High‐copy genome integration and stable production of p‐coumaric acid via a POT1‐mediated strategy in Saccharomyces cerevisiae

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.