在无天然游离质粒产甘油假丝酵母中非整合表达合成咖啡酸

【背景】咖啡酸（3，4-二羟基肉桂酸）是一种有多种生物活性和药用价值的天然酚类化合物，产甘油假丝酵母（Candida glycerinogenes）具有咖啡酸前体代谢途径，高耐酸且生长与发酵速率快，是潜在高产咖啡酸的底盘细胞，但无游离载体将影响咖啡酸合成的深入研究。【目的】探索在无天然游离质粒的C. glycerinogenes中构建操作更简便、表达能力更强的游离载体合成咖啡酸的可行性。【方法】筛选自主复制序列（autonomously replicating sequence，ARS），构建适用于C. glycerinogenes合成咖啡酸的游离载体，并通过改造其ARS位置、标记基因URA5启动子长度、基因表达元件和利用Kozak序列优化表达并合成咖啡酸。【结果】构建的5个分别含不同ARS的载体中，pTGAPU-CA-AOX1t-KLARS在C. glycerinogenes中能自我复制并表达合成咖啡酸的基因，而且当ARS位于目的基因表达元件上游、URA5启动子截短250 bp，或分别采用Kozak序列与终止子URA5t后，咖啡酸产量较改造前均有明显提升，最高产量为初始产量的3.73倍，达29.1 mg/L，高于前期整合表达产量。【结论】在C. glycerinogenes中非整合表达合成咖啡酸且优于整合表达，为今后利用游离载体改造咖啡酸合成代谢途径提供了新工具，同时为其他无游离质粒菌株构建非整合表达体系提供参考。

Synthesis of caffeic acid by episomal expression in Candida glycerinogenes without natural episomal plasmids

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.