Study and modification of the genetic and metabolic configuration of Actinobacteria from CO2-rich waste gasses.

NASHMIAS M.E.; ZUBIMENDI J.P.; GALLEGOS A.L.; TORRES TEJERIZO, G.; SILVA R.A.; HERNÁNDEZ M.A.; ALVAREZ H.M.; QUELAS J.I.

Los Cocos, Córdoba

Congreso; XVII Sociedad Argentina de Microbiología General (SAMIGE); 2022

Sociedad Argentina de Microbiología General (SAMIGE)

CO2 represents the major component of greenhouse gas emissions associated with waste gases (WG), for this reason CO2 capture is one of the current challenges. The use of microorganisms with the natural ability to capture CO2 and other environmental gases is proposed among the processes with potential applicability and sustainability to turn waste gases into useful chemicals. This study focuses on an oligotrophic actinobacterium, Rhodococcus aetherivorans L13, that has potential for the development of a bioprocess using WG (with higher amounts of CO2) as a carbon source to transformCO2 into triacylglycerides (TAG) and/or polyhydroxyalkanoates (PHA). We first sequenced R. aetherivorans L13 genome, with 6.4 Mpb and 6,328 open reading frames. Next, we analyzed the global transcriptome by RNAseq, comparing oligotrophic (WG) versus heterotrophic conditions. This study revealed 2,179 genes with changes in their expression (fold change ≥ 2 ≤). Next, we reconstructed metabolic pathways in order to visualize oligotrophic metabolism and choose oligotrophic pregulated native genes with the aim of enhancing metabolic pathways for better assimilation of CO2 by overexpressing them in the same cellular background. We point out genes involved in: i) oxidoreduction, to increase/regenerate NADH/NADPH pools (genes encoding a putative Proline dehydrogenase and a L glutamate semialdehyde dehydrogenase), ii) carboxylation of intermediates, to increase CO2 assimilation/fixation (genes encoding a Ribulose-phosphate3-epimerase, Transketolase, Aldehyde dehydrogenase, a putative oligotrophic metabolism regulator, a highly upregulated aldR transcriptional regulator, and their neighbor MFS-type transporter). We cloned all these genes in a thiostrepton inducible vector (pTip-QC2). In multiple efforts, no transformants of R. aetherivorans L13 were observed, so another phylogenetically close species transformation was proposed: R. erythropolis ATCC15960, able to fix/assimilate CO2 in oligotrophic conditions and with pTip-QC2 efficiency transformation reported so far. Thus, transformed strains of R. erythropolis were obtained with the empty vector (reference) and the vector with each selected gene. Gene overexpression was measured by RT-qPCR, evaluating fold change with and without induction (mediated by thiostrepton), in liquid minimal medium cultures without addition of any organic carbon source with shaking at 200 rpm. Preliminary results showed a differential behavior in the overexpression of four genes encoding the Ribulose-phosphate3-epimerase, aldehyde dehydrogenase, transcriptional regulator and MFS-type transporter. In a preliminary assay, we obtain more cell biomass than reference (dry weight) overexpressing aldehyde dehydrogenase and aldR transcriptional regulator, enzymes that were involved in first reactions of CO2 fixation. These overexpressing strains are candidates for further characterizing their TAG/PHA production.