Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network.

Phosphopantetheinyl transferases (PPTases) play essential roles in both primary metabolisms and secondary metabolisms via post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). In this study, an industrial FK506 producing strain Streptomyces tsukubaensis L19, together with Streptomyces avermitilis, was identified to contain the highest number (five) of discrete PPTases known among any species thus far examined. Characterization of the five PPTases in S.

An acyltransferase domain of FK506 polyketide synthase recognizing both an acyl carrier protein and coenzyme A as acyl donors to transfer allylmalonyl and ethylmalonyl units.

Unlabelled: Acyltransferase (AT) domains of polyketide synthases (PKSs) usually use coenzyme A (CoA) as an acyl donor to transfer common acyl units to acyl carrier protein (ACP) domains, initiating incorporation of acyl units into polyketides. Two clinical immunosuppressive agents, FK506 and FK520, are biosynthesized by the same PKSs in several Streptomyces strains. In this study, characterization of AT4FkbB (the AT domain of the fourth module of FK506 PKS) in transacylation reactions showed that AT4FkbB recognizes both an ACP domain (ACPT csA) and CoA as acyl donors for transfer of a unique allylmalonyl (AM) unit to an acyl acceptor ACP domain (ACP4FkbB), resulting in FK506 production.

Two bacterial group II phosphopantetheinyl transferases involved in both primary metabolism and secondary metabolism.

It is known that bacterial group II phosphopantetheinyl transferases (PPTases) usually phosphopantetheinylate acyl carrier proteins (ACPs) involved in the secondary metabolism. For example, a bacterial group II PPTase SchPPT has been known to phosphopantetheinylate only ACPs involved in secondary metabolism, such as scn ACP0-2 and scn ACP7. In this study, we found two bacterial group II PPTases, Hppt and Sppt, could phosphopantetheinylate not only scn ACP0-2 and scn ACP7, but also sch FAS ACP, an ACP involved in primary metabolism.

Characterization and evolutionary implications of the triad Asp-Xxx-Glu in group II phosphopantetheinyl transferases.

Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu.

DptR2, a DeoR-type auto-regulator, is required for daptomycin production in Streptomyces roseosporus.

Daptomycin, a novel cyclic lipopeptide antibiotic against Gram-positive bacteria, is produced by Streptomyces roseosporus. Though its biosynthetic mechanism, structural shuffling and fermentation optimization have been extensively studied, little is understood about its production regulation at the transcriptional levels. Here we reported that dptR2, encoding a DeoR-type regulator located close to the daptomycin biosynthesis gene cluster in S.

Proteasome involvement in a complex cascade mediating SigT degradation during differentiation of Streptomyces coelicolor.

In Streptomyces coelicolor, the ECF sigma factor SigT negatively regulates cell differentiation, and is degraded by ClpP protease in a dual positive feedback manner. Here we further report that the proteasome is required for degradation of SigT, but not for degradation of its anti-sigma factor RstA, and RstA can protect SigT from degradation independent of the proteasome. Meanwhile, deletion of the proteasome showed reduced production of secondary metabolites, and the fermentation medium from wild type could promote SigT degradation.

[Construction of a temperature-sensitive and replication-defective T-vector and its application for gene knockout in Salmonella pullorum].

Knockout is an important method for gene function study, while vector is the core of gene knockout. In order to obtain an effective vector for rapid construction of mutant and essentiality identification of the corresponding gene, we constructed a recombinant plasmid named plDM-T based on the temperature-sensitive and replication- defective plasmid plDM1 by inserting an Xcm I adapter into the EcoR I and Pst I sites of pIDM1. Digesting with Xcm I, pIDM-T can be prepared as a linear T-vector for PCR products cloning and be used to knockout the corresponding gene in the genome with insertion duplication mutagenesis.