Glucaric Acid Biosensor:Mechanism And Application In The Screening Of GA High-yielding Engineering Strains

Glucaric acid(GA)is a highly functionalized compound,widely used in different fields such as chemical industry,medicine and food industry.At present,the production of gluconic acid is mainly produced by chemical oxidation.,under drastic reaction conditions,suffering from low yield,expensive catalysts and generating numerous toxic by-products difficult to purify and harmful to environmental.These methods were limited in application,and thus the limitation of GA production and widely application of multi-function GA.With the development of synthetic biology,it is an effective way and an inevitable choice of green manufacturing in the future that engineering cells be used as miniature factorys,using natural boilogical synthetic pathways to produce high value products.The Prather team of the Massachusetts institute of technology constructed the glucaric acid synthesis pathway in E.coli and improved the yield of gluconic acid by a series of improvements.In the study of the microbial synthesis pathway of GA,researchers found that the inositol oxidase MIOX was the restriction enzyme for the synthesis pathway.Moreover,the yield cannot exceed 5 g/L,due to the restriction of the acid resistance capacity of E.coli.therefore,it is preferable to replace the host E.coli by S.cerevisiae or other species.However,the lack of effective method of high-throughput screening has become one of the problems in the study of high yield GA.In recent years,the team of George M.Church has constructed a series of biosensors that produce different fluorescence intensity based on the concentration of target products.And one of them is a biosensor for GA based on CdaR protein and a 521 bp promoter.However,the mechanism of the GA biosensor is not clear,and its components are all derived from E.coli,that restricted its application in other species.In view of the above problems,this project has carried out the following researches:(1)The GA biosensor mechanism is preliminarily revealed that GA concentration regulates the binding position of the GFP upstream operating sequence F1-521 where CdaR protein combined with,and then influences the transcription of downstream reporter gene.At first,it is determined that the binding positions of CdaR protein to F1-521 under different GA concentrations;then,based on the feature that GA biosensor can produce fluorescence response to GA and a series of nucleotide mutation,the HTH structure on CdaR combined with F1-521 was predicted and verified;finally,the function mechanism of the biosensor was analyzed with the characteristics of the upstream core sequence of GFP.This study laied a foundation for further optimizing the use of GA biosensors in the E.coli system and made possible the application of the GA biosensor into yeast and other species.(2)Based on GA biosensor system,one-pot two-strain method was established,and its effectiveness of high throughput screening of high-yielding GA engineering strains in E.coli and S.cerevisiae was tested.Metabolite biosensor system consists of two components:the synthetic pathway and the corresponding biosensor.There are two forms of observation methods:one is putting two components into one cell,named as one-pot one-strain,the other is putting them into two cells respectively,called as one-pot two-strain.This study shows that one-pot two-strain method has higher repeatability and sensitivity than one-pot one-strain method.And as a result of GA biosensor plasmid pJKR-H-cdaR can only be used in E coli system,one-pot two-strain method could be applied in S.cerevisiae system has has a wider range of application than one-pot one-strain method.(3)Three mutants with significantly improved enzyme activity were obtained by using one-pot two-strain method.Then it is determined that two mutations,D82Y and S173N,improved MIOX activity after in vitro activity of MIOX with singlie point mutation was measured,and the activity of MIOX was increased 3 times by D82Y and 1.5 times by S173N.The combination of D82Y and S173N increased GA yield about 3 times compared with EA31.(4)We added INO1 or NOX or both of them to the GA synthesis pathway composed of MIOX and Udh and introduced these new ways into S.cerevisiae.Then G A production of the series of S.cerevisiae strains were detected via one-pot two-strain method.In this work,the introduction of INO1 increases the substrate of GA synthesis by converting glucose to MI,the introduction of NOX controled by different promoters alleviated the metabolic burden of the bacteria.In conclusion,GA yield was improved about 75%by INO1 and GPD2-NOX and was improved about 35%by INO1 and TEF1-NOX.