Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China.
ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China.
Biotechnol Bioeng. 2023 Feb;120(2):536-552. doi: 10.1002/bit.28286. Epub 2022 Nov 23.
Lutein, as a carotenoid with strong antioxidant capacity and an important component of macular pigment in the retina, has wide applications in pharmaceutical, food, feed, and cosmetics industries. Besides extraction from plant and algae, microbial fermentation using engineered cell factories to produce lutein has emerged as a promising route. However, intra-pathway competition between the lycopene cyclases and the conflict between cell growth and production are two major challenges. In our previous study, de novo synthesis of lutein had been achieved in Saccharomyces cerevisiae by dividing the pathway into two stages (δ-carotene formation and conversion) using temperature as the input signal to realize sequential cyclation of lycopene. However, lutein production was limited to microgram level, which is still too low to meet industrial demand. In this study, a dual-signal hierarchical dynamic regulation system was developed and applied to divide lutein biosynthesis into three stages in response to glucose concentration and culture temperature. By placing the genes involved in δ-carotene formation under the glucose-responsive ADH2 promoter and genes involved in the conversion of δ-carotene to lutein under temperature-responsive GAL promoters, the growth-production conflict and intra-pathway competition were simultaneously resolved. Meanwhile, the rate-limiting lycopene ε-cyclation and carotene hydroxylation reactions were improved by screening for lycopene ε-cyclase with higher activity and fine tuning of the P450 enzymes and their redox partners. Finally, a lutein titer of 19.92 mg/L (4.53 mg/g DCW) was obtained in shake-flask cultures using the engineered yeast strain YLutein-3S-6, which is the highest lutein titer ever reported in heterologous production systems.
叶黄素作为一种具有强抗氧化能力的类胡萝卜素，是视网膜黄斑色素的重要组成部分，在医药、食品、饲料和化妆品行业有广泛的应用。除了从植物和藻类中提取，利用工程细胞工厂进行微生物发酵生产叶黄素已成为一种很有前途的途径。然而，类胡萝卜素环化酶之间的途径内竞争和细胞生长与生产之间的冲突是两个主要挑战。在我们之前的研究中，通过将叶黄素生物合成途径分为两个阶段（δ-胡萝卜素形成和转化），并使用温度作为输入信号，实现番茄红素的顺序环化，从而在酿酒酵母中实现了叶黄素的从头合成。然而，叶黄素的产量仅限于微克水平，这仍然太低，无法满足工业需求。在这项研究中，开发了一种双信号层次动态调控系统，并将其应用于根据葡萄糖浓度和培养温度将叶黄素生物合成分为三个阶段。通过将参与 δ-胡萝卜素形成的基因置于葡萄糖响应型 ADH2 启动子下，将参与 δ-胡萝卜素转化为叶黄素的基因置于温度响应型 GAL 启动子下，同时解决了生长-生产冲突和途径内竞争问题。同时，通过筛选具有更高活性的番茄红素 ε-环化酶，并对 P450 酶及其氧化还原伴侣进行精细调控，提高了限速的番茄红素 ε-环化和类胡萝卜素羟化反应。最终，在使用工程酵母菌株 YLutein-3S-6 的摇瓶培养中获得了 19.92 mg/L（4.53 mg/g DCW）的叶黄素产量，这是异源生产系统中报道的最高叶黄素产量。
