CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production.
CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production.
复制标题
DOI：
10.1186/s12934-016-0623-3
复制
发表时间：
2017-01-17
影响因子：
6.4
通讯作者：
Koffas MA
中科院分区：
文献类型：
--
作者：
Cress BF;Leitz QD;Kim DC;Amore TD;Suzuki JY;Linhardt RJ;Koffas MA
花色素苷是一类颜色鲜艳的糖基化类黄酮色素，其使花和果实宿主组织具有主要为红色、橙子、紫色和蓝色的色调。尽管所有花色苷都表现出pH响应性光化学变化，但核心花色苷骨架上的独特结构装饰除了提高稳定性和独特的药理学性质外，还会导致显着的颜色变化。在这项工作中，我们首次报道了在微生物生产系统中将重建的植物花色素苷途径从（+）-儿茶素延伸到O-甲基化花色素苷，这一努力需要内源性代谢物UDP-葡萄糖和S-腺苷-L-甲硫氨酸（SAM或SAM Met）的同时共选择。将不同植物来源的花青素O-甲基转移酶（AOMT）同源基因与矮牵牛花色素合成酶（PhANS）和拟南芥花色素3-O-葡萄糖基转移酶（At 3GT）在大肠杆菌中共表达。葡萄属葡萄AOMT（VvAOMT 1）和香仙客来'Kaori-no-mai' AOMT（CkmOMT 2）是产生芍药苷3′-O-甲基化产物（P3 G）的最有效AOMT，其最高滴度分别为2.4和2.7 mg/L。在调节质粒拷贝数和优化VvAOMT 1和CkmOMT 2表达条件后，使用VvAOMT 1将产量进一步提高至23 mg/L。最后，CRISPRi被用来沉默转录抑制因子MetJ，以解除甲硫氨酸生物合成途径的调控，并提高SAM对矢车菊素3-O-葡萄糖苷（C3 G）（P3 G的生物合成前体）的O-甲基化的可用性。MetJ阻遏导致51 mg/L的最终滴度（在放大至摇瓶时为56 mg/L），这代表了相对于非靶向CRISPRi对照菌株的2倍改进和总体21倍改进。大肠利用丰富且相对便宜的黄酮醇前体（+）-儿茶素，对大肠杆菌菌株进行工程改造，以生产特殊的花青素P3 G。此外，dCas 9介导的metJ转录抑制减轻了限制性SAM池大小，增强了甲基化花青素苷产物的滴度。虽然P3 G和其他O-甲基化花青素苷色素的微生物生产可能作为天然食品和饮料着色剂对食品工业是有价值的，但我们预计，在此构建的菌株也将证明可用于观赏植物工业，作为评估推定的花青素苷O-甲基转移酶以追求定制花色素组合物的平台。本文的在线版本（doi：10.1186/s12934-016-0623-3）包含补充材料，可供授权用户使用。
Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-l-methionine (SAM or AdoMet). Anthocyanin O-methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase (PhANS) and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT). Vitis vinifera AOMT (VvAOMT1) and fragrant cyclamen ‘Kaori-no-mai’ AOMT (CkmOMT2) were found to be the most effective AOMTs for production of the 3′-O-methylated product peonidin 3-O-glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved to 23 mg/L using VvAOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O-methylation of cyanidin 3-O-glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall. An E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O-methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O-methyltransferases in pursuit of bespoke flower pigment compositions. The online version of this article (doi:10.1186/s12934-016-0623-3) contains supplementary material, which is available to authorized users.