Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-forest Biomass, Nanjing Forestry University, Nanjing, 210037, China; Jiangsu Key Laboratory of Biomass-based Green Fuels and Chemicals, Nanjing Forestry University, Nanjing, 210037, China; College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China.
Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-forest Biomass, Nanjing Forestry University, Nanjing, 210037, China; Jiangsu Key Laboratory of Biomass-based Green Fuels and Chemicals, Nanjing Forestry University, Nanjing, 210037, China; College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China.
Metab Eng. 2021 Jul;66:60-67. doi: 10.1016/j.ymben.2021.04.008. Epub 2021 Apr 15.
Geraniol is a valuable monoterpene extensively used in the fragrance, food, and cosmetic industries. Increasing environmental concerns and supply gaps have motivated efforts to advance the microbial production of geraniol from renewable feedstocks. In this study, we first constructed a platform geraniol Escherichia coli strain by bioprospecting the key enzymes geranyl diphosphate synthase (GPPS) and geraniol synthase (GES) and selection of a host cell background. This strategy led to a 46.4-fold increase in geraniol titer to 964.3 mg/L. We propose that the expression level of eukaryotic GES can be further optimized through fusion tag evolution engineering. To this end, we manipulated GES to maximize flux towards the targeted product geraniol from precursor geranyl diphosphate (GPP) via the utilization of fusion tags. Additionally, we developed a high-throughput screening system to monitor fusion tag variants. This common plug-and-play toolbox proved to be a robust approach for systematic modulation of protein expression and can be used to tune biosynthetic metabolic pathways. Finally, by combining a modified E1* fusion tag, we achieved 2124.1 mg/L of geraniol in shake flask cultures, which reached 27.2% of the maximum theoretical yield and was the highest titer ever reported. We propose that this strategy has set a good reference for enhancing a broader range of terpenoid production in microbial cell factories, which might open new possibilities for the bio-production of other valuable chemicals.
香叶醇是一种有价值的单萜，广泛应用于香料、食品和化妆品行业。日益增长的环境问题和供应缺口促使人们努力从可再生原料中通过微生物生产香叶醇。在这项研究中，我们首先通过生物勘探关键酶香叶基二磷酸合酶（GPPS）和香叶醇合酶（GES）和选择宿主细胞背景，构建了一个平台香叶醇大肠杆菌菌株。这一策略使香叶醇的产量增加了 46.4 倍，达到 964.3mg/L。我们提出，通过融合标签进化工程，可以进一步优化真核 GES 的表达水平。为此，我们通过利用融合标签来最大化从前体香叶基二磷酸（GPP）到目标产物香叶醇的通量来操纵 GES。此外，我们开发了一种高通量筛选系统来监测融合标签变体。这种通用的即插即用工具包被证明是一种系统调节蛋白质表达的有效方法，并可用于调整生物合成代谢途径。最后，通过结合改良的 E1*融合标签，我们在摇瓶培养中实现了 2124.1mg/L 的香叶醇，达到了最大理论产量的 27.2%，这是迄今为止报道的最高产量。我们提出，该策略为增强微生物细胞工厂中更广泛的萜类化合物生产提供了良好的参考，这可能为其他有价值的化学品的生物生产开辟新的可能性。
