背景:丁香酸(SA)是一种高价值的天然化合物,具有多种生物活性和广泛的应用,常见于水果中,蔬菜,和草药。SA主要通过化学合成生产,尽管如此,这些化学方法有很多缺点,例如相当大的设备要求,苛刻的反应条件,昂贵的催化剂,和许多副产品。因此,在这项研究中,通过使用工程化的全细胞设计和开发了一种用于SA生产的新型生物转化途径。
结果:一种来自乙氧菌(DesAOMT)的O-甲基转移酶,优先催化邻苯二酚类似物的间羟基上的甲基转移反应,已确定。当S-腺苷甲硫氨酸(SAM)用作甲基供体时,表达DesAOMT的全细胞可以将没食子酸(GA)转化为SA。我们在大肠杆菌中构建了多酶级联反应,含有内源性莽草酸激酶(AroL)和分支酸裂解酶(UbiC),以及来自荧光假单胞菌的对羟基苯甲酸羟化酶突变体(PobA**),和DesAOMT;SA是通过全细胞催化由莽草酸(SHA)生物合成的。底盘细胞的代谢系统也影响了SA生物合成的效率,阻断分支酸盐代谢途径提高了SA的产量。当辅因子NADPH的供应优化时,SA的滴度达到133μM(26.2mg/L)。
结论:总体而言,我们通过使用静止或生长的全细胞在大肠杆菌中设计了用于SA生物合成的多酶级联。这项工作确定了O-甲基转移酶(DesAOMT),可以催化GA的甲基化产生SA。包含在工程大肠杆菌中表达的四种酶的多酶级联,用于从SHA合成SA。菌株的代谢系统和生物转化条件影响催化效率。本研究为SA的生物合成提供了一条新的绿色路线。
BACKGROUND: Syringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells.
RESULTS: An O-
methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA**) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 μM (26.2 mg/L).
CONCLUSIONS: Overall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-
methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.