The surfactin biosynthetic complex of Bacillus subtilis: COM domain-mediated biocombinatorial synthesis, and single step purification of native multi-modular NRPSs and multi-enzyme complexes
Most biosynthetic templates for the assembly of peptide natural products are composed of two or more nonribosomal peptide synthetases (NRPSs). For example, the surfactin biosynthetic complex consists of three NRPSs (SrfA-A, SrfA-B, and SrfA-C), which are encoded by the polycistronic srfA operon with...
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|Summary:||Most biosynthetic templates for the assembly of peptide natural products are composed of two or more nonribosomal peptide synthetases (NRPSs). For example, the surfactin biosynthetic complex consists of three NRPSs (SrfA-A, SrfA-B, and SrfA-C), which are encoded by the polycistronic srfA operon within the chromosome of the producer strain Bacillus subtilis. According to the molecular logic employed by NRPS assembly lines, the biosynthesis of a defined product relies on the proper, well-orchestrated interaction between partner-enzymes (i.e. SrfA-A/SrfA-B, and SrfA-B/SrfA-C), and the prevention of futile interactions between non-partner enzymes (i.e. SrfA-A/SrfA-C). Based on most recent in vitro studies, these selective interactions between NRPSs are controlled by the interplay of communication-mediating (COM) domains, located at the C- and N-termini of the corresponding donor and acceptor enzymes.
In the first part of this study, the potential of COM domains was exploited for the directed reprogramming of the surfactin biosynthetic complex, and the setting up of an in vivo system for the true biocombinatorial synthesis of lipopeptides. To this end, the first COM domain pair, facilitating the selective interaction between SrfA-A and SrfA-B, was substituted against various cognate, mis-cognate and non-cognate COM domain pairs. The consequences of these manipulations were then analyzed by means of HPLC and high-resolution MS. These experiments verified that COM domain pairs of the tyrocidine biosynthetic complex retain their functionality and selectivity even in the context of a heterologous host and NRPS system. Furthermore, utilization of a designated non-cognate COM domain pair allowed for an intended skipping of the second NRPS SrfA-B, the enforcement of a productive interaction between the natural non-partner enzymes SrfA-A and SrfA-C, and thus the directed synthesis of a shortened lipotetrapeptide product. In another experiment, all donor and acceptor enzymes of the biosynthetic complex were equipped with the same set of cognate COM domain pair. The resulting abrogation of the selectivity-barrier led to the establishment of an so-called universal communication system, and afforded the envisioned biocombinatorial synthesis of two lipopeptide products. All these experiments verified – for the first time in vivo, and within the context of a natural NRP assembly line – the decisive role of COM domains for the control of protein-protein communication between NRPSs.
The second objective of this work was the establishment of a gentle method for the purification of NRPSs and multi-enzymatic NRPS complexes. The approach taken was based on the utilization of polyol-responsive monoclonal antibodies (PR-mAb), which are able to release their bound antigen under gentle, non-denaturating conditions, in the presence of polyols. PR-mAbs were originally developed and used for the purification of the E. coli RNA polymerase holo-enzyme complex, including low-affinity bound -factors. Among others, these studies led to the identification of a antigen/antibody pair epitope/NT73. Within the scope of this work, the coding sequence of this epitope tag was fused to the 3’-end of the srfA-A gene within the chromosome of B. subtilis. Subsequently, the encoded SrfA-A-epi protein could be purified from cleared crude extracts of the resulting mutant using immunoaffininty chromatography. SDS-PAGE and MS/MS analyses, as well as biochemical characterizations unequivocally verified the purification of the epitop-tagged SrfA-A protein in active holo-form, as well as co-purification of SrfA-B (molecular weight of the dimeric complex: approx. 803 kDa). Under the conditions tested, the third NRPS, SrfA-C, as well as additional proteins, associated with the surfactin complex, could not be detected.|
|Physical Description:||144 Pages|