Open Access

Mohammad Yoonus Khan

• Gluconobacter oxydans, an acetic acid bacterium with distinct oxidative metabolic pathways, offers significant industrial potential due to its ability to produce valuable chemicals such as Lsorbose, dihydroxyacetone and gluconic acid. This work aims to advance genetic engineering techniques for G. oxydans, with focus on the development and optimization of regulatable gene expression systems. WeGluconobacter oxydans, an acetic acid bacterium with distinct oxidative metabolic pathways, offers significant industrial potential due to its ability to produce valuable chemicals such as Lsorbose, dihydroxyacetone and gluconic acid. This work aims to advance genetic engineering techniques for G. oxydans, with focus on the development and optimization of regulatable gene expression systems. We examine the evolution from constitutive to inducible gene expression systems in G. oxydans and highlight the adaptation of the L-arabinose-dependent AraC-ParaBAD and L-rhamnose-dependent RhaS-PrhaBAD systems from Escherichia coli. The study identifies specific challenges in these systems, including high inducer concentrations and medium acidification for AraC-ParaBAD, and regulatory anomalies for RhaS-PrhaBAD, such as unexpected expression by L-rhamnose. Additionally, we introduce novel photocaged inducers, 6-Nitropiperonyl-β-L-arabinopyranoside and 6-Nitropiperonyl-β-L-rhamnopyranoside, for lightcontrolled gene expression, despite encountering issues with solubility and UV-induced effects. The study further also includes the construction of integrative plasmids for the targeted genomic restoration of the key genes gdhM and sldAB in G. oxydans BP.9, and the genomic integration of PGOX0384-rhaT in G. oxydans 621H Δupp. The study includes a detailed analysis of reporter gene expression modulation using both conventional and caged inducers. These findings contribute to the advancement of genetic tools for G. oxydans, offering a pathway to more efficient and sustainable biotechnological applications.…