Park Beom Gi, Kim Junyeob, Kim Eun-Jung, Kim Yechan, Kim Joonwon, Kim Jin Young, Kim Byung-Gee
School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea.
Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
Front Bioeng Biotechnol. 2021 Feb 22;9:624838. doi: 10.3389/fbioe.2021.624838. eCollection 2021.
As a means to develop oleaginous biorefinery, was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.
作为开发油脂生物炼制的一种手段，利用进化代谢工程和用于细胞色素P450（CYP）表达的合成FadR启动子，从葡萄糖生产ω-羟基棕榈酸。首先，构建一个基础菌株，使用代谢工程策略从葡萄糖生产游离脂肪酸（FFA）。随后，通过甲磺酸乙酯（EMS）诱导的随机诱变和荧光激活细胞分选（FACS）筛选，筛选出改善的FFA过量生产者。此外，设计了含有用于CYP表达的细菌FadR结合序列的合成启动子，以响应FFA浓度的激增，从而激活ω-羟基化途径，与培养第一天相比，从培养第三天起转录活性提高了14倍。然后，筛选内源性物质，并在开发的菌株中使用合成FadR启动子进行表达，以生产ω-羟基棕榈酸。通过实施合成FadR启动子，可以有效地解耦细胞生长和生产阶段。最后，在分批发酵中，我们证明了在氮限制培养基中使用FmeN3-TR1-alk5生产160 mg/L的ω-羟基棕榈酸。本研究展示了在产油酵母中使用具有细菌转录调节因子（即FadR）结合序列的合成启动子生产ω-羟基脂肪酸的一个出色例子。
