The Construction And Optimization Of High Crocetin Producing Saccharomyces Cerevisiae

Crocetin,naturally exists in Crocus sativus L,has great potential applications in medical and food fields.Microbial production of crocetin has received increasing concern in recent years.However,only a patent from EVOVA Inc.and a report from Lou et al.have illustrated the feasibility of microbial biosynthesis of crocetin,but there was no specific titer data reported so far.Saccharomyces cerevisiae is generally regarded as food safety and productive host,and manipulation of key enzymes is critical to balance metabolic flux,consequently improve output.Therefore,to promote crocetin production in S.cerevisiae,all the key enzymes,such as Crt Z,CCD and ALD should be engineered combinatorially.In this study,the existing ?-carotene producing strain in our laboratory was used as the host,by combinatorial screening of Crt Zs and CCDs from different sources,screening the endogenous and exogenous ALD,and overexpression of CCD2 and Syn_ALD,the titer of crocetin was improved to 1219 ?g/L from 240 ?g/L.The crocetin yield per unit OD600 was further enhanced by 1.6 folds after deleting competition pathway gene.The evolution,structure and cofactor of restriction enzyme CCD were systematically analyzed,and the relationship between structure and function was studied.The Crt Z-CCD fusion was constructed in this study to explore the effect of the fusion order and expression way of the gene on crocetin production.The yield of crocetin was increased to 1646 ?g/L by fusion overexpressed Crt Z-CCD.Eventually,plackett-Burman design,the fastest-rise experiment,and Box-Behnken response surface methodology(RSM)was performed to optimize the fermentation medium,and the titer of crocetin reached to 1951 ?g/L,which is the highest crocetin titer at shake flask level reported in eukaryotic cell.In this study,the synthetic pathway of crocetin was optimized by the way of suitability research,restriction enzyme properties analysis and optimization of fermentation medium,which provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.