Abstract Glycerol is the main byproduct of the biodiesel production process and can be converted into valuable products by Yarrowia lipolytica. Betulinic acid is a pentacyclic triterpene with a variety of pharmacological properties. Herein we report heterologous biosynthesis of betulinic acid in Y. lipolytica by co-expressing lupeol synthase opAtLUP1, NADPH-cytochrome P450 monooxygenase opCYP716A12, and NADPH-cytochrome P450 reductase opAtCPR1. Initially, the engineered Y. lipolytica produced 0.32 mg/L betulinic acid. This titer was increased to 9.41 mg/L following P450 enzyme fusion and overexpression of key genes from the upstream mevalonate (MVA) pathway. Substitution of glycerol for glucose further enhanced betulinic acid production to 16.98 mg/L, 1.8-fold higher than the titer obtained with glucose. 26.53 mg/L betulinic acid was obtained with 40 g/L glycerol as the sole carbon source, which was comparable to the titer obtained from Saccharomyces cerevisiae with 50 g/L glucose in shake flask cultivation. Glycerol improved betulinic acid titer by increasing the expression of key genes in the MVA pathway, and by increasing the supply of acetyl-CoA. Our study highlights the possible applications of glycerol as carbon source in the production of valuable terpenoids by engineered Y. lipolytica.

中文翻译：

---

甘油改善工程化解脂耶氏酵母中桦木酸的异源生物合成

摘要 甘油是生物柴油生产过程中的主要副产品，可通过解脂耶氏酵母转化为有价值的产品。桦木酸是一种具有多种药理特性的五环三萜。在此，我们报告了通过共表达羽扇豆醇合酶 opAtLUP1、NADPH-细胞色素 P450 单加氧酶 opCYP716A12 和 NADPH-细胞色素 P450 还原酶 opAtCPR1 在解脂耶氏酵母中进行桦木酸的异源生物合成。最初，工程化解脂耶氏酵母产生 0.32 mg/L 桦木酸。在 P450 酶融合和上游甲羟戊酸 (MVA) 途径的关键基因过表达后，该滴度增加至 9.41 mg/L。用甘油代替葡萄糖进一步提高桦木酸产量至 16.98 mg/L，比用葡萄糖获得的效价高 1.8 倍。26. 以 40 g/L 甘油为唯一碳源获得 53 mg/L 桦木酸，与在摇瓶培养中用 50 g/L 葡萄糖从酿酒酵母中获得的滴度相当。甘油通过增加 MVA 途径中关键基因的表达和增加乙酰辅酶 A 的供应来提高桦木酸滴度。我们的研究强调了甘油作为碳源在通过工程化解脂耶氏酵母生产有价值的萜类化合物中的可能应用。