3个植物源苯甲醇酰基转移酶合成乙酸肉桂酯的研究

Biosynthesis of cinnamyl acetate by three plant-derived benzyl acyltransferases in engineered Escherichia coli

摘要:

[背景] 乙酸肉桂酯是一种重要的香料化合物，在化妆品和食品工业上具有广泛的应用，传统的生产方法主要依靠植物提取和化学合成。[目的] 通过筛选不同植物源的酰基转移酶，利用大肠杆菌从头合成乙酸肉桂酯。[方法] 首先，通过在苯丙氨酸高产菌BPHE中表达异源基因苯丙氨酸解氨酶（Phenylalanine Ammonia-Lyase from Arabidopsis thaliana，AtPAL）、对羟基肉桂酰辅酶A连接酶（Hydroxycinnamate：CoA Ligase from Petroselinum crispum，Pc4CL）和肉桂酰辅酶A还原酶（Cinnamyl-CoA Reductase from Arabidopsis thaliana，AtCCR），并结合大肠杆菌自身的内源性醇脱氢酶（Alcohol Dehydrogenases，ADHs）或醛酮还原酶（Aldo-Keto Reductases，AKRs）的催化作用构建了从苯丙氨酸到肉桂醇的生物合成途径。然后，苯甲醇苯甲酰转移酶（Benzyl Alcohol O-Benzoyltransferase from Nicotiana tabacum，ANN09798；Benzyl Alcohol O-Benzoyltransferase from Clarkia breweri，ANN09796）或苯甲醇乙酰转移酶（Benzyl Alcohol Acetyltransferase from Clarkia breweri，BEAT）被引入到上述重组大肠杆菌中发酵培养生产乙酸肉桂酯。最后，在大肠杆菌中过表达乙酰辅酶A合成酶（Acetyl Coenzyme A Synthetase，ACS）来提高底物乙酰辅酶A的量。[结果] 探讨了3个植物源苯甲醇酰基转移酶生物合成乙酸肉桂酯的能力，并应用于合成乙酸肉桂酯的细胞工厂，最终使乙酸肉桂酯最高产量达到166.9±6.6 mg/L。[结论] 植物源苯甲醇酰基转移酶具有一定的底物宽泛性，能以肉桂醇为底物，催化合成乙酸肉桂酯。首次利用植物源的苯甲醇酰基转移酶合成乙酸肉桂酯，为微生物细胞工厂以葡萄糖作为碳源生产乙酸肉桂酯提供参考。

Abstract:

[Background] Cinnamyl acetate is an important flavor compound and widely used in cosmetics and food industries. The traditional production methods include direct extraction from plants and chemical synthesis. [Objective] In this work, we aim to achieve de novo biosynthesis of cinnamyl acetate in Escherichia coli by screening benzyl alcohol O-acyl transferases from different plants and constructing biosynthetic pathway for cinnamyl acetate. [Methods] First, a biosynthetic pathway of aglycon cinnamyl alcohol from phenylalanine was constructed in the high-phenylalanine-producing E. coli strain named BPHE by expressing the enzymes phenylalanine ammonialyase (PAL), hydroxycinnamate:CoA ligase (4CL), and cinnamyl-CoA reductase and endogenous alcohol dehydrogenases or aldo-keto reductases in E. coli. Subsequently, the benzyl alcohol O-benzoyltransferase from Nicotiana tabacum (ANN09798) or benzyl alcohol O-benzoyltransferase from Clarkia breweri (ANN09796) or benzyl alcohol acetyltransferase from Clarkia breweri (BEAT) were introduced into the above recombinant E. coli strain to produce cinnamyl acetate. We further improved the acetyl-CoA production by overexpressing endogenous acetyl-CoA synthetase (ACS) in E. coli. [Results] We investigated the ability of three plant-derived benzyl alcohol acyltransferase to biosynthesize cinnamyl acetate, which were further applied for synthesizing cinnamyl acetate in E. coli. The production of cinnamyl acetate by the engineered E. coli reached 166.9±6.6 mg/L. [Conclusion] Plant derived benzyl alcohol acyltransferase demonstrate flexibility to a wide range of substrates and can catalyze the synthesis of cinnamyl acetate by using cinnamyl alcohol as substrate. This study provides a foundation for microbial production of cinnamyl acetate and its derivatives using glucose as the renewable carbon source.