Evidence for the formation of ScbR/ScbR2 heterodimers and identification of one of the regulatory targets in Streptomyces coelicolor

The homologous transcriptional regulators ScbR and ScbR2 have previously been identified as γ-butyrolactone (GBL) and antibiotic receptors, respectively. They regulate diverse physiological processes in Streptomyces coelicolor in response to GBL and antibiotic signals. In this study, ScbR and ScbR2 proteins were shown to interact using a bacterial two-hybrid system where adenylate cyclase activity was reconstituted in Escherichia coli BH101. These ScbR/ScbR2 interactions in S. coelicolor were then demonstrated by co-immunoprecipitation. The ScbR/ScbR2 heterodimer was shown to co-exist with their ScbR and ScbR2 respective homodimers. When potential operator targets in S. coelicolor were investigated, the heterodimer was found to bind in the promoter region of sco5158, which however was not a target for ScbR or ScbR2 homodimers. These results revelaed a new mechanism 25 of regulation by ScbR and ScbR2 in S. coelicolor

Development of a synthetic oxytetracycline-inducible expression system for streptomycetes using de novo characterized genetic parts

Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (KD = 1.7 × 10–10 M) and oxytetracycline induced dissociation of the OtrR/DNA complex in a concentration-dependent manner. On the basis of these genetic parts, a synthetic inducible expression system Potr* was optimized. Induction of Potr* with 0.01–4 μM of oxytetracycline triggered a wide-range expression level of gfp reporter gene in different Streptomyces species. Benchmarking Potr* against the widely used constitutive promoters ermE* and kasOp* revealed greatly enhanced levels of expression when Potr* was fully induced. Finally, Potr* was used as a tool to activate and optimize the expression of the silent jadomycin biosynthetic gene cluster in Streptomyces venezuelae. Altogether, the synthetic Potr* presents a new versatile tool for fine-tuning gene expression in streptomycetes

Overproduction and identification of butyrolactones SCB1-8 in the antibiotic production superhost Streptomyces M1152

Gamma-butyrolactones (GBLs) are signalling molecules that control antibiotic production in Streptomyces bacteria. The genetically engineered strain S. coelicolor M1152 was found to overproduce GBLs SCB1-3 as well as five novel GBLs named SCB4-8. Incorporation experiments using isotopically-labelled precursors confirmed the chemical structures of SCB1-3 and established those of SCB4-8

Identification of Alp1U and Lom6 as epoxy hydrolases and implications for kinamycin and lomaiviticin biosynthesis

International audienceThe naturally occurring diazobenzofluorenes, kinamycins, fluostatins and lomaiviticins, possess highly oxygenated A-rings, via which the last forms a dimeric pharmacophore. However, neither the A-ring transformation nor the dimerization mechanisms have been explored thus far. Here we propose a unified biosynthetic logic for the three types of antibiotics and verify one key reaction via detailed genetic and enzymatic experiments. Alp1U and Lom6 from the kinamycin and lomaiviticin biosynthesis, respectively, are shown to catalyse epoxy hydrolysis on a substrate that is obtained by chemical deacetylation of a kinamycin-pathway-derived intermediate. Thus, our study provides the first evidence for the existence of an epoxy intermediate in lomaiviticin biosynthesis. Furthermore, our results suggest that the dimerization in the lomaiviticin biosynthesis proceeds after dehydration of a product generated by Lom6

Cloning, expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from Thermoanaerobacter tengcongensis

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP(+) (k(cat)/K-m = 2618 mM(-1) s(-1), k(cat) = 249 s(-1), K-m = 0.10 +/- 0.01 mM) as cofactor, although NAD(+) (k(cat)/K-m = 138 mM(-1) s(-1), k(cat) = 604 s(-1), K-m = 4.37 +/- 0.56 mM) could also be accepted. The K-m values of glucose-6-phosphate were 0.27 +/- 0.07 mM and 5.08 +/- 0.68 mM with NADP(+) and NAD(+) as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8-9.0 for NADP(+) and at pH 7.0-8.6 for NAD(+) while the optimal temperature was 80 degrees C for NADP(+) and 70 degrees C for NAD(+). This was the first observation that the NADP(+)-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD(+)-linked and growth (75 degrees C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase