Directed Evolution Of Phosphoketolase And Applications Of 3-hydroxypropionic Acid Production

With the increasing depletion of fossil fuel consumption and environmental pollution,research on the synthesis of biochemicals replacing petroleum-based chemicals has received more attention.Saccharomyces cerevisiae,as a eukaryotic model strain,has become an attractuve platform strain for the production of biochemicals.Here,we focused on the directed evolution of phosphoketolase for production of 3-hydroxypropionic acid in S.cerevisiae.3-Hydroxypropionic acid is of great value as a bulk chemical.Our group have worked on the rewiring of central carbon metabolism of S.cerevisiae,and using 3-hydroxypropionic acid as a show case.In this process,we have found the potential application of the phosphoketolase pathway.However,this pathway also faces the limition of low enzyme activity.Here we used the directed evolution to screen for optimal mutations of phosphoketolase to improve the phosphoketolase activity.At present,we found that Leu504Met mutation site can enhance the enzyme activity.At the same time,we analyzed the mechanism of MCR,a key enzyme for 3-hydroxypropionic acid production.We have selected the genome integration method to express the N-terminal side of MCR,together with the plasmid method to express the C-terminal side MCR,and increased the 3-HP titer from 0.1 g/L to 1.5 g/L.Finally,we integrated the phosphoketolase mutation into the high 3-HP production strain,and increased the final titer to 2.2 g/L.In this work,we obtained both a high production of 3-hydroxypropionic acid and the reduced carbon loss.