Mellor Silas B, Behrendorff James B Y H, Ipsen Johan Ø, Crocoll Christoph, Laursen Tomas, Gillam Elizabeth M J, Pribil Mathias
Section for Plant Biochemistry, Department of Plant and Environmental Science, University of Copenhagen, Frederiksberg, Denmark.
School of Biology and Environmental Science, Queensland University of Technology, Brisbane, QLD, Australia.
Front Plant Sci. 2023 Jan 9;13:1049177. doi: 10.3389/fpls.2022.1049177. eCollection 2022.
Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco () chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.
光合细胞器具有将太阳能转化为新陈代谢所需化学能的能力，这为工程化小分子生物合成提供了诱人的特性。将生化生产直接与作为能量和底物来源的光合同化作用相结合的可能性，引起了代谢工程师的兴趣。具体而言，植物中发现的化学多样性通常依赖于细胞色素P450介导的羟基化反应，这些反应依赖于催化所需的还原剂供应，并且常常导致复杂分子异源生产的代谢瓶颈。通过将P450酶导向植物叶绿体，可以巧妙地应对此类氧化还原前提条件。在本研究中，我们通过将工程化的靛蓝生物合成途径靶向烟草叶绿体，探索了植物光合机制驱动P450依赖的靛蓝前体吲哚-β-D-葡萄糖苷（靛苷）形成的能力。我们表明，当由光合还原力驱动时，属于人类CYP2家族的天然和工程变体在叶绿体中均具有催化活性，并优化构建体设计以提高生产力。然而，虽然增加色氨酸的供应会导致吲哚积累增加，但它并不能提高靛苷的生产力，这表明P450活性限制了整体生产力。不同氧化还原伙伴的共表达也不能提高生产力，这表明还原力的供应不是瓶颈。最后，动力学测量表明，不同的氧化还原伙伴被光系统I有效还原，但植物铁氧还蛋白提供了最高的光依赖P450活性。这项研究证明了光合作用支持P450依赖的代谢途径的内在能力。因此，无论酶的来源如何，植物和光合微生物都特别适合工程化P450依赖的代谢途径。我们的发现对光合宿主中用于生产高价值化学品或用于药理学研究的药物代谢物的代谢工程具有启示意义。
