ABSTRACT
Sigma factors and sigma factor-related mechanisms control antibiotic production in Streptomyces. In this contribution, the orf21 gene was overexpressed in the wild-type strain of Streptomyces clavuligerus ATCC2764, yielding S. clavuligerus/pIORF21, to further evaluate its regulatory effect on clavulanic acid (CA) biosynthesis under different culture medium conditions. The orf21 overexpression, regulated under the constitutive promoter ermE*, led to 2.6-fold increase in CA production in GSPG medium, and a 1.8-fold decrease using ISP medium. As for GYM and MYM media, S. clavuligerus/pIORF21 strain showed higher aerial mycelium production compared to control. Glycerol uptake rate profile was affected by orf21 overexpression. Furthermore, in GSPG, S. clavuligerus/pIORF21 slightly increased the expression of adpA and gcas genes, whilst, in ISP, the claR gene expression was drastically reduced, which is consistent with a decreased CA production, observed in this medium. These findings suggest the protein encoded by the orf21 gene plays a role in the regulation of CA biosynthesis as a response to the nutritional composition of the medium.
ABSTRACT
The performance of software tools for de novo transcriptome assembly greatly depends on the selection of software parameters. Up to now, the development of de novo transcriptome assembly for prokaryotes has not been as remarkable as that for eukaryotes. In this contribution, Rockhopper2 was used to perform a comparative transcriptome analysis of Streptomyces clavuligerus exposed to diverse environmental conditions. The study focused on assessing the incidence of software parameters on software performance for the identification of differentially expressed genes as a final goal. For this, a statistical optimization was performed using the Transrate Assembly Score (TAS). TAS was also used for evaluating the software performance and for comparing it with related tools, e.g., Trinity. Transcriptome redundancy and completeness were also considered for this analysis. Rockhopper2 and Trinity reached a TAS value of 0.55092 and 0.58337, respectively. Trinity assembles transcriptomes with high redundancy, with 55.6% of transcripts having some duplicates. Additionally, we observed that the total number of differentially expressed genes (DEG) and their annotation greatly depends on the method used for removing redundancy and the tools used for transcript quantification. To our knowledge, this is the first work aimed at assessing de novo assembly software for prokaryotic organisms.
ABSTRACT
Background: Clavulanic acid (CA), a ß-lactamase inhibitor, is industrially produced by the fermentation of Streptomyces clavuligerus. The efficiency of CA production is associated with media composition, culture conditions and physiological and genetic strain characteristics. However, the molecular pathways that govern CA regulation in S. clavuligerus remain unknown. Methods and Results: Here we used RNA-seq to perform a comparative transcriptome analysis of S. clavuligerus ATCC 27064 wild-type strain grown in both a favorable soybean-based medium and in limited media conditions to further contribute to the understanding of S. clavuligerus metabolism and its regulation. A total of 350 genes were found to be differentially expressed between conditions; 245 genes were up-regulated in favorable conditions compared to unfavorable. Conclusion: The up-regulated expression of many regulatory and biosynthetic CA genes was positively associated with the favorable complex media condition along with pleiotropic regulators, including proteases and some genes whose biological function have not been previously reported. Knowledge from differences between transcriptomes from complex/defined media represents an advance in the understanding of regulatory paths involved in S. clavuligerus' metabolic response, enabling the rational design of future experiments.
ABSTRACT
In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.