Optical Regulation And Optimization Of L-isopulegol Synthesis Pathway

l-isopulegol is widely used as a spice in medicine,food,beverage,household chemicals and cosmetics.In recent years,the demand for l-isopulegol has been increasing and naturally extracted menthol has not been able to meet the growing demand.Thus the economic significance of developing new synthetic methods for the l-isopulegol production is increasing.In our work,we designed a biosynthetic pathway for the production of l-isopulegol.The enzyme cascade reaction is divided into three steps.The enzyme ? GeDH(adhp)oxidizes the substrate geraniol to geranyl aldehyde,which is reduced to citronellal by the action of enzyme? OYE2p(ER3),and finally l-isopulegol is produced by the action of a cyclase.However,we found that the activity of the oxidase adhp in the first step was about 10 times higher than the reductase in the second step,so that the geranyl aldehyde formed in the first step could not be completely converted into citronellal in time,which result in a large accumulation of NADH.The NADH is utilized by enzyme ?(adhp)to convert the intermediate geranyl aldehyde and citronellal into by-products such as geraniol and citronellol,which reduces the efficiency of the multi-enzyme cascade and synthesis of the product.Here,we fous on the problem of enzyme mismatch between adhp and ER3.We use the light-sensitive protein AsLOV2 domain to insert into the unconservative sites of the adhp protein.The conformational change of the AsLOV2 domain under blue light illumination would cause structural perturbation of the natural protein.Thereby the adhp activity is inhibited by blue light excitation and the coenzyme NADH produced in the first step is only used by ER3,which minimizes the coenzyme spillover and reduces the occurrence of side reactions so that the cascade reaction system can more smoothly move toward the target product.The research includes the following aspects:1.Screening insertion sites of the AsLOV2 domain.Firstly,the insertion sites around catalytic active center of the protein adhp under the premise of not interfering with the protein structure was determined by crystal structure analysis and sequence alignment.Then the ten allosteric sites exposed on the surface of adhp proteins were screened and the AsLOV2 domain was successfully inserted into these ten sites to construct fusions named alovl-10,respectively.We screened alov9 from these 10 fusions used in next study.2.We achieve accurate reversible regulation of the protein alov9 on several factors,including optical inhibition,illumination time,light intensity,illumination distance,cofactor FMN concentration.3.We determine the photocontrol conditions for the extracellular catalytic reaction.Firstly,the optimum temperature and pH value of enzyme 1(adhp)and enzyme 2(OYE2p)reaction system were screened to determine the optimal reaction system.Then we explore the ratio of extracellular reaction of enzymeyl(adhp)and enzyme?(OYE2p).Finally,we determine the formation of each component during the reaction process by analyzing the reaction process curve,such as substrate consumption,product,by-product formation.The yield of by-product was reduced by a maximum of 54%compared to the blank group,while the yield of the product citronellal was increased by 31%.4.Extracellular optical catalysis experiments.During the course of the extracellular reaction,the reaction system was subjected to 470 nm blue light illumination.We hypothesized that the activity of alov9 protein was inhibited by blue light excitation and the reductase ? ER3 activity of the second step was matched,In summary,we successfully achieved photocontrol activity of adhp oxidase by using the light-sensitive protein AsLOV2 and solved the problem of enzyme mismatch in the process of multi-enzyme cascade.The formation of by-products was reduced,while the yield of citronellal was increased.Inserting the light-sensitive protein into the vicinity of the active site of the target protein to control the original enzyme,which is an innovative use of the light-sensitive protein.We believe this strategy will provide new ideas and methods for other metabolic regulation research.