achieved compared to 0.18 g/L/h in the control without zinc supplementation.More importantly,micronutrient Zn²⁺ contributed to the enhanced butanol tolerance of as high as 18 g/L butanol compared to that of 15 g/L in the control without Zn²⁺ supplementation.Furthermore,synergy of Zn²⁺ and CaCO3 on xylose utilization and butanol tolerance was also investigated.LC-ESI/MS analysis for the key intracellular metabolites indicated that Zn²⁺-mediated regulation might act on the central carbon metabolism associated with glycolysis,acidogenesis and solventogenesis at global levels.At the early fermentation stage under the Zn²⁺ supplementation condition,accumulation of glycolysis-derived metabolites such as G6P,F6P,FBP and PEP was drastically decreased,which consequently lowered the level of branch node intermediates AcCoA,AcAcCoA and BuCoA,but ATP and NADH levels were significantly increased,leading to carbon flux redistribution and an earlier initiation of solventogenesis for intracellular redox rebalance.Global transcriptional analysis was further performed to elucidate the Zn²⁺-mediated response in the central carbon,energy and redox metabolism.Under the Zn²⁺ supplementation condition,expression of multiple genes responsible for sugar?disaccharides/hexose/pentose?transport and metabolism were upregulated.Especially,glucose-PTS related genes CAC0570?glcG,12.25-fold?,CAC1353 and CAC1354,fructose-specific operon fru and 14 genes associated with xylose/arabinose transport and metabolism were significantly upregulated.On the other hand,the expression levels of 13 genes involved in glycolysis,acidogenesis and solventogenesis were also differentially upregulated while genes hydA for H2 biosynthesis,abrB,ctsR and hrcA related to transcription regulator were downregulated.In order to validate the Zn²⁺-mediated regulatory mechanism,genetic engineered strains were constructed by overexpressing g/cG,CAC 1353 and CAC 1354 in Cacetobutylicu.respectively.After overexpressing CAC1353 or CAC1354,cell growth and glucose utilization were slowed down with an extended lag phase observed.Surprisingly,overexpressing glcG led to more efficient ABE fermentation with fermentation time shortened to 28 h with butanol production and productivity increased to 13.9 g/L and 0.50 g/L/h respectively.Moreover,overexpressing glcG also contributed to enhanced formate,acetate,butyrate and butanol tolerance of C.acetobutylicum.Especially,under 7.5 g/L acetate stress condition,as high as 16.4 g/L butanol was achieved by C.acetobutylicum L7-glcG?with glcG overexpression?.Based on LC-ESI/MS analysis,overexpressing glcG might result in a cascade effect on the central carbon flux and cofactor production/regeneration associated with energy and redox metabolism?ATP and NADH?at global levels,suggesting glcG was substantially involved in the Zn²⁺-mediated regulatory mechanism for ABE fermentation by C.acetobutylicum.For batch ABE fermentation using the enzymatic hydrolysate of pretreated com stover,butanol productivity as high as 0.44 g/L/h was achieved by C.acetobutylicum with glcG overexpression compared to only 0.18 g/L/h achieved by the control strain,making ABE fermentation from lignocellulosic biomass more economically competitive.