High-level De novo biosynthesis of arbutin in engineered Escherichia coli
High-level De novo biosynthesis of arbutin in engineered Escherichia coli
复制标题
工程大肠杆菌中熊果苷的高水平从头生物合成
DOI：
10.1016/j.ymben.2017.06.001
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发表时间：
2017-07-01
影响因子：
8.4
通讯作者：
Yan, Yajun
中科院分区：
文献类型：
--
作者：
Shen, Xiaolin;Wang, Jia;Yan, Yajun
熊果苷是一种存在于多种植物中的对苯二酚糖苷类化合物。由于其众所周知的皮肤美白特性以及抗氧化、抗微生物和抗炎活性，它被广泛用于制药和化妆品行业。目前，熊果苷的生产主要采用植物提取法或酶法，产品收率低，加工成本高。在这项工作中，我们建立了一个人工途径在大肠杆菌高水平生产熊果苷从简单的碳源。首先，4-羟基苯甲酸1-羟化酶从近平滑假丝酵母CBS 604和葡萄糖基转移酶从萝芙木serpentina的特点是在体外酶测定。将这两个基因导入E.大肠杆菌发酵葡萄糖，熊果苷产量为54.71mg/ L。通过增强莽草酸途径基因，进一步将碳流重定向到熊果苷生物合成途径，使熊果苷产量达到3.29 g/L，与初始菌株相比增加了60倍。通过对培养基中葡萄糖浓度的优化，熊果苷摇瓶发酵效价达到4.19 g/L，比出发菌株提高了77倍。本工作建立了从简单碳源出发的熊果苷的从头生物合成方法，为莽草酸途径衍生物的生物合成提供了一个可推广的策略。在我们的工程菌株中实现的高滴度也表明熊果苷的工业规模生物制造的潜力。
Arbutin is a hydroquinone glucoside compound existing in various plants. It is widely used in pharmaceutical and cosmetic industries owing to its well-known skin-lightening property as well as anti-oxidant, anti-microbial, and anti-inflammatory activities. Currently, arbutin is usually produced by plant extraction or enzymatic processes, which suffer from low product yield and expensive processing cost. In this work, we established an artificial pathway in Escherichia coli for high-level production of arbutin from simple carbon sources. First, a 4-hydroxybenzoate 1-hydroxylase from Candida parapsilosis CBS604 and a glucosyltransferase from Rauvolfia serpentina were characterized by in vitro enzyme assays. Introduction of these two genes into E. coli led to the production of 54.71 mg/ L of arbutin from glucose. Further redirection of carbon flux into arbutin biosynthesis pathway by enhancing shikimate pathway genes enabled production of 3.29 g/L arbutin, which is a 60-fold increase compared with the initial strain. Final optimization of glucose concentration added in the culture medium was able to further improve the titer of arbutin to 4.19 g/L in shake flasks experiments, which is around 77-fold higher than that of initial strain. This work established de novo biosynthesis of arbutin from simple carbon sources and provided a generalizable strategy for the biosynthesis of shikimate pathway derived chemicals. The high titer achieved in our engineered strain also indicates the potential for industrial scale bio-manufacturing of arbutin.