Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Jinnan District, Tianjin 300350, China.
Georgia Tech Shenzhen Institute, Tianjin University, Tangxing Road 133, Nanshan District, Shenzhen 518071, China.
ACS Synth Biol. 2021 Jun 18;10(6):1531-1544. doi: 10.1021/acssynbio.1c00081. Epub 2021 Jun 8.
The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the production of nerol and borneol in prene-overproducing . We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPS mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPS variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.
无环橙花醇和双环龙脑都是广泛应用于食品、化妆品和制药行业的单萜醇。新兴的合成生物学使微生物生产成为一种有前途的替代方法，可有效地、可持续地供应单萜醇。在这项研究中，我们结合代谢和植物单萜合酶工程来提高过量生产前体香叶基二磷酸（NPP）的微生物中橙花醇和龙脑的产量。我们设计了生长正交的香叶基二磷酸（NPP）作为单萜醇生物合成的通用前体，并共表达来自 的橙花醇合酶（GmNES）以产生橙花醇，或共表达来自 的截短的莰烯二磷酸合酶（LdtBPPS）以生产龙脑。此外，通过基于结构模拟的 LdtBPPS 定点突变，我们筛选出多个显著提高无环橙花醇或双环龙脑产量的变体，其中 LdtBPPS 突变体在龙脑合成方面优于野生型 LdtBPPS，而 LdtBPPS 变体在橙花醇生产方面优于 GmNES。随后，我们过表达内源性 Nudix 水解酶 NudJ 以促进前体的去磷酸化，并提高葡萄糖合成橙花醇和龙脑的产量。最后，在优化发酵工艺后，工程菌株 ENO2 在摇瓶中产生了 966.55mg/L 的橙花醇，菌株 ENB57 产生了 87.20mg/L 的龙脑，达到了迄今为止微生物中橙花醇和龙脑的最高报道产量。这项工作展示了一种用于微生物生产天然萜类醇的组合工程策略。
