RESUMO
Bacitracin is a broad-spectrum polypeptide antibiotic, which is formed by 11 amino acids residues. Precursor amino acids supply might be the limit factor during bacitracin fermentation. First, our results demonstrated that increasing Ile and Leu supplies were regarded as the efficient strategies for the enhanced titer of bacitracin. Then, the amino acid permease YhdG, which was identified as the BCAA permease, was deleted and overexpressed in DW2, respectively. Our results showed that knocking out of permease YhdG could improve bacitracin production remarkablely. The bacitracin titer of the yhdG deficient strain DW2ΔyhdG reached 917.35 U/mL by flask fermentation, increased by 11% compared with that of DW2. In addition, the bacitracin titer was decreased by 25% in the YhdG overexpressed strain. Meanwhile, the intracellular concentrations of BCAA were higher than DW2 during the biosynthesis of bacitracin. The above results suggested that the permease YhdG might act as an exporter for branched chain amino acids in B. licheniformis DW2. Taken together, the increasing intracellular concentrations of branched chain amino acids by deleting amino acid permease YhdG could improve bacitracin titer. This study provided a new strategy for high-level production of bacitracin.
RESUMO
The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.