Resveratrol is a plant secondary metabolite with diverse, potential health-promoting benefits. Due to its nutraceutical merit, bioproduction of resveratrol via microbial engineering has gained increasing attention and provides an alternative to unsustainable chemical synthesis and straight extraction from plants. However, many studies on microbial resveratrol production were implemented with the addition of water-insoluble phenylalanine or tyrosine-based precursors to the medium, limiting in the sustainable development of bioproduction. Here we present a novel coculture platform where two distinct metabolic background species were modularly engineered for the combined total and de novo biosynthesis of resveratrol. In this scenario, the upstream Escherichia coli module is capable of excreting p-coumaric acid into the surrounding culture media through constitutive overexpression of codon-optimized tyrosine ammonia lyase from Trichosporon cutaneum (TAL), feedback-inhibition-resistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroGfbr) and chorismate mutase/prephenate dehydrogenase (tyrAfbr) in a transcriptional regulator tyrR knockout strain. Next, to enhance the precursor malonyl-CoA supply, an inactivation-resistant version of acetyl-CoA carboxylase (ACC1S659A,S1157A) was introduced into the downstream Saccharomyces cerevisiae module constitutively expressing codon-optimized 4-coumarate-CoA ligase from Arabidopsis thaliana (4CL) and resveratrol synthase from Vitis vinifera (STS), and thus further improve the conversion of p-coumaric acid-to-resveratrol. Upon optimization of the initial inoculation ratio of two populations, fermentation temperature, and culture time, this co-culture system yielded 28.5 mg/L resveratrol from glucose in flasks. In further optimization by increasing initial net cells density at a test tube scale, a final resveratrol titer of 36 mg/L was achieved. This is first study that demonstrates the use of a synthetic E. coli–S. cerevisiae consortium for de novo resveratrol biosynthesis, which highlights its potential for production of other p-coumaric-acid or resveratrol derived biochemicals.

中文翻译：

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通过大肠杆菌-酿酒酵母共培养物的模块化工程从头生产白藜芦醇。

白藜芦醇是一种植物次生代谢物，具有多种潜在的健康促进功效。由于其营养保健价值，通过微生物工程生物生产白藜芦醇受到越来越多的关注，并为不可持续的化学合成和从植物中直接提取提供了替代方案。然而，许多关于微生物白藜芦醇生产的研究都是在培养基中添加不溶于水的苯丙氨酸或酪氨酸前体来进行的，限制了生物生产的可持续发展。在这里，我们提出了一个新颖的共培养平台，其中两种不同的代谢背景物种被模块化设计，用于白藜芦醇的总生物合成和从头生物合成。在这种情况下，上游大肠杆菌模块能够通过来自皮肤毛孢子菌 (TAL) 的密码子优化的酪氨酸氨裂合酶（TAL）的组成型过表达（反馈抑制抗性 3-脱氧-d-）将对香豆酸分泌到周围的培养基中。转录调节因子 tyrR 敲除菌株中的阿拉伯庚酮酸 7-磷酸合酶 (aroGfbr) 和分支酸变位酶/预苯酸脱氢酶 (tyrAfbr)。接下来，为了增强前体丙二酰辅酶A的供应，将乙酰辅酶A羧化酶的失活抗性版本（ACC1S659A，S1157A）引入下游酿酒酵母模块中，该模块组成性表达来自拟南芥（4CL）的密码子优化的4-香豆酸辅酶A连接酶）和来自葡萄（STS）的白藜芦醇合酶，从而进一步提高对香豆酸向白藜芦醇的转化。通过优化两个群体的初始接种比例、发酵温度和培养时间，该共培养系统从烧瓶中的葡萄糖中产生了 28.5 mg/L 白藜芦醇。通过在试管规模上增加初始净细胞密度进行进一步优化，最终白藜芦醇滴度达到 36 mg/L。这是第一项展示合成大肠杆菌的使用的研究。酿酒酵母联盟致力于白藜芦醇从头生物合成，该联盟强调了其生产其他对香豆酸或白藜芦醇衍生生物化学品的潜力。