Surveys of non-ribosomal peptide and polyketide assembly lines in fungi and prospects for their analysis in vitro and in vivo

Abstract

With many bioactive non-ribosomal peptides and polyketides produced in fungi, studies of their biosyntheses are an active area of research. Practical limitations of working with mega-dalton synthetases including cell lysis and protein extraction to recombinant gene and pathway expression has slowed understanding of many secondary metabolic processes relative to bacterial counterparts. Recent advances in accessing fungal biosynthetic machinery are beginning to change this. Here we describe the successes of some studies of thiotemplate biosynthesis in fungal systems, along with very recent advances in chemical tagging and mass spectrometric strategies to selectively study biosynthetic conveyer belts in isolation, and within a few years, in endogenous fungal proteomes.

Fig. 1

Basic enzymatic domains and their reactions for non-ribosomal peptide synthesis; A: adenylation, T: thiolation, and C: condensation. Non-experts can refer to Appendix #1 for detailed descriptions of function.

Fig. 2

Basic enzymatic domains and their reactions for polyketide synthesis; AT: acyltransferase, T: thiolation, and KS: ketosynthase. Non-experts can refer to Appendix #1 for detailed descriptions of function.

Fig. 3

Some typical NRPS tailoring domains and their catalyzed reactions; A^MT: N-methyl transferase (preferentially found in fungi), E: epimerase, and TE: thioesterase.

Fig. 4

Some typical PKS tailoring domains and their catalyzed reactions; KR: ketoreductase, DH: dehydratase, and ER: enoyl reductase. R = ribose-ADP(P). The KR and ER domains are NADPH dependent enzymes.

Fig. 5

Overview of fungal vs. bacterial NRPSs. NRPSs can be divided into three types, linear (a), iterative (b) and non-linear (c). Fungal NRPSs generally follow a single megasynthetase logic, whereas bacterial NRPS pathways are generally spread over a greater number of polypeptides. Fungal NRPSs are also rich in A-^MT domains, a feature rarely seen in bacterial NRPSs. CsS: cyclosporine synthetase; EnS: enniatin synthetase; Pes1: synthetase encoded by pes1, TycA, B, C: tyrocidine synthetase A, B, C; GrsA, B: gramicidin S synthetase A, B; SyrB1, E: syringomycin synthetase B1, F.

Fig. 6

Overview of fungal vs. bacterial type I PKSs. Type I PKSs can be divided into three types, non-reducing (nr), partially-reducing (pr) and highly-reducing (hr). Fungal type I PKSs generally follow an iterative logic using a single AT, KS, and T iteratively, whereas bacterial type I PKS use modules of PKS machinery sequentially. PksA, polyketide synthase A from aflatoxin biosynthesis; 6-MSAS, 6-methyl salicylic acid cynthase; TENS, tenellin synthase; DEBS, 6-deoxyerythronolide B synthase.

Fig. 7

Some fungal- and bacterial-derived compounds, 1 –10.

Fig. 8

Overview of mass spectrometric techniques for analyzing activity in vitro. A) The activity based screening method is an in vitro assay for determining substrate specificity of NRPSs using a complex substrate pool and an accurate mass shift (Δm) as the readout. FTMS enables resolution and accurate assignment of covalently-bound intermediates. B) The Ppant ejection assay simplifies complex mass spectra of peptides or intact proteins by selectively ejecting phosphopantetheine and phosphopantetheine bound intermediates allowing for correct intermediate assignment even at high intact mass.

Fig. 9

Overview of proteomic techniques for detecting the expression of thiotemplate enzymes in vivo. A) The PrISM workflow incorporates the “Ppant ejection assay” for the selective identification of thiotemplate enzymes. The workflow utilizes tandem mass spectrometric data of thiotemplate enzymes as a starting point for the cloning and analysis of thiotemplate pathways. B) OASIS utilizes thiotemplate-specific metabolic labeling with affinity tags to allow for enrichment and identification of thiotemplate pathways.