Hanamghar Sayali S, Mellor Silas Busck, Mikkelsen Lisbeth, Crocoll Christoph, Motawie Mohammed Saddik, Russo David A, Jensen Poul Erik, Zedler Julie A Z
Synthetic Biology of Photosynthetic Organisms, Matthias Schleiden Institute for Genetics, Bioinformatics and Molecular Botany, Friedrich Schiller University Jena, 07743 Jena, Germany.
Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg, Denmark.
ACS Synth Biol. 2025 Mar 21;14(3):867-877. doi: 10.1021/acssynbio.4c00800. Epub 2025 Mar 2.
Plants produce a large array of natural products of biotechnological interest. In many cases, these compounds are naturally produced at low titers and involve complex biosynthetic pathways, which often include cytochrome P450 enzymes. P450s are known to be difficult to express in traditional heterotrophic chassis. However, cyanobacteria have shown promise as a sustainable alternative for the heterologous expression of P450s and light-driven product biosynthesis. In this study, we explore strategies for improving plant P450 stability and membrane insertion in cyanobacteria. The widely used model cyanobacterium sp. PCC 6803 was chosen as the host, and the well-studied P450 CYP79A1 from the dhurrin pathway of was chosen as the model enzyme. Combinations of the P450 fused with individual elements (e.g., signal peptide, transmembrane domain) or the full length cyanobacterial, thylakoid-localized, protein PetC1 were designed. All generated CYP79A1 variants led to oxime production. Our data show that strains producing CYP79A1 variants with elements of PetC1 improved thylakoid targeting. In addition, chlorophyll-normalized oxime levels increased, on average, up to 18 times compared to the unmodified CYP79A1. These findings offer promising strategies to improve heterologous P450 expression in cyanobacteria and can ultimately contribute to advancing light-driven biocatalysis in cyanobacterial chassis.
植物产生大量具有生物技术应用价值的天然产物。在许多情况下，这些化合物在天然状态下产量较低，并且涉及复杂的生物合成途径，其中常常包括细胞色素P450酶。已知P450酶难以在传统的异养宿主中表达。然而，蓝细菌已显示出有望作为P450酶异源表达和光驱动产物生物合成的可持续替代方案。在本研究中，我们探索了提高植物P450酶在蓝细菌中的稳定性和膜插入的策略。选择广泛使用的模式蓝细菌集胞藻PCC 6803作为宿主，并选择来自高粱氰苷途径中研究充分的P450酶CYP79A1作为模式酶。设计了将P450与单个元件（例如信号肽、跨膜结构域）或全长蓝细菌类囊体定位蛋白PetC1融合的组合。所有产生的CYP79A1变体都导致了肟的产生。我们的数据表明，产生带有PetC1元件的CYP79A1变体的菌株改善了类囊体靶向。此外，与未修饰的CYP79A1相比，叶绿素标准化的肟水平平均提高了18倍。这些发现为改善蓝细菌中异源P450酶的表达提供了有前景的策略，并最终有助于推动蓝细菌底盘中的光驱动生物催化。
