Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

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 CO2- and CO-utilizing microorganisms in biotechnological processes to produce value-added compounds from synthesis gas (mixtures of CO2, CO, and H2) 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 CO2, CO, and H2 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 (CO2 fixation via Calvin-Benson-Bassham cycle), for CO metabolism (CO dehydrogenase), and for H2 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. carboxidovoransvia 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.