Engineering anaerobic electron flow through heterologous rhodoquinone synthesis in model microbial and photosynthetic...

Anaerobic conditions facilitate bioproduction by enabling diverse metabolic pathways; however, they disrupt redox balance due to the accumulation of reduced cofactors, limiting metabolic efficiency. Rhodoquinone (RQ), a low-redox-potential quinone, supports electron transport under anaerobic conditions. Unlike menaquinone, RQ is synthesized from ubiquinone through a single enzymatic reaction catalyzed by rhodoquinone biosynthesis protein A (RquA), making it a simple, adaptable metabolic engineering tool. In this study, RQ was synthesized in the menaquinone-deficient Escherichia coli ΔmenA strain via heterologous gene expression of rquA from Euglena gracilis. The engineered strain tripled succinate production under anaerobic conditions compared with the control strain. Redox analysis showed a decreased NADH/NAD⁺ ratio, reflecting improved electron flow under oxygen-limited conditions. Introducing rquA into a strain with high succinate production further increased succinate yields, confirming compatibility with existing metabolic modifications. To explore broader applications, rquA from Rhodospirillum rubrum was expressed in Cyanidioschyzon merolae mitochondria using a construct with a C. merolae mitochondrial targeting signal. Quinone analysis confirmed RQ synthesis, and the engineered strains produced more succinate anaerobically relative to the controls. Although redox cofactor ratios in C. merolae remained stable, rotenone sensitivity indicated altered mitochondrial electron transport under anaerobic conditions. These findings demonstrate that RQ synthesis enhances anaerobic metabolism in bacterial and eukaryotic systems, providing a versatile tool for metabolic engineering under oxygen-limited conditions.