Aims: To overcome the defective unstable production of p-coumaric acid (p-CA) using episomal plasmids and simultaneously achieve genetic stability and high-copy integration in Saccharomyces cerevisiae.
Methods and results: Two-micron plasmids were used to obtain high titres of p-CA, but p-CA production was decreased significantly in a nonselective medium after 72 h. To overcome the defect of unstable p-CA production during fermentation, delta integration with the triosephosphate isomerase gene from Schizosaccharomyces pombe (POT1) was employed as a selection marker to integrate heterologous p-CA synthesis cassette, and the high-level p-CA-producing strain QT3-20 was identified. In shake flask fermentation, the final p-CA titre of QT3-20 reached 228.37 mg L-1 at 168 h, 11-fold higher than integrated strain QU3-20 using URA3 as the selective marker, and 9-fold higher than the best-performing episomal expression strain NKE1. Additionally, the p-CA titre and gene copy number remained stable after 100 generations of QT3-20 in a nonselective medium.
Conclusion: We achieved high-copy genome integration and stable heterologous production of p-CA via a POT1-mediated strategy in S. cerevisiae.
Significance and impact of study: With superior genetic stability and production stability in a nonselective medium during fermentation, the high-level p-CA-producing strain constructed via POT1-mediated delta integration could serve as an efficient platform strain, to eliminate the threat of unstable and insufficient supply for future production of p-CA derivatives, make downstream processing and biosynthesis much simpler.
Keywords: POT1; fermentation; metabolic engineering strategy; p-coumaric acid; yeast cell factory.
© 2022 Society for Applied Microbiology.