Abstract:[Background] Caffeic acid (3,4-dihydroxycinnamic acid), a natural phenolic compound, has various biological activities and great medicinal value. Candida glycerinogenes, which harbors the caffeic acid precursor metabolic pathway, is a potential high caffeic acid-yielding chassis cell owing to the high acid tolerance, fast growth, and high fermenting rate. However, it has no episomal vectors, which limits the in-depth research on caffeic acid synthesis. [Objective] To explore whether it is feasible to construct episomal vectors with stronger expression of caffeic acid in C. glycerinogenes without natural episomal plasmids in a simpler way. [Methods] Autonomously replicating sequences (ARSs) were selected to establish the episomal plasmid for the synthesis of caffeic acid in C. glycerinogenes, and the plasmid was further optimized by modifying its ARS position, the marker gene promoter length, elements for gene expression, and using Kozak sequence. [Results] Of the five vectors constructed containing different ARSs, pTGAPU-CA-AOX1t-KLARS was able to self-replicate and express the genes for the synthesis of caffeic acid in C. glycerinogenes. After the vector was modified in different ways, such as ARS located upstream of the target gene expression element, URA5 promoter truncated by 250 bp, using Kozak sequence or URA5 terminator, the yield of caffeic acid was significantly higher. The maximum yield after the modification was 3.73 times higher than the initial yield, reaching 29.1 mg/L. [Conclusion] Caffeic acid was synthesized by episomal expression in C. glycerinogenes, with better results than that by integrated expression, which provides a new tool for future modification of the caffeic acid anabolic pathway with episomal vectors and a reference for the construction of episomal expression systems in other strains without episomal plasmids.
