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

Abstract

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 S. cerevisiae, using codon-optimized TAL (coTAL, encoding tyrosine ammonia lyase) from Rhodobacter capsulatus, coC3H (encoding p-coumaric acid 3-hydroxylase) and coCPR1 (encoding cytochrome P450 reductase 1) from Arabidopsis thaliana in 2 μ multi-copy plasmids to produce caffeic acid from glucose. Then, integrated expression of coTAL via delta integration with the POT1 gene (encoding triose phosphate isomerase) as selection marker and episomal expression of coC3H, coCPR1 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 coC3H and coCPR1 into the chromosome of high p-coumaric acid yielding strain QT3-20. The strain D9 constructed via 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 de novo synthesis of caffeic acid in S. cerevisiae 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 S. cerevisiae.

Enzyme and Pathway Engineering for Improved Betanin Production in Saccharomyces cerevisiae

“…The delta loci was often used for multicopy integration to enhance the expression of the target genes . After sequential optimization of the precursor l -tyrosine pathway for betanin biosynthesis, we proceeded to overexpress the OfCYP76AD8M10 , CcCYP76AD4 M465D , and MjCDGT genes at the delta loci of strain BEW7.…”

“…The delta loci was often used for multicopy integration to enhance the expression of the target genes. 32 After sequential optimization of the precursor L-tyrosine pathway for betanin biosynthesis, we proceeded to overexpress the Of CY-P76AD8M10, CcCYP76AD4 M465D , and MjCDGT genes at the delta loci of strain BEW7. Forty-five clones were randomly selected for fermentation, and the highest titer of betanin was 56.9 mg/L with clone 26 (Figure 3D), which was designated as strain BEW8 with 68% higher than that of strain BEW7.…”

Simple phenylpropanoids: recent advances in biological activities, biosynthetic pathways, and microbial production

“…141 Recently, the multi-copy integration expression strategies based on delta sites were applied to integrate the genes of the CaA pathway into yeast and resulted in an increase of CaA production by 50 times compared to that produced by the initial multi-copy-plasmid expression. 142 Some other carbon sources and the whole-cell biocatalyst strategy have also been successfully used for CaA synthesis. 143,144 By introducing the xylose assimilation pathway into Candida glycerinogenes, the advantage of using mixed sugars as carbon sources showed that the optimized strain eventually obtained 1.2-fold higher CaA than that using glucose.…”