Electron transfer engineering of artificially designed cell factory for
complete biosynthesis of steroids.

¹ Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
² Science Center for Future Foods, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
³ Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
⁴ Jiangsu Provisional Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

ABSTRACT: Biosynthesis of steroids by artificially designed cell factories often involves numerous nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzymes that mediate electron transfer reactions. However, the unclear mechanisms of electron transfer from regeneration to the final delivery to the NADPH-dependent active centers limit systematically engineering electron transfer to improve steroids production. Here, we elucidate the electron transfer mechanisms of NADPH-dependent enzymes for systematically engineer electron transfer of Saccharomyces cerevisiae, including step-by-step engineering the electron transfer residues of 7-Dehydrocholesterol reductase (DHCR7) and P450 sterol side chain cleaving enzyme (P450scc), electron transfer components for directing carbon flux, and NADPH regeneration pathways, for high-level production of the cholesterol (1.78 g/L) and pregnenolone (0.83 g/L). The electron transfer engineering (ETE) process makes the electron transfer chains shorter and more stable which significantly accelerates deprotonation and proton coupled electron transfer process. This study underscores the significance of ETE strategies in steroids biosynthesis and expands synthetic biology approaches.

【研究背景】

类固醇合成涉及多个依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的酶促反应，这些反应通过电子传递链从NADPH再生到催化活性中心。然而，电子传递机制尚未完全阐明，限制了系统性工程改造以提高类固醇产量的能力。文章提出了一种新的合成生物学策略——电子传递工程（ETE），通过优化NADPH依赖酶的电子传递残基、电子传递组件和NADPH再生途径，实现高效的类固醇生产。研究以酵母为平台，聚焦于胆固醇（Cho）和孕烯醇酮（Prn）的合成，选择了DHCR7和P450scc作为关键酶，分别代表两种典型的电子传递模式。

ETE策略的提出是对传统代谢工程和辅因子工程的补充，强调从电子再生到最终传递的整个链条的系统优化。这种策略的创新性在于不仅关注辅因子供应，还深入到酶内电子传递路径的微观改造，为后续实验设计奠定了理论基础。选择DHCR7和P450scc作为研究对象，突出了研究的代表性和可推广性。

【方法与结论】

研究团队以甾体类化合物胆固醇和孕烯醇酮的关键合成步骤中C7=C8双键还原及侧链剪切为例，首先利用计算模拟结合突变表征解析了DHCR7的电子传递机制并进一步指导了电子传递工程改造其内部电子传递链，将胆固醇从头合成产量由31 mg/L提升至191 mg/L，且动力学模拟和QMMM共同证实改造后DHCR7拥有更短和更稳定的电子传递链；随后，筛选得到了Rehmannia glutinosa来源的RgCYP87A3，孕烯醇酮的产量达94 mg/L，并利用QMMM解析其剪切胆固醇侧链进程，发现去质子化和质子耦合电子转移（PCET）具有较高能垒且被认为是限速步骤，进而在催化口袋中引入酸性残基以加速去质子化和PCET进程，孕烯醇酮产量提升至243 mg/L；最后，通过前体代谢途径强化和引入定位线粒体的人源ADR-ADX为ERG25供应电子，进而促使下游甾体代谢产物的空间位置与同定位于线粒体的DHCR7的空间位置重合，调整碳通量流向胆固醇。此外，进一步通过增强NADPH的合成和循环途径，以及连接肽拉近CYP和CPR的物理距离（图8A），胆固醇和孕烯醇酮在5 L发酵罐中的最高产量分别达到1.78 g/L和0.83 g/L。

本研究阐明了两种NADPH依赖酶在甾体生物合成中的电子传递机制，并系统设计了从电子再生到最终传递至催化中心的全过程，实现了甾体的高效积累。因此，电子传递工程为构建高效甾体及天然产物合成的细胞工厂提供了一种先进且高效的全新策略。

图1 酿酒酵母中构建胆固醇合成通路