Developing Multi-Copy Chromosomal Integration Strategies for Heterologous Biosynthesis of Caffeic Acid in .

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 , using codon-optimized (, encoding tyrosine ammonia lyase) from , (encoding -coumaric acid 3-hydroxylase) and (encoding cytochrome P450 reductase 1) from in 2 μ multi-copy plasmids to produce caffeic acid from glucose. Then, integrated expression of delta integration with the gene (encoding triose phosphate isomerase) as selection marker and episomal expression of , 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 and into the chromosome of high -coumaric acid yielding strain QT3-20. The strain D9 constructed 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 synthesis of caffeic acid in 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 .