The Construction And Optimization Of High Production Of Caffeic Acid In Saccharomyces Cerevisiae

Caffeic acid naturally existing in many plants has a wide range of medicinal potential.As Saccharomyces cerevisiae is currently recognized as an engineering host with high safety and productivity,this study used Saccharomyces cerevisiae as a substrate cell for the systematic rational design and optimization of caffeic acid heterologous synthetic pathways in order to obtain high-yield caffeic acid yeast strains.Since key cytochrome P450 enzymes generally do not function well enough in S.cerevisiae cells,the engineered biosynthesis of plant-derived natural products in genetically modified microorganisms faces many challenges.In this study,BY4741,an frequently-used strain of Saccharomyces cerevisiae,was used as the substrate cell.Two part of enzymes 4-hydroxyphenylacetic acid 3-hydroxylase,HpaB and HpaC,were recruited from several microbial sources to construct the whole functional 4-hydroxyphenylacetic acid 3-hydroxylase in Saccharomyces cerevisiae.In combination with tyrosine ammonia lyase,two enzymes from different sources exhibit significant ability to produce caffeic acid from the substrate L-tyrosine.We obtained the most compatible enzyme combination of HpaB from Pseudomonas aeruginosa(PahpaB)and HpaC from Salmonella enterica(SehpaC),and the production of caffeic acid was up to 40 times higher than the combination of HpaB and HpaC both from Escherichia coli and achieved biosynthesis of caffeic acid in eukaryotic yeast during shake flasks culture.The highest production of caffeic acid was 289.4 mg/L ± 4.6 mg/L.This means that we have effectively improved the compatibility and expression ability of the heterologous enzymes with the yeast substrate.By protein homology-modeling analysis we found six key amino acid residues around the flavin adenine dinucleotide(FAD)binding domain in HpaB and thought they might had a key effect on enzyme activity and the entire metabolic pathway.We have established an effective approach to construct a compatible synthetic biological system for the synthesis of non-native hydroxyphenylpropanoids caffeic acid.