Aims The aim of this work was to clarify the mechanism of monounsaturated fatty acid (MUFA) synthesis in Bradyrhizobium TAL1000 and the effect of high temperature on this process. Methods and Results Bradyrhizobium TAL1000 was exposed to a high growth temperature and heat shock, and fatty acid composition and synthesis were tested. To determine the presence of a possible desaturase, a gene was identify and overexpressed in Escherichia coli. The desaturase expression was detected by RT‐PCR and Western blotting. In B. TAL1000, an aerobic mechanism for MUFA synthesis was detected. Desaturation was decreased by high growth temperature and by heat shock. Two hours of exposure to 37°C were required for the change in MUFA levels. A potential ∆9 desaturase gene was identified and successfully expressed in E. coli. A high growth temperature and not heat shock reduced transcript and protein desaturase levels in rhizobial strain. Conclusions In B. TAL1000, the anaerobic MUFA biosynthetic pathway is supplemented by an aerobic mechanism mediated by desaturase and is down‐regulated by temperature to maintain membrane fluidity under stressful conditions. Significance and Impact of the Study This knowledge will be useful for developing strategies to improve a sustainable practice of this bacterium under stress and to enhance the bioprocess for the inoculants' manufacture.

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Desaturases, key enzymes in the metabolism of fatty acids, regulate the physical and biochemical properties of membranes. They adjust the composition of saturated and unsaturated fatty acids in response to changes in the environmental. We demonstrated the existence of Δ9 desaturase activity in epimastigotes of the Trypanosoma cruzi Tulahuen strain. In the present study, showed that this enzyme has an approximate molecular mass of 50kDa and a pI value of approximately 9. In order to characterize the Δ9 desaturase of Trypanosoma cruzi, (TcΔ9DES) we have cloned, sequenced and expressed in Escherichia coli. The gene consists of 1300bp and encodes a peptide of 433 amino acids with a molecular weight of 50kDa. Analysis of the amino acid sequence revealed three clusters of histidine and two hydrophobic regions, characteristic of membrane-bound desaturases. Gene expression studies showed that TcΔ9DES was overexpressed as an active protein. Fatty acid analysis showed that the expressed protein was confirmed to be functional with Δ9 desaturase activity. This enzyme changed the fatty acid profile of TcΔ9DES-expressing E. coli, decreasing the levels of palmitic (16:0) and stearic (18:0) acids and enhancing palmitoleic (16:1Δ9) and monounsaturated 18 carbons fatty acids. When [1-14C]palmitic or [1-14C]stearic acid was used as substrate, TcΔ9DES-expressing E. coli exhibited high desaturase activity associated with increased levels of monounsaturated fatty acids, suggesting that the TcΔ9DES enzyme was actively expressed in E. coli. To check the commitment of TcΔ9DES against sterol biosynthesis inhibitors we tested the activity under ketoconazole effect. Native TcΔ9DES, showed a significant activity inhibition. Since TcΔ9DES has shown active participation under different environmental factors, among them, ketoconazole, we consider that it plays a critical role in the metabolism of the parasite.

Fetal bovine serum (FBS) is an important factor in the culture of Trypanosoma cruzi, since this parasite obtains and metabolizes fatty acids (FAs) from the culture medium, and changes in FBS concentration reduce the degree of unsaturation of FAs in phosphoinositides. When T. cruzi epimastigotes were cultured with 5% instead of 10% FBS, and stearic acid was used as the substrate, (9) desaturase activity decreased by 50%. Apparent K (m) and V (m) values for stearic acid, determined from Lineaweaver-Burk plots, were 2 microM and 219 pmol/min/mg of protein, respectively. In studies of the requirement for reduced pyridine nucleotide, (9) desaturase activity reached a maximum with 8 microM NADH and then remained constant; the apparent K (m) and V (m) were 4.3 microM and 46.8 pmol/min/mg of protein, respectively. The effect of FBS was observed only for (9) desaturase activity; (12) desaturase activity was not affected. The results suggest that decreased FBS in culture medium is a signal that modulates (9) desaturase activity in T. cruzi epimastigotes.