Liu Kun, Wang Feng-Qing, Liu Ke, Zhao Yunqiu, Gao Bei, Tao Xinyi, Wei Dongzhi
State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
Bioresour Bioprocess. 2022 Sep 1;9(1):93. doi: 10.1186/s40643-022-00575-7.
Progesterone is one of the classical hormone drugs used in medicine for maintaining pregnancy. However, its manufacturing process, coupled with organic reagents and poisonous catalysts, causes irreversible environmental pollution. Recent advances in synthetic biology have demonstrated that the microbial biosynthesis of natural products, especially difficult-to-synthesize compounds, from building blocks is a promising strategy. Herein, overcoming the heterologous cytochrome P450 enzyme interdependency in Mycolicibacterium neoaurum successfully constructed the CYP11A1 running module to realize metabolic conversion from waste phytosterols to progesterone. Subsequently, progesterone yield was improved through strategies involving electron transfer and NADPH regeneration. Mutant CYP11A1 (mCYP11A1) and adrenodoxin reductase (ADR) were connected by a flexible linker (L) to form the chimera mCYP11A1-L-ADR to enhance electron transfer. The chimera mCYP11A1-L-ADR, adrenodoxin (ADX), and ADR-related homolog ARH1 were expressed in M. neoaurum, showed positive activity and produced 45 mg/L progesterone. This electron transfer strategy increased progesterone production by 3.95-fold compared with M. neoaurum expressing mCYP11A1, ADR, and ADX. Significantly, a novel inorganic-biological hybrid system was assembled by combining engineered M. neoaurum and InP nanoparticles to regenerate NADPH, which was increased 84-fold from the initial progesterone titer to 235 ± 50 mg/L. In summary, this work highlights the green and sustainable potential of obtaining synthetic progesterone from sterols in M. neoaurum.
孕酮是医学上用于维持妊娠的经典激素类药物之一。然而，其制造过程连同有机试剂和有毒催化剂会造成不可逆转的环境污染。合成生物学的最新进展表明，利用天然产物的微生物生物合成，特别是由构件合成难以合成的化合物，是一种很有前景的策略。在此，通过克服新金色分枝杆菌中异源细胞色素P450酶的相互依赖性，成功构建了CYP11A1运行模块，以实现从废弃植物甾醇到孕酮的代谢转化。随后，通过涉及电子传递和NADPH再生的策略提高了孕酮产量。突变型CYP11A1（mCYP11A1）和肾上腺皮质铁氧化还原蛋白还原酶（ADR）通过柔性接头（L）连接形成嵌合体mCYP11A1-L-ADR，以增强电子传递。嵌合体mCYP11A1-L-ADR、肾上腺皮质铁氧化还原蛋白（ADX）和ADR相关同源物ARH1在新金色分枝杆菌中表达，显示出阳性活性并产生了45mg/L的孕酮。与表达mCYP11A1、ADR和ADX的新金色分枝杆菌相比，这种电子传递策略使孕酮产量提高了3.95倍。值得注意的是，通过将工程化的新金色分枝杆菌与InP纳米颗粒结合组装了一种新型无机-生物混合系统来再生NADPH，其从初始孕酮滴度增加了84倍，达到235±50mg/L。总之，这项工作突出了从新金色分枝杆菌中的甾醇获得合成孕酮的绿色和可持续潜力。
