Mycobacterium marinum CAR (MmCAR) is one of the most widely used CARs as the key enzyme for the synthesis of aldehydes, alcohols and further products from the respective carboxylic acids. Herein, we describe the first functionally secreted 131 kDa CAR and its isolated A-domain using Komagataella phaffii and a methanol-free constitutive expression strategy. Precipitated and lyophilized MmCAR (500 µg) was isolated from the culture supernatant and showed no decrease in activity for piperonylic acid (80% conversion), even when stored for up to 3 weeks at 4°C. Lyophilized MmCAR precipitate gave 48% yield of E/Z-nonanal-4-nitrobenzoyloxime from the reduction of nonanoic acid and in-situ derivatization with O-4-nitrobenzoyl-hydroxylamine. Furthermore, K. phaffii could successfully secrete the MmCAR adenylation domain. Its activity was confirmed by the amidation of benzoic acid with n-hexylamine. Neither enzyme variant was glycosylated by the yeast. In summary, functional CAR can be secreted by K. phaffii and used for cell free conversion of carboxylic acids to various products.

海分枝杆菌CAR ( Mm CAR) 是应用最广泛的 CAR 之一，作为合成醛、醇和相应羧酸的其他产品的关键酶。在此，我们使用Komagataella phaffii和无甲醇组成型表达策略描述了第一个功能性分泌的 131 kDa CAR 及其分离的 A 结构域。从培养物上清液中分离出沉淀和冻干的Mm CAR (500 µg)，即使在 4°C 下储存长达 3 周，胡椒酸的活性也没有降低（80% 转化率）。冻干的Mm CAR沉淀物通过壬酸的还原和用O -4-硝基苯甲酰基-羟胺的原位衍生化得到48%产率的E/Z-壬醛-4-硝基苯甲酰肟。此外，K. phaffii可以成功分泌Mm CAR 腺苷酸化结构域。其活性通过苯甲酸与正己胺的酰胺化得到证实。这两种酶变体都没有被酵母糖基化。总之，功能性CAR可以由K.phaffii分泌并用于将羧酸无细胞转化为各种产物。