Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis

Abstract

Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically dissect this pathway, we have reconstituted it in Bacillus subtilis. Successful MP1 production required promoter engineering and the reassembly of essential biosynthetic genes in a modular plasmid. The resulting system allows for rapid pathway manipulation, including protein tagging and gene deletion. We find that 8 processing proteins are sufficient for the production of MP1 and that the tailoring enzyme TclS catalyzes a C-terminal reduction step that distinguishes MP1 from its sister compound micrococcin P2. IMPORTANCEThe emergence of antibiotic resistance is one of the most urgent human health concerns of our day. A crucial component in an integrated strategy for countering antibiotic resistance is the ability to engineer pathways for the biosynthesis of natural and derivatized antimicrobial compounds. In this study, the model organism B. subtilis was employed to reconstitute and genetically modularize a 9-gene system for the biosynthesis of micrococcin, the founding member of a growing family of thiopeptide antibiotics.

Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

“…S3-S8). Loss of micrococcin formation in GST-TclE strains harboring individual mutations was then verified by evaluating the bioactivity of methanol extracts against a Staphylococcus aureus indicator strain (13).…”

“…1A), an archetypal thiopeptide, contains six thiazoles, two Thr-derived Dhb residues, and a central pyridine ring formed from two Ser-derived Dha residues. It is additionally oxidatively decarboxylated at the C-terminal Thr residue, initially to a ketone to form micrococcin P2 (MP2), which may be rereduced to an alcohol, yielding micrococcin P1 (MP1) (13). Micrococcin/thiocillin (tcl) biosynthetic gene clusters have been identified in Macrococcus caseolyticus strain 115 (Mc) and Bacillus cereus ATCC 14579 (Bc) (14,15).…”

“…Mc requires only 9 of its 12 tcl genes to synthesize MP1 (SI Appendix, Fig. S1) (13), and these genes can be easily manipulated as synthetic gene clusters (Fig. 1B) in our heterologous Bacillus subtilis producer strain expressing the precursor peptide TclE and the eight biosynthetic proteins TclIJKLMNPS (Fig.…”

“…In vitro studies of thiopeptide biosynthetic components have provided information about functional sets of enzymatic processing (11,12), but complete reconstitution remains a challenging endeavor. As an alternative approach, we recently reported an in vivo heterologous expression system that allows both efficient genetic changes in the core and biosynthetic enzymes and isolation and characterization of biochemical intermediates in micrococcin biosynthesis (13).…”

“…Our findings highlight strict sequential order of biochemical conversions leading to the micrococcin product and pave the way toward using this understanding to engineer antimicrobial compounds with clinically useful properties. dehydrogenases, are proposed to catalyze the C-terminal oxidative decarboxylation (TclP) and subsequent rereduction (TclS) (13).…”