Background:In vitro reconstitution of an artificial metabolic pathway has emerged as an alternative approach to conventional in vivo fermentation-based bioproduction.Particularly,employment of thermophilic and hyperth...Background:In vitro reconstitution of an artificial metabolic pathway has emerged as an alternative approach to conventional in vivo fermentation-based bioproduction.Particularly,employment of thermophilic and hyperthermophilic enzymes enables us a simple preparation of highly stable and selective biocatalytic modules and the construction of in vitro metabolic pathways with an excellent operational stability.In this study,we designed and constructed an artificial in vitro metabolic pathway consisting of nine(hyper)thermophilic enzymes and applied it to the conversion of glycerol to lactate.We also assessed the compatibility of the in vitro bioconversion system with methanol,which is a major impurity in crude glycerol released from biodiesel production processes.Results:The in vitro artificial pathway was designed to balance the intrapathway consumption and regeneration of energy and redox cofactors.All enzymes involved in the in vitro pathway exhibited an acceptable level of stability at high temperature(60℃),and their stability was not markedly affected by the co-existing of up to 100 mM methanol.The one-pot conversion of glycerol to lactate through the in vitro pathway could be achieved in an almost stoichiometric manner,and 14.7 mM lactate could be produced in 7 h.Furthermore,the in vitro bioconversion system exerted almost identical performance in the presence of methanol.Conclusions:Many thermophilic enzymes exhibit higher stability not only at high temperatures but also in the presence of denaturants such as detergents and organic solvents than their mesophilic counterparts.In this study,compatibilities of thermophilic enzymes with methanol were demonstrated,indicating the potential applicability of in vitro bioconversion systems with thermophilic enzymes in the conversion of crude glycerol to value-added chemicals.展开更多
Bio-based chemical production has drawn attention regarding the realization of a sustainable society.In vitro metabolic engineering is one of the methods used for the bio-based production of value-added chemicals.This...Bio-based chemical production has drawn attention regarding the realization of a sustainable society.In vitro metabolic engineering is one of the methods used for the bio-based production of value-added chemicals.This method involves the reconstitution of natural or artificial metabolic pathways by assembling purified/semi-purified enzymes in vitro.Enzymes from distinct sources can be combined to construct desired reaction cascades with fewer biological constraints in one vessel,enabling easier pathway design with high modularity.Multiple modules have been designed,built,tested,and improved by different groups for different purpose.In this review,we focus on these in vitro metabolic engineering modules,especially focusing on the carbon metabolism,and present an overview of input modules,output modules,and other modules related to cofactor management.展开更多
基金supported in part by the Japan Science and Technology Agency,PRESTO/CREST programsupported by the Japan Society for the Promotion of Science,KAKENHI Grant(26450088).
文摘Background:In vitro reconstitution of an artificial metabolic pathway has emerged as an alternative approach to conventional in vivo fermentation-based bioproduction.Particularly,employment of thermophilic and hyperthermophilic enzymes enables us a simple preparation of highly stable and selective biocatalytic modules and the construction of in vitro metabolic pathways with an excellent operational stability.In this study,we designed and constructed an artificial in vitro metabolic pathway consisting of nine(hyper)thermophilic enzymes and applied it to the conversion of glycerol to lactate.We also assessed the compatibility of the in vitro bioconversion system with methanol,which is a major impurity in crude glycerol released from biodiesel production processes.Results:The in vitro artificial pathway was designed to balance the intrapathway consumption and regeneration of energy and redox cofactors.All enzymes involved in the in vitro pathway exhibited an acceptable level of stability at high temperature(60℃),and their stability was not markedly affected by the co-existing of up to 100 mM methanol.The one-pot conversion of glycerol to lactate through the in vitro pathway could be achieved in an almost stoichiometric manner,and 14.7 mM lactate could be produced in 7 h.Furthermore,the in vitro bioconversion system exerted almost identical performance in the presence of methanol.Conclusions:Many thermophilic enzymes exhibit higher stability not only at high temperatures but also in the presence of denaturants such as detergents and organic solvents than their mesophilic counterparts.In this study,compatibilities of thermophilic enzymes with methanol were demonstrated,indicating the potential applicability of in vitro bioconversion systems with thermophilic enzymes in the conversion of crude glycerol to value-added chemicals.
基金Research in our laboratory was supported in part by the Japan Science and Technology Agency(PRESTO,CREST,and A-STEP programs)and the Japan Society for the Promotion of Science(KAKENHI program).
文摘Bio-based chemical production has drawn attention regarding the realization of a sustainable society.In vitro metabolic engineering is one of the methods used for the bio-based production of value-added chemicals.This method involves the reconstitution of natural or artificial metabolic pathways by assembling purified/semi-purified enzymes in vitro.Enzymes from distinct sources can be combined to construct desired reaction cascades with fewer biological constraints in one vessel,enabling easier pathway design with high modularity.Multiple modules have been designed,built,tested,and improved by different groups for different purpose.In this review,we focus on these in vitro metabolic engineering modules,especially focusing on the carbon metabolism,and present an overview of input modules,output modules,and other modules related to cofactor management.
