紫苏酸已被研究作为抗癌和抗菌药物。紫苏酸的生产引起了人们的广泛关注。同时，热带假丝酵母是一种非常规的二倍体酵母，其最显着的特征是其代谢烷烃或脂肪酸以促进生长和增殖的能力。因此，本工作首先通过表达留兰香L-柠檬烯合酶基因LS_Ms，合成了热带念珠菌中紫苏酸的前体（L-柠檬烯）。编码tLS_Ms截短版本的基因的表达增加了L-柠檬烯的产量，滴度比热带念珠菌GJR-LS-01增加了2.78倍。与细胞质表达相比，tLS_Ms 基因的区室化表达抑制了热带念珠菌中 L-柠檬烯的产生。与区室化表达（线粒体或过氧化物酶体）相比，七个前体合成基因的细胞质过表达显着增强了热带念珠菌中 L-柠檬烯的产量，在热带念珠菌 GJR-tLS-01 中增加了 31.7 倍。在细胞质中过表达突变基因ERG20WW的热带念珠菌GJR-EW-tLS-04中，L-柠檬烯效价显着升高，是对照的11.33倍。与对照相比，L-柠檬烯对 60 g/L 葡萄糖的效价增加了 1.40 倍。最后，丹参细胞色素P450酶基因CYP7176和拟南芥NADPH细胞色素P450还原酶基因CPR在热带念珠菌GJR-EW-tLS-04C中异源表达，用于合成紫苏酸，滴度达到106.69mg/L。在 5 L 发酵罐中。这是在工程微生物中从头合成紫苏酸的第一份报告。结果还表明，热带假丝酵母可以有效地产生其他化学物质。要点： • 关键基因的细胞质表达有利于热带假丝酵母L-柠檬烯的生产。 • 紫苏酸首先是在工程微生物中从头合成的。 • 5 L 发酵罐中紫苏酸的滴度达到 106.69 mg/L。© 2024。作者。

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid's precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis. KEY POINTS: • Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis. • Perillic acid was first synthesized de novo in engineered microorganisms. • The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.© 2024. The Author(s).