AUTHOR=Qi Hang , Yu Long , Li Yuanzi , Cai Miao , He Jiaze , Liu Jiayu , Hao Luyao , Xu Haijin , Qiao Mingqiang 

TITLE=Developing Multi-Copy Chromosomal Integration Strategies for Heterologous Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.851706

DOI=10.3389/fmicb.2022.851706

ISSN=1664-302X

ABSTRACT=<p>Caffeic acid, a plant-sourced phenolic compound, has a variety of biological activities, such as antioxidant and antimicrobial properties. The caffeic acid biosynthetic pathway was initially constructed in <italic>S. cerevisiae</italic>, using codon-optimized <italic>TAL</italic> (<italic>coTAL</italic>, encoding tyrosine ammonia lyase) from <italic>Rhodobacter capsulatus</italic>, <italic>coC3H</italic> (encoding <italic>p</italic>-coumaric acid 3-hydroxylase) and <italic>coCPR1</italic> (encoding cytochrome P450 reductase 1) from <italic>Arabidopsis thaliana</italic> in 2 μ multi-copy plasmids to produce caffeic acid from glucose. Then, integrated expression of <italic>coTAL via</italic> delta integration with the <italic>POT1</italic> gene (encoding triose phosphate isomerase) as selection marker and episomal expression of <italic>coC3H</italic>, <italic>coCPR1</italic> using the episomal plasmid pLC-c3 were combined, and caffeic acid production was proved to be improved. Next, the delta and rDNA multi-copy integration methods were applied to integrate the genes <italic>coC3H</italic> and <italic>coCPR1</italic> into the chromosome of high <italic>p</italic>-coumaric acid yielding strain QT3-20. The strain D9 constructed <italic>via</italic> delta integration outperformed the other strains, leading to 50-fold increased caffeic acid production in optimized rich media compared with the initial construct. The intercomparison between three alternative multi-copy strategies for <italic>de novo</italic> synthesis of caffeic acid in <italic>S. cerevisiae</italic> suggested that delta-integration was effective in improving caffeic acid productivity, providing a promising strategy for the production of valuable bio-based chemicals in recombinant <italic>S. cerevisiae</italic>.</p>