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.
Highlights
Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound, widely occurring in plants, and the precursor of other natural products, such as chlorogenic acid, rosmarinic acid and caffeic acid phenethyl ester (Wu et al, 2011; David et al, 2015)
There has been considerable research effort applied to relieving retro-inhibition and redirecting carbon flux from glucose into aromatic amino acids (AAA) metabolism to maximize the pool of L-tyrosine (Tyr) and L-phenylalanine (Phe) (Huccetogullari et al, 2019; Liu Q. et al, 2019)
Episomal expression strains were constructed not affected by optimization of the L-tyrosine metabolic pathway, whereas the introduction of the caffeic acid hetero-synthesis pathway lowered the growth rates of NKP4-6, compared with NKC6 (Figure 2)
Summary
Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound, widely occurring in plants, and the precursor of other natural products, such as chlorogenic acid, rosmarinic acid and caffeic acid phenethyl ester (Wu et al, 2011; David et al, 2015). With the development of synthetic biology and metabolic engineering, heterologous biosynthesis of caffeic acid in microorganisms (e.g., Escherichia coli, Saccharomyces cerevisiae) provides a potential alternative source (Cao et al, 2020). As the precursors of caffeic acid in the plant phenylpropanoid pathway, aromatic amino acids (AAA; i.e., L-phenylalanine, L-tyrosine) can be produced by microorganisms through the shikimic acid pathway (Krivoruchko and Nielsen, 2015; Figure 1). Phenylalanine ammonia lyase (PAL) and two cytochrome P450 monooxygenases, cinnamate4-hydroxylase (C4H) and p-coumarate 3-hydroxylase (C3H) participate in the synthesis of caffeic acid from L- Phe (Kim et al, 2011; Figure 1). P-coumaric acid (pCA), the direct precursor of caffeic acid in plants, can be generated by microbial tyrosine ammonia lyase (TAL) from L-Tyr directly, instead of by C4H and PAL from L-Phe (Hernandez-Chavez et al, 2019). Jones et al (2017) achieved the highest caffeic acid titer (1.03 g/L) from a simple carbon source using EcHpaBC combined with optimization of culture conditions
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