Metabolic Engineering of Escherichia coli for High-Level Production of Salicin

Abstract

Salicin is a notable phenolic glycoside derived from plants including Salix and Populus genus and has multiple biological activities such as anti-inflammatory and antiarthritic, anticancer, and antiaging effects. In this work, we engineered production of salicin from cheap renewable carbon resources in Escherichia coli ( E. coli ) by extending the shikimate pathway. We first investigated enzymes synthesizing salicylate from chorismate. Subsequently, carboxylic acid reductases (CARs) from different resources were screened to achieve efficient reduction of salicylate. Third, glucosyltransferases from different sources were selected for constructing cell factories of salicin. The enzymes including salicylate synthase AmS from Amycolatopsis methanolica , carboxylic acid reductase CARse from Segniliparus rotundus, and glucosyltransferase UGT71L1 from Populous trichocarpa were overexpressed in a modified E. coli strain MG1655-U7. The engineered strain produced 912.3 ± 12.7 mg/L salicin in 72 h of fermentation. These results demonstrated the production of salicin in a microorganism and laid significant foundation for its commercialization for pharmaceutical and nutraceutical applications.

植物苯丙素类天然产物生物合成研究进展

“…水杨酸是一种重要的植物激素， 广泛存在于杨柳 科植物中， 对植物的系统或局部防御反应有一定的调 节作用。此外，据报道水杨酸还能够调节光合作用、 呼吸作用、营养生长、种子萌发、开花、衰老等多种 过程， 从而影响植物的生长发育 [24][25][26][27] 。 乙酰水杨酸 (阿 司匹林)一直是治疗各种医学病症的重要药物 [28] ，广 泛应用于控制发热和疼痛， 以及治疗心血管疾病 [28][29][30] 和皮肤病如痤疮、水泡和瘙痒 [31] 。 目前研究认为植物中可能存在两种水杨酸合成 途径：异分支酸合酶(isochorismate synthase，ICS) 途径(主要部分)和 PAL 途径 [32,33] (图 2) 。在 ICS 途径中，在叶绿体中异分支酸合酶(ICS)首先将分 支酸转化为异分支酸 [33] ，随后异分支酸通过 MATE (multidrug and toxin extrusion)转运蛋白从叶绿体进 入细胞质，经氨基转移酶催化生成异分支酸-9-谷氨 酸(isochorismate-9-glutamate ， ICS-Glu ) 。 ICS-Glu 不稳定， 推测可自发或经酰基转移酶催化分解生成水 杨酸。该途径中已鉴定的酶包括：MATE 转运蛋白， EDS5(enhanced disease susceptibility 5) ，它能够将 异分支酸从叶绿体转运到细胞质 [33] ；PBS3(avrPphB susceptible 3)编码的氨基转移酶，负责催化 L-谷氨 酸 与 异 分 支 酸 生 成 ICS-Glu [32] ； EPS1 ( enhanced Pseudomonas susceptibility 1)是 BAHD 酰基转移酶 家族蛋白， 具有 ICS-Glu 裂解酶活性，可以加速 ICS-Glu 分解产生水杨酸 [33,34] 。 除 ICS 途径外，推测植物在细胞质中利用 PAL 途径也能合成少部分水杨酸。 有同位素示踪实验表明 在烟草中苯丙氨酸可以经肉桂酸或苯甲酸转化为水 杨酸 [35] 。 但也有同位素示踪实验发现在拟南芥中苯丙 氨酸不能生成水杨酸， 而是合成其同分异构体对羟基 苯甲酸(p-hydroxybenzoic acid，PHB) [36] 。PAL 途 径合成水杨酸的相关酶还有待进一步鉴定。 微生物中也报道了一条水杨酸合成途径， 嗜甲基 拟无枝酸菌(Amycolatopsis methanolica)的 水杨酸合 A c c e p t e d https://engine.scichina.com/doi/10.1360/SSV-2024-0029 成酶(salicylic acid synthase，AmS)可以直接利用分 支酸生成水杨酸 [37] 。 本实验室前期在改良的大肠杆菌 MG1655-U7 中过表达了 AmS，同时引入来源于塞格 尼氏杆菌( Segniliparus rotundus ) 的 羧酸还原酶 (carboxylic acid reductase，CAR)和来源于毛果杨 ( Populous trichocarpa ) 的 葡萄糖基转移酶 (glucosyltransferase，UGT71L1) ，成功实现了水杨 苷的微生物合成 [38]…”

Biyofarmasötik Keşif, Geliştirme ve Üretimin Güncel Paradigması Olarak Mikroorganizmaların Metabolik Mühendisliği: Sentetik Biyolojinin Katkıları

“…Zhank vd. (2022) yaptıkları çalışmada shikimate yolunu genişleterek E. coli'de ucuz yenilenebilir karbon kaynaklarından salisin üretimini gerçekleştirmişlerdir [29]. Yapılan bir diğer çalışmada ise glikozu verimli bir şekilde bütil asetata dönüştüren, metabolik olarak tasarlanmış bir E. coli suşu geliştirilmiştir [30].…”