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

[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±6.6mg/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。

[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(4 CL),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.