Expanding the biocatalytic toolbox with a new reductase from Leptospira biflexa

ZOCCHI, SOFÍA B.; CECCOLI, ROMINA D.; BIANCHI, DARIO A.; RIAL, DANIELA V.

Rosario

Congreso; LIX Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2023

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

The search for ecofriendly ways to synthesize chemical substances has led to the incorporation of biocatalysts, such as pure enzymes, whole microorganisms or plant-derived materials, into synthetic routes. This usually results in operational benefits due to the high efficiency, exquisite selectivity and specificity of enzymatic reactions. Since most enzymes are active under mild conditions, their compatibility with the environment is an advantage. The main interest of our group is to develop sustainable processes based on the design of biocatalysts with potential applications in the field of chemical synthesis. The Old Yellow Enzymes (OYEs) are flavoproteins capable of catalyzing the asymmetric reduction of C=C bonds activated by an electron-withdrawing group, such as an aldehyde or a ketone. They have been used for the preparation of a wide variety of compounds, many of them of great interest for the pharmaceutical industry (1,2). In order to access OYEs that could significantly increase the toolbox of redox biocatalysts, we searched for sequences coding for this type of enzymes using bioinformatics methods. We identified the sequence of a possible OYE in the genome of Leptospira biflexa and cloned it into an expression vector. The recombinant protein was produced in Escherichia coli cells transformed with the constructed plasmid and its soluble expression was determined by polyacrylamide gel electrophoresis under denaturing conditions. The ability of the new biocatalyst to reduce activated C=C bonds of α,β-unsaturated ketones was evaluated by in vivo biotransformation assays using resting recombinant whole cells as biocatalyst and a set of α,β-unsaturated ketones as substrate candidates. E. coli cells transformed with the expression vector were used as control. Bioconversions were analyzed by gas chromatography coupled to mass spectrometry. Under the conditions tested, this biocatalyst was able to reduce the α,β-unsaturated ketone 1-phenyl-1-hexen-3-one to the corresponding saturated ketone. Therefore, this work provides a new biocatalyst with carbon-carbon double bond reduction activity for potential applications in chemical synthesis.1) Parmeggiani, F.; Brenna, E.; Colombo, D.; Gatti, F. G.; Tentori, F.; Tessaro, D. ChemBioChem, 2022, 23, e202100445. 2) Shi, Q.; Wang, H.; Liu, J.; Li, S.; Guo, J.; Li, H.; Jia, X.; Huo, H.; Zheng, Z.; You, S.; Qin, B. Appl. Microbiol. Biotechnol., 2020, 104, 8155-8170.