Study On Metabolic Engineering Of Saccharomyces Cerevisiae For Production Of Glucaric Acid

Glucaric acid,a kind of dicarboxylic acid present in nature,had application potential in pharmaceutical and polymer industry and was called one of the top value-added chemicals from biomass.Production of glucaric acid relied mainly on chemical oxidation,including nitric acid oxidation and TEMPO oxidation.However,chemical production had low selectivity and caused huge pollution to the environment.With the development of metabolic engineering and synthetic biology,metabolic engineering of microbes for production of glucaric acid provided a novel strategy for researchers.This study used Saccharomyces cerevisiae as chassis,and constructed glucaric acid synthetic pathway via expressing myo-inositol oxygenase miox4 gene from Arabidopsis Thaliana and Uronate dehydrogenase udh gene from Pseudomonas syringae.Given that MIOX4 was rate-limiting enzyme in this pathway and episomal plasmid expression in S.cerevisiae was instable,miox4 and udh were inserted into multi-copy delta site in yeast genome in this study,which increased exogenous gene copies.In addition,through fed-batch fermentation and fermentation optimization,the highest glucaric acid titer reached 6.01 g·L^-1.The results were as follows:?1?Construction of episomal plasmid expression S.cerevisiae strain for production of glucaric acid.Opi1 gene was knocked out to generate BY4741opi1?strain.pY26-miox4-6×HIS plasmid was constructed and transformed into the above strain and SDS-PAGE confirmed the successful expression of miox4.Then pY26-miox4-udh plasmid was constructed and transformed into BY4741opi1?strain.The presence of glucaric acid was detected via liquid chromatography-mass spectrometry and then 0.54 g·L^-11 of glucaric acid was determined via high performance liquid chromatography,suggesting glucaric acid production in S.cerevisiae.?2?Integration of miox4 and udh into delta site of the yeast genome to increase glucaric acid titer.miox4 and udh were both integrated into multi-copy delta site in yeast genome and then a strain with high titer was selected.Quantitive real-time PCR detection showed that relative gene transcription level of delta site constitutive expression was 5 times higher than that of episomal plasmid expression.MIOX4enzymatic activity assay showed that the MIOX4 activity was higher and more stable in delta site constitutive expression than that of episomal plasmid expression.It also revealed that myo-inositol was significant to maintain MIOX4 activity.?3?Fermentation optimization for production of glucaric acid.Through optimization of medium component and fed-batch fermentation conditions,the best culture medium and fermentation condition were set as follows:YPD medium supplemented with 10.8 g·L-1 myo-inositol,5 g·L^-1 glucose was fed at 24 h and 48 h respectively,and agitation rate and air flow rate were set as 700 r·min^-1 and 3L·min^-1,respectively.The highest glucaric acid titer reached 6.01 g·L^-1.