Study On The Key Enzyme MIOX4 Of Glucaric Acid Synthesis And The Glucaric Acid Biosensor

Glucaric acid is an important value-added chemical,which is widely used in food,medicine and health care,textile and clothing.In our previous study,a series of metabolic engineering strategies has been adopted to improve the production of glucaric acid in Saccharomyces cerevisiae.The production of glucaric acid in the engineering strain of K4-D_ZWF1 in our laboratory reached to10.85 g·L^-1 in a 5 L fermenter.However,the low enzyme activity and poor stability of MIOX4 still cannot meet the application requirements.Therefore,it is of great significance to screen out MIOX4 with higher enzyme activity by the method of directed evolution.In addtion,the screening of mutant libraries in directed evolution is a key issue and the construction of glucaric acid biosensor in S.cerevisiae has not been reported yet.It is of great interest to develop a general method for constructing biosensors to identify their indicated chemical substances in eukaryotes.This paper mainly studies the following three aspects:（1）The inositol oxygenase MIOX4 was modified by directed evolution to improve its catalytic activity.S.cerevisiae was used as a chassis microorganism in this study.The key amino acid sequence of MIOX4 from Arabidopsis thaliana genome was randomly mutated by semi-rational evolution-error-prone PCR method.Using the production of glucaric acid as a screening index,the high-throughput screening of MIOX4 mutants was carried out using the Escherichia coli glucaric acid biosensor.The experimental results showed that 4 potential MIOX4 mutants（S133R,K88R,E72V,T40P）were screened from the mutant library,which increased the glucaric acid production by 53%,30%,21%and 17%,respectively,compared with the unmutated strains.Through structural analysis of the mutant MIOX4（S133R）,it was found that the mutant site R133 increased the steric distance between the site and the substrate inositol,thereby reducing the steric hindrance and improving the catalytic ability of the substrate inositol.（2）The glucaric acid-responsive promoter elements were analysed in S.cerevisiae by RNA-Seq technology and the glucaric acid biosensor was constructed based on these results.Through transcriptome sequencing,the expression of 36 genes was determined to be significantly up-regulated by glucaric acid.The GFP reporter plasmids were then constructed by using S.cerevisiae BY4741opi1Δas the host strain,and it was found that two promoters of P_YCR012W and P_YGL009C had the most significant response to glucaric acid.By examining their responses to different concentrations of glucaric acid,it was found that P_YCR012W had a dose-dependent effect on glucaric acid,and its linear relationship was also better than P_YGL009C.Therefore,P_YCR012W can be used as the response element for constructing glucaric acid biosensor.（3）The glucaric acid-responsive transcription factors were identified by truncating the glucaric acid-responsive promoter elements.By sequentially truncating 100 bp of the upstream region of the 5’site of P_YCR012W,it was found that when the truncation length exceeded 300 bp,the fluorescence dropped sharply,and the induction of the reporter gene in response to glucaric acid was inhibited.These results suggest that the-564 to-464 bp region in P_YCR012W may contain the DNA binding sites that responds to glucaric acid activators.By predicting the transcription factor binding sites in this region and then annalysing the response of P_YCR012W-GFP to glucaric acid in the mutants of knocking out transcription factor-encoding genes.It was found that two transcription factors,Ash1p and Cbf1p,might be involved in the glucaric acid response.In conclusion,a MIOX4（S133R）mutant was screened by directed evolution,which increased the production of glucaric acid by 53%compared with the wild type.A glucaric acid biosensor was constructed using the components of S.cerevisiae,and it was found that two proteins,Ash1p and Cbf1p,may be related to the response of glucaric acid.This study lays the foundation for the subsequent construction of more sensitive glucaric acid biosensor.