Cloning and expressing the genes encoding Glycerol dehydratase (GDHt) and 1,3-Propanediol dehydrogenase (1,3-PDDH) in E. coli

Abstract: The biodiesel sector has witnessed tremendous growth in recent years, leading to a significant increase in glycerol production as a by-product. Addressing the challenge of glycerol valorization is crucial for the sustainability of the biodiesel industry. One promising solution is the conversion of glycerol into more valuable chemicals, such as 1,3-propanediol (1,3-PDO). Microorganisms offer several advantages for this conversion process, including their diverse metabolic pathways and enzymatic capabilities. Key enzymes involved in the 1,3-PDO production pathway include glycerol dehydratase (GDHt), glycerol dehydratase reactivase (GDHtR), and 1,3-propanediol dehydrogenase (1,3-PDDH). The aim of the present research project was to bio-transform glycerol into 1,3-propanediol by cloning and expressing GDHt and 1,3-PDDH in E. coli. Additionally, the specific activity of GDHt enzyme and the necessity of GDHtR were investigated. The selected genes dhaB, orfX, and orfZ from Klebsiella pneumoniae were utilized for the synthesis of GDHt, GDHtR, and 1,3-PDDH, respectively. Initial measurements of GDHt’s activity revealed detectable levels, which increased upon the addition of GDHtR, indicating the influence of GDHtR on GDHt's activity. However, the interpretation of activity values is complicated by potential interferences from aldehydes or metabolites present in crude cell extracts. To address these complexities, enzyme assays using purified enzymes were performed, but no statistically significant data were obtained. Further investigations are required to optimize the measurement of GDHt activity and address the potential influence of the cofactor B12. Coexpression of GDHt and 1,3-PDDH enzymes, resulted in a reduction of cofactor NADH, indicating the potential conversion of glycerol into 1,3-PDO. Nevertheless, the formation of the product could not be detected, and the decline in NADH could not be solely attributed to the coupled-enzymatic reaction. Additional investigations are required to elucidate possible interferences originating from B12 absorption and cellular components within crude cell extracts that may affect the observed absorbance. Future prospects involve optimizing experimental conditions, utilizing purified enzymes and alternative substrates, and thoroughly investigating potential interferences in activity measurements. These efforts will contribute to the development of sustainable and efficient strategies of enhancing the efficiency of glycerol bio-transformation processes, facilitating the production of 1,3PDO—a valuable compound with diverse industrial applications.