Genetic Engineering of Oligotropha carboxidovorans Strain OM5-A Promising Candidate for the Aerobic Utilization of Synthesis Gas.

Due to climate change and worldwide pollution, development of highly sustainable routes for industrial production of basic and specialty chemicals is critical nowadays. One possible approach is the use of CO ₂ - and CO-utilizing microorganisms in biotechnological processes to produce value-added compounds from synthesis gas (mixtures of CO ₂ , CO, and H ₂ ) or from C1-containing industrial waste gases. Such syngas fermentation processes have already been established, e.g. , biofuel production using strictly anaerobic acetogenic bacteria. However, aerobic processes may be favorable for the formation of more costly (ATP-intensive) products. Oligotropha carboxidovorans strain OM5 is an aerobic carboxidotrophic bacterium and potentially a promising candidate for such processes. We here performed RNA-Seq analysis comparing cells of this organism grown heterotrophically with acetate or autotrophically with CO ₂ , CO, and H ₂ as carbon and energy source and found a variety of chromosomally and of native plasmid-encoded genes to be highly differentially expressed. In particular, genes and gene clusters encoding proteins required for autotrophic growth (CO ₂ fixation via Calvin-Benson-Bassham cycle), for CO metabolism (CO dehydrogenase), and for H ₂ utilization (hydrogenase), all located on megaplasmid pHCG3, were much higher expressed during autotrophic growth with synthesis gas. Furthermore, we successfully established reproducible transformation of O. carboxidovorans via electroporation and developed gene deletion and gene exchange protocols via two-step recombination, enabling inducible and stable expression of heterologous genes as well as construction of defined mutants of this organism. Thus, this study marks an important step toward metabolic engineering of O. carboxidovorans and effective utilization of C1-containing gases with this organism.