Fructose and glucosamine metabolisms in Rhodococci: from basic knowledge to biotechnological applications

IGLESIAS AA; ALVAREZ HM; ESCOBAR M; ASENCION DIEZ MD

Mendoza

Congreso; LVIII Annual SAIB Meeting; 2022

Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular

The oleaginousbehavior constitutes the basis for several rhodococcal species to be consideredas potential biofactories for oiled compound productions. Oleaginicity analysisunder different carbon and nitrogen supplies is a critical task to identifyscenarios for the utilization of Rhodococci in the treatment of environmentalpollutants. To elucidate Rhodococcusjostii and Rhodococcus fascians metabolicbehavior we grew both bacteria in different sole carbon sources at different concentrations.We used minimal saline medium (MSM) complemented with ammonium chloride asnitrogen source, where MSM0, MSM0.1 and MSM1 contained no NH4Cl; 0.1or 1g/L NH4Cl, respectively. It was extensively reported that highnitrogen availability (such as MSM1) increases biomass production, while lowernitrogen concentrations trigger lipid accumulation in Rhodococci. Amongstthe different sugars analyzed as carbon sources, we found that both R. jostii and R. fascians grew efficiently in fructose (Fru) or glucosamine(GlcN). Thus, the aim of our current work is to deepen the understanding regardingthe metabolic steps involved in Fru and GlcN metabolisms, and their possible interconnection.Fru was assayed at 0.1, 1, 3 and 10 % w/v in eitherMSM0, MSM0.1 or MSM1 for R. jostii. No growth was detected in MSM0,demonstrating the essentiality of a nitrogen source. When grown in MSM0.1 with0.1% w/v Fru, OD600 was 1.5, while with all other Fru concentrations, growingvalues reached OD600 of 6. On the other hand, when grown in MSM1, R. jostiireached OD600 units of 2, 11, 56 and 45 for 0.1, 1, 3 and 10 % w/v Fru,respectively. Results suggest an interplay between Fru consumption and nitrogenavailability. R. jostii growth in 1, 3 y 10% w/v GlcN was between 6 and8 in MSM, thus indicating that high biomass production seems to be linked toFru metabolism. We then analyzed same Fru concentrations for R. fasciansgrowth in MSM1. Curiously, the OD600 values were up to 10-11. Then, we aretempted to speculate regarding different regulatory mechanisms involved in Fru metabolismbetween different rhodococcal species. To understand the differentbehaviors described above, we approached the biochemical characterization of putativegenes related to Fru and GlcN consumption. We focused on enzymes belonging tothe Fru-6P/Fru-1,6-P2 interconversion node (e.g. PPi and/or ATPdependent phosphofructokinases and Fru-1,6-P2 phosphatase) and tothe link between Fru-6P and GlcN-6P (GlcN-6P synthase and Glc-6P deaminase). Genesencoding the enumerated enzymes were amplified, cloned and expressed in Escherichiacoli for their purification and kinetic characterization. These preliminaryresults provide new insights to further understand rhodococcal metabolism.  The latter, would allow to harness theirpotential as microbial biofactories starting from agricultural disposals(generally rich in sugars like Fru), or as biocatalysts providers forbiotechnological processes.