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. Author manuscript; available in PMC: 2012 Jan 1.
Published in final edited form as: Fungal Genet Biol. 2010 Jun 30;48(1):49–61. doi: 10.1016/j.fgb.2010.06.012

Surveys of Non-Ribosomal Peptide and Polyketide Assembly Lines in Fungi and Prospects for Their Analysis in vitro and in vivo

Bradley S Evans a,b, Sarah J Robinson c, Neil L Kelleher b,d,*
PMCID: PMC2965788  NIHMSID: NIHMS218214  PMID: 20601041

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.

Keywords: fungal metabolism, nonribosomal peptide synthetases, polyketide synthases, thiotemplate biosynthesis, secondary metabolism, proteomics, mass spectrometry, Fourier-Transform mass spectrometry

1.0 Introduction

The sustained examination of medicinally relevant natural products and their biosyntheses is driven by the need for new medicines that will selectively inhibit targets implicated in human disease. Drug discovery and synthesis are often inspired by new molecular scaffolds discovered in nature that can be used to probe and provide new information about biological targets. In an effort to maximize the diversity of natural compounds isolated and to minimize the isolation of well-studied compounds, fungal systems offer a significant jump from the `simpler' world of natural product discovery in bacteria. Highly potent fungal molecules such as lovastatin, aflatoxin and fuminosin are reviewed, along with protein-based analysis techniques using mass spectrometry (MS) to evaluate their biosynthetic proteins and/or their small molecule products. Along with facile genome sequencing, such tools promise to increase the rate at which we can access and understand the ultra-large genes involved in production of non-ribosomal peptides and polyketides.

Biosynthetic products span from essential primary metabolites such as fatty acids that make up the cell membrane to polyketides (PKs) and nonribosomal peptides (NRPs) that constitute pigments, antibiotics and siderophores. An often exploited mechanism of natural product genesis is thiotemplate biosynthesis, which allows for indirect encoding of ketide and peptide metabolite biosynthesis by a protein template. Thiotemplating refers to the covalent tethering of intermediates to a unique cofactor, 4'-phosphopantetheine, through a labile thioester bond. These tethered intermediates are arranged along an assembly line of enzymes that are usually a multi-active site, mega-enzyme (type I), a series of interacting enzymes (type II), or (for polyketide synthases (PKSs)) a single active site enzyme that acts repeatedly to form the final product (type III). The assembly lines of nonribosomal peptide synthetases (NRPSs) can be further subdivided into linear (a), iterative (b) and non-linear (c) [1]. The fungal type I iterative PKSs (IPKSs) are classified as either highly-reducing (hr), partially reducing (pr), or non-reducing (nr) [2]. This assembly line style of production lends itself to study by large molecule MS to characterize the proteins involved in the biosynthesis as well as the covalently-attached intermediate at each biosynthetic step, when recombinant proteins are available and activity reconstituted.

Regardless of type, enzymes involved in thiotemplate biosynthesis have enormous primary structures and can form some of the largest protein complexes within cells, sometimes larger than the ribosome [3]. The logic of thiotemplating offers two advantages – 1) substrate/intermediate shuttling through the pathway – intermediates cannot diffuse away, 2) potentially toxic intermediates are not allowed to affect the producing organism – a mechanism of host resistance. In short, thiotemplate biosynthesis enables efficient and safe biosynthesis of highly active secondary metabolites. Substrates are activated for entry into thiotemplate pathways by acyl-CoA ligases, acyl transferases (fatty acids and polyketides), or adenylation enzymes (nonribosomal peptides). The energy of activation of each monomer is conserved throughout the pathway by transthioesterification along the assembly line until the final product is released. Biosynthetic mechanisms of NRPs and PKs share many common elements, most important covalent tethering of intermediates to the assembly line. This commonality allows for techniques specifically targeting this quality to be applied generally to thiotemplate systems. Thiotemplate biosynthesis has been studied extensively in bacteria and through this work many techniques have been developed that are applicable to fungal systems. Fungal systems have historically been difficult to study in vitro due to problems with cloning and expression of the large and sometimes intron containing genes and for this reason, progress in the fungal world has lagged behind that of bacterial counterparts. Progress on that front has been reviewed elsewhere [4]. For this reason we first review the progress made on bacterial thiotemplate systems before moving to the more recent advances in fungal systems.

2.0 Mechanisms of Biosynthesis

For those familiar with NRPSs and PKSs, we direct you to the text below straight away; for those interested in some background reading, please see Appendix #1. The basic enzymatic functions at the core of NRPSs – adenylation (A), thiolation (T) and condensation (C) are displayed in Fig. 1, the basic functions of PKSs – acyl transferase (AT), thiolation (T) and ketosynthase (KS) are displayed in Fig. 2.

Fig. 1.

Fig. 1

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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.

Fig. 2

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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.

2.1 NRP Biosynthesis

In addition to these three basic functions for peptide elongation in NRPSs, product release can be achieved by hydrolysis of the last thioester with or without macrocyclization [5] as seen in Fig. 3. Also there are several other tailoring activities in NRPS assembly lines, as seen in Fig. 3. These include C- and N-methyltransferases, N-formyltransferases, amino transferases, L-D epimerases, oxidases, reductases, cyclases, and halogenases among others [6]. NRPS pathways may also be integrated into hybrid NRPS-PKS pathways, as seen with emericellamide [7], and the final products may be decorated with lipid and/or carbohydrate moieties. Given these extensive biotransformations in NRPSs, the peptide products exhibit a diverse array of structures and biological effects.

Fig. 3.

Fig. 3

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

2.2 PK Biosynthesis

PKs, unlike NRPs must undergo extensive on assembly-line transformations in order to achieve complex structural content. The presence of the optional ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER) functions after initial activation allows for reduced C-C bonds and chemical stability for linear or macrocyclic PKs (Fig. 4). Stereocenters introduced by the action of KRs that are not further reduced by DH and/or ER enzymes contribute to the diversity in structure PK products possess [8]. Absence of theses reducing functions results in highly reactive poly-carbonyl backbones that undergo aromatization and cyclization guided by product template (PT) domains [9,10]. C-methyl transferases (C-MT) and utilization of alternate acyl-CoAs such as methyl-malonyl- CoA, results in addition of branched structures to the backbone. Additionally, like NRPs, PKs can undergo post assembly line tailoring by O-methyl transferases, glycosylases and others [11].

Fig. 4.

Fig. 4

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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.

2.3 Differences Between Fungal and Bacterial Thiotemplate Pathways

While there are no well-defined rules regarding differences between fungal and bacterial NRPSs and PKSs, there are some general trends that distinguish them (Fig. 5, 6). First, fungal pathways tend to be mostly large megasynthetases (type I) while bacterial pathways tend more toward modularity (type II). Bacterial NRPSs generally terminate by the action of a thioesterase (TE) domain whereas fungal NRPSs tend to terminate by the action of a terminal condensation domain. Another distinguishing characteristic of fungal NRPSs is that many fungal A-domains have an inserted N-methyltransferase domain [12]. Fungal type I PKSs tend to follow the iterative model; each domain acts repeatedly activating and modifying the same monomers. Serial use of the same set of enzymatic functions can result in polyketides dominated by one structural motif; these polyketides are characterized as non-reduced or highly reduced due the presence or absence of PKS reducing functions in the synthase. In addition, presence of KR or KR and DH domains (but not ER domains) results in an intermediate class of partially reduced polyketides. Bacterial type I PKSs act modularly; each domain acts once. Large polyketides require multiple large enzymes for biosynthesis in bacteria, because each domain functions once in the bacterial systems. A benefit of modularity is it allows for mixing of non-reducing and reducing modules. Both bacteria and fungi have numerous hybrid NRPS-PKS pathways. Both fungal and bacterial pathways are often clustered together on the genome, but fungal pathways may be split onto separate chromosomes [13]. Generally, fungal thiotemplate genes do not contain introns or only contain a few introns, however as more fungal genomes are sequenced it has become apparent this is not always the case [14].

Fig. 5.

Fig. 5

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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.

Fig. 6

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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.

2.4 Fungal PKs

As described above, polyketides are synthesized by acetyl building blocks, with common variation established by methylation, oxidation, and reduction. Commonality between NRPs and PKs is the compelling range of bioactivities they exhibit. One of the most broadly studied, and most potent mycotoxic compound classes are the aflatoxins. Aflatoxins are isolated from A. nomius, A. tamari, A. flavus and A. parasiticus and share an analogous biosynthetic route with A. nidulans derived sterigmatocystin (1) (Fig. 7). The potent liver carcinogenic properties exhibited by 1 and the aflatoxins have impelled the review and publication of a significant body of literature [15–19]. In terms of the tailoring potential of fungal PKs, both aflatoxin B1 (2) and compound 1 utilize the above mentioned tailoring domains to derivatize the starter, fatty acid synthase derived, hexanoic acid. Significant protein study has characterized the role of a pathway-specific Zn2Cys6 transcription activator that is common to both 1 and 2, but not a necessary characteristic of these biosynthetic pathways [20]. This Zn2Cys6 transcription factor (AflR) was the first pathway-specific fungal natural product formation factor identified. A second mycotoxic compound of mixed NRPS/PKS biogenesis is ochratoxin A (3), produced by both Penicilli and Aspergilli generas. This unique dihydroisocoumarin phenylalanine compound has been studied relative to its B analogue (Cl in 3 is replaced with H in ochratoxin B) and determined that chlorination is the penultimate biosynthetic step [21]. In addition to the negative effects induced by ochratoxin and aflatoxin compound classes, numerous other PKs including T-toxins [22], fumonisins [23], citrinin [24], aurofusarin [25], patulin [26], and dothistromin [27] are closely regulated and actively studied to limit exposure and to prevent severe mycotoxic effects. In addition to their negative mycotoxic properties, fungal PKs have been flagged as a notable source of medicinally applicable natural products. Most noteworthy is the lovastatin/compactin compound class. Lovastatin (4) has been commercially developed as a hypolipidemic drug via HMG-CoA reductase inhibition. In summary, fungal-derived PKs induce a broad range of harmful and beneficial biological responses and continue to drive the future studies of this principal compound class.

Fig. 7.

Fig. 7

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

2.5 Fungal NRPs

Fungal-derived NRPs exhibit a number of pharmacologically relevant properties including both antibiotic (penicillins) and possible anticancer agents such as gliotoxin [28] as well as those like HC-toxin exhibiting destructive mycotoxic properties [29]. The significant bioactivity of these compounds prompts elucidation of their biosynthetic mechanisms. Prominent NRPS systems in fungi are now well-understood including the β-lactam antibiotics: penicillins and cephalosporins. Both compound classes share initial linear tripeptide biosynthesis of ACV (5) from L-α-aminoadipic acid, L-cysteine and L-valine (the latter epimerized to D-valine). Mechanistic studies have confirmed a two-step β-lactam formation which precedes thiazolidine ring formation as seen in penicillin N (6, [30]). Diverse biosynthetic routes ensue from compound 6 to the resulting penicillin or cephalosporin analogues. An equally significant fungal NRP that spurred extensive biosynthetic study was cyclosporin A (7), an immunosuppressant used to prevent allotransplantation rejection under the trademarked name Sandimmune®. Early evaluation of the undecapeptide gene cluster of 7 led to isolation of one enzyme responsible for adenylation and N-methylation of seven out of 11 amino acids, later designated cyclosporin synthetase [31]. This was not only one of the earliest fungal NRPS breakthroughs, but also one of the largest synthetases identified (1.6 MDa) and demonstrated the extensive degree of N-methylation exhibited in fungal NRPs. Beyond peptide diversity induced by tailoring activities, the use of domains or modules more than once in an assembly line adds variation to linear fungal derived NRPs. One example of this that led to an early understanding of iterative biosynthesis was ennaitin A (8), a cyclohexadepsipeptide consisting of D-2-hydroxyisovaleryl – N-methyl-L-leucine [32]. Enniatin A (8) is biosynthesized in three iterative condensations of dipeptidol building blocks [33,34] with an intramolecular reaction mechanism [35] forming 8. Together, compounds 1 –10 (Fig. 7) represent variations in the potential of fungi to produce pharmacologically relevant and biosynthetically diverse natural products.

Recent advances in mass spectrometry for protein characterization have propelled biochemical analysis of thiotemplate systems. Mass spectrometry and activity-based profiling approaches have been primarily applied to the investigation of bacterial systems, but there have been three recent and high profile examples of mass spectrometry being used to study important fungal systems, lovastatin, aflatoxin and fumonisin.

3. Protein Level Analysis of Thiotemplate Enzymes

3.1 Thiotemplate protein studies in bacteria

The path to implementation of proteomic-like techniques with fungi builds from bioinformatic, genomic and mass spectrometric methods already established for bacteria. Bioinformatic analyses often provide successful prediction of adenylation domain specificity; however, in vitro analysis should always be performed to fully characterize a biosynthetic pathway (particularly for orphan gene clusters where the metabolite produced is unknown). The traditional methods used to analyze substrate specificity of adenylation domains have relied on the pyrophosphate exchange assay [36–38]. While these methods are sensitive, they cannot be used to determine substrate specificity of adenylation domains in a complex or undefined mixture such as the native metabolome. In addition, analysis may be hampered by unavailability of radiolabeled substrates. Radiolabeling also has a deficit in that it does not report on any further transformations to the substrate once tethered to the carrier protein by the activating module or other enzymes present in a reaction mixture. The ability to rapidly and accurately analyze the intermediates of a thiotemplate pathway is of particular importance for fungal systems because the current models of adenylation domain specificity do not include a well-defined fungal nonribosomal code [39].

A bacterial example of domain specificity investigation by Dorrestein, et al. [40] included the presentation of nine adenylation-carrier protein di-domain proteins with a pool of substrates of defined content in addition to a full metabolome, followed by identification of intermediates in seven pathways. An example of the workflow is shown in Fig. 8A. Interpretation of specificity was accomplished using high resolution mass spectrometry for proteins or peptides with covalently tethered phosphopantetheine (Ppant) bound intermediates and discrimination between potential intermediates differing in mass by as little as two daltons. Due to the labile nature of the Ppant arm, selective ejection of the complex from the peptide or enzyme containing it in the mass spectrometer is used in a technique known as the “Ppant ejection assay” [41]. Using MS, gentle fragmentation of a mixture containing phosphopantheinylated peptides leads to identification of T-domain active sites and the identity of the intermediate(s) bound to them. Since the focus of analysis is now the small molecule cofactor and bound intermediate(s), accurate precursor mass assignment is no longer a prerequisite for accurately determining the identity of the bound intermediate (Fig. 8B). Dorrestein, et al. pioneered this technique for in vitro characterization by partially elucidating seven systems and extending the analysis to a 126 kDa protein and its bound intermediate. When performed on a Fourier-Transform mass spectrometer (FTMS), the Ppant ejection assay discerns between intermediates differing by one dalton as exemplified by the discrimination between acetoacetate and β-aminobutyrate loaded carrier proteins from the mycosubtilin pathway of B. subtilis [42].

Fig. 8.

Fig. 8

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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.

In addition to determining substrate specificity of activating domains, the Ppant ejection assay can be used to monitor transformations occurring to the covalently bound substrate. Dorrestein, et al. were able to monitor the dehydration and subsequent rehydration of a polyketide intermediate in vitro. Notable was the use of a bench-top mass spectrometer, demonstrating how this type of analysis can be performed in laboratories with access to standard mass spectrometers. The ability of the Ppant ejection assay to shift analysis towards low mass is significant because at masses above 70 kDa or so, intact mass alone cannot discriminate between intermediates that are close (<2 Da) in mass due to complex isotoptic distributions and low signals at high MW. Lee, et al. extended analysis in this high mass regime to determine the substrate specificity of a 108 kDa synthetase in a multiplexed assay [43]. Since 2006, about a dozen labs have employed the Ppant ejection assay using a variety of mass spectrometers to characterize thiotemplate systems in vitro [44–49].

Mass spectrometric tools for the in vitro characterization of recombinantly produced enzymes are now well developed. The advent of a technique called PrISM (for Proteomic Investigation of Secondary Metabolism, see Fig. 9A) now enables the investigation of thiotemplate systems closer to the in vivo level [50]. Bumpus, et al. have shown that identification of T-domain active site peptides can be carried out on crude lysates of recombinantly produced enzymes in addition to full proteomic mixtures of bacterial enzymes at the native expression level. Indeed, the Ppant ejection assay can be used to accurately identify phosphopantetheinylated peptides in full proteome-scale digests. The Ppant ejection assay is very robust because the mass of the Ppant ion is measured so accurately (i.e., <2 part-per-million error) that the detection of thiotemplate biosynthetic pathways can be highly selective. These features allow for the Ppant ejection assay to be used in the PrISM workflow for monitoring expression of targeted systems and for the discovery of thiotemplate systems (even with no genome sequence). PrISM has been demonstrated in the proteome of an unsequenced Bacillus for the identification of a NRPS and hybrid NRPS-PKS gene clusters and association of the small molecule products to their biosynthetic enzymes. This study led to the identification of a new kurstakin lipopeptide.

Fig. 9.

Fig. 9

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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.

Labeling of carrier proteins by in vivo phosphopantetheinylation or metabolic incorporation of CoA analogues is an approach complementary to PrISM. La Clair et al. synthesized a variety of CoA analogues for chemoenzymatic labeling of carrier proteins using the promiscuous phosphopantetheinyl transferase Sfp from B. subtilis [51]. Chemical conjugation of fluorescent probes to CoA enabled quantitative and qualitative assessment of in vivo phosphopantetheinylation as well as detection of carrier protein containing recombinant enzymes at levels below that detectable by standard SDS-PAGE analysis. Biotinylated CoA analogues were also synthesized and used to pull down nondenatured carrier protein containing enzymes. Meier et al. extended this work by synthesizing a collection of bioorthogonal panthetheine analogues for in vivo labeling of carrier proteins [52]. Advantages of moving towards using smaller panthetheine analogues as opposed to CoA analogues are ease of synthesis, stability of the compounds and increased efficiency of metabolic incorporation due to easier transport across membranes. The panthetheine analogues synthesized were shown to be incorporated into CoA analogues both in the in vitro reconstituted CoA biosynthetic pathway as well as into the endogenous CoA biosynthetic pathway of E. coli. The authors used the in vitro and in vivo synthesized CoA analogues to successfully modify VibB from the vibriobactin pathway of V. cholerae recombinantly expressed in E. coli with purified or co-expressed Sfp. In a follow-up study, Meier et al. synthesized fluorescent acyl transferase, ketosynthase and thioesterase activity based probes and combined them with previously synthesized carrier protein probes [53]. The combined set of probes was shown to be active in vivo for labeling a purified PKS (PikAIII, pikromycin from S. venezuale) and a NRPS TE domain (Tyc-TE, tyrocidine from B. brevis). The probe set was used in vivo to profile fatty acid biosynthesis proteins from cultured human cells and SrfAC, an NRPS involved in surfactin biosynthesis in B. subtilis. These kind of reagents have helped established a new method termed OASIS (for Orthogonal Active Site Identification System, Fig. 9B), which is a new method that couples the metabolic labeling of thiotemplate enzymes with mass spectrometry based proteomics [54]. OASIS was successful in enriching, identifying and profiling proteins of all four NRPS and PKS pathways from B. subtilis, and holds promise for discovery of thiotemplate systems in unsequenced genomes. In conclusion, a number of proteomic techniques have been developed and used to identify mechanisms of bacterial NRPS biosynthesis.

3.2 Thiotemplate protein studies in fungi

The techniques mentioned above have been used to elucidate fungal PKS pathways for two extensively studied compound classes: aflatoxins and lovastatins. Aflatoxins are one of the most studied compound classes produced by A. flavus and A. parasiticus due to their carcinogenic and toxic properties. The biosynthesis of aflatoxins employs a multidomain nonreducing iterative polyketide synthase (IPKS). The biosynthetic pathway has been well studied [55,56], but information regarding individual domains within the aflatoxin IPKS (PksA) has been limited. Most recently, a deconstructive approach to PksA of aflatoxin B1 (2) using heroic efforts for cloning and expression, followed by LC-FTMS resulted in characterization of individual domains and the advanced intermediates assembled within each domain [10]. The relative abundance of seven acylated intermediates, extended in a processive fashion, was established by FTMS in the absence and presence of a product template (PT) domain, which is common among nonreducing IPKSs but functionally ill-defined prior to this report [9]. Specifically, the PT domain unites with ketosynthase and thioesterase domains to irreversibly drive formation of the correct cyclization, aromatization or dehydration product [10]. The use of tandem mass spectrometry (or MSn) for structural evaluation of sub-stoichiometric intermediates also confirmed the architecture and length of ring systems constructed due to the fragmentation of linear units surrounding rings, without intra-ring fragmentation.

Lovastatins from A. terreus and other fungi have been extensively studied and developed for their hypolipidemic properties, producing a great depth of knowledge regarding their biosynthesis. One relatively understudied mechanism was the release of lovastatin, which does not proceed via the expected off-loading TE domain. Therefore, Xie et al. examined the rate of lovastatin acid (9) formation/off-loading by LC/MS to determine the role of two interacting domains LovF and LovD. Previous work by Kennedy et al. determined the biosynthetic product of Lov domains up to LovD was monacolin J, which is the final biosynthetic product in the absence of the final domain, LovF [57]. In the presence of LovF, the α-S-methylbutyrate side chain (highlighted) of 9 was added, which led to completion of 9. To further probe this relationship, Xie et al. used the Ppant ejection assay (vide supra) to confirm the localization of the α-S-methylbutyrate side chain of 6 on LovF (345.1843 Da, Pant arm plus α-methylbutyrate). Next, monacolin J and LovD were added to α-methylbutyrate-loaded LovF and transfer of the diketide side chain from LovF to monacolin J was catalyzed by a dissociated acyltransferase of LovD, resulting in 9. Further, LC-MS was used to evaluate the rate of formation of compound 9 as well as the presence of lovastatin analogues when specific cofactors were excluded [58]. Another thorough study involved the reconstitution of the lovastatin nonaketide synthase LovB and its partner enoyl reductase enzyme LovC and provided the basis for rules dictating highly reducing IPKSs [59]. Ma et al. engineered a S. cerevisiae strain to efficiently express both LovB and LovC domains that were used for complete characterization of their synthetic processes until completion of dihydromonacolin L. MS techniques, including the Ppant ejection assay, were used to determine the identity and level of phosphopantetheinylation of the LovB domain and to monitor and verify intermediate formation [59].

In addition to these lovastatin and aflatoxin studies, a study of fumonisin (10) biosynthesis, from Fusarium also utilized MS product confirmation. Selective alkyl chain addition to the ACP domain was monitored via Ppant ejection, and the mechanism of product release was detrmined to be pyridoxal 5' phosphate (PLP) dependent. Termination was identified to proceed via PLP dependent decarboxylative condensation of L-alanine and acyl-S-ACP with greatest product formation observed for C18-S-ACP. This study by Gerber et al. discloses a new highly reducing IPKS termination mechanism catalyzed by non-standard thioesterase/cyclase chain release enzymes [60]. Utilizing contemporary mass spectrometry, this study contributes to the specific knowledge of fumonisin biosynthesis and the general mechanisms possible for thiotemplate transformations.

4.0 Outlook

These recent studies of fungal PKS proteins are stepping stones towards the implementation of the aforementioned proteomics approaches to natural product elucidation techniques. Mechanism of compound formation as well as early previews of compound structures can be accessed through measurements of biosynthetic proteins directly. Previous challenges including fungal protein purification, isolation, identification and quantitation as well as cell lysis have been recently reviewed with the most efficient techniques to overcome these challenges presented elsewhere [61]. With proteomics maturing rapidly for full proteome coverage, thought now to be defined as detecting over 4000 of the ~6300 genes in the fungal proteomic-benchmark system, S. cerevisiae [62], it is time to apply this expanded tool set to understanding secondary metabolism in fungi.

One example where the reviewed proteomics tools could be put to good use is in the connection of secondary metabolites with their respective biosynthetic genes. For example, A. nidulans is known to produce 22 NRP and PK products, but has the genetic capacity for even more. Analysis of the A. nidulans FGSC A4 genome reveals 26 PKSs and 24 NRPSs [63]. Thus far only 6 PKSs responsible for biosynthesis of monodictyphenone, asperfuranone, orsellinic acid/F9775, asperthicin, napthopyrone, and sterigmatocystin, [64–69] and 4 NRPSs responsible for the biosynthesis of terrequinone, aspyridones, emericellamide and pennicillin [7,70–72] of A. nidulans have been characterized, although it is clear from the detection and characterization of numerous peptide and polyketide products that many more can be expressed. These compounds along with all other compounds covered in this review, their source and bioactivity are listed in Table 1. It is estimated that there are roughly 1.5 million species of fungi [73], but only a small fraction have been sampled by either genomic or proteomic techniques; clearly there is a wealth of fungal biodiversity to be discovered, elucidated and exploited. With the expectation that many if not most of the 1.5 million fungal species will be unculturable, we can expect that metaproteomics [74] will play an eventual role in teasing out the important and complex biosynthetic pathways of fungi.

Table 1.

Compounds covered in this review

Compound Source Type Bioactivity Reference
aflatoxins fungal polyketide toxin [15–19,55,56]
sterigmatocystin fungal polyketide toxin [69]
ochratoxin fungal polyketide toxin [21]
T-toxins fungal polyketide toxin [22]
fumonisins fungal polyketide toxin [23,60]
citrinin fungal polyketide toxin [24]
aurofusarin fungal polyketide toxin [25]
patulin fungal polyketide toxin [26]
dothistromin fungal polyketide toxin [27]
lovastatin fungal polyketide hypolipidemic [57–59]
compactin fungal polyketide hypolipidemic [75]
penicillins fungal/bacterial peptide antibacterial [30,72,76]
gliotoxin fungal polyketide toxin [28]
HC-toxin fungal peptide toxin [29]
cephalosporins fungal/bacterial peptide antibacterial [76]
cyclosporin A fungal peptide immunosuppressant [31]
ennaitin A fungal peptide toxin [32–35]
mycosubtilin bacterial peptide antifungal [42]
kurstakin bacterial peptide antifungal [50]
vibriobactin bacterial peptide siderophore [52]
pikromycin bacterial polyketide antibacterial [53]
tyrocidin bacterial peptide antibacterial [53]
surfactin bacterial peptide antibacterial/antifungal [53]
monodictyphenone fungal polyketide unknown [64]
asperfuranone fungal polyketide antitumor [65]
orsellinic acid/F9775 fungal polyketide protease inhibitor [66]
asperthicin fungal polyketide toxin [67]
napthopyrone fungal polyketide pigment [68]
terrequinone fungal peptide antitumor [70]
aspyridones fungal hybrid toxin [71]
emericellamide fungal hybrid antibacterial [7]
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5.0 Acknowledgements

The authors thank the National Institutes of Health (GM 067725) for support of our work in natural products discovery and biosynthesis.

Appendix #1

A domain set of A-T-C comprises one module of a NRPS and contains all the functions necessary for incorporation of one monomer [77]. Adenylation domains are the “gatekeepers” of the NRPS assembly line. These enzymes are composed of ~500 amino acids and belong to the AMP-binding super-family of enzymes. Entry of monomers is regulated by specific domains for each monomer and requires ATP for activation. A-domains contain a conserved D-K pair that is responsible for recognition of the amino and carboxyl ends of an amino acid and for coordination of the Mg2+ and ATP cofactors. Substrate specificity of the A-domain is derived from the residues lining the amino acid binding pocket. Well-characterized systems can be used to predict the specificity of uncharacterized A-domains, which is a driving force behind proteomic based NRPS elucidations [1,78,79]. The adenylation domain catalyzes adenylation of the amino acid substrate carboxyl with AMP. The aminoacyl adenylate intermediate is short-lived and is a substrate for a transesterification to the free thiol of the T-domain active site.

The smallest domains, referred to as thiolation or just carrier domains, are ~100 amino acid stretches that carry intermediates and owe their name to the free thiol of their phosphopantetheine (Ppant) cofactor. The T-domain active site is well conserved and contains the GG(H/D)SL phosphopantetheine attachment site motif. The sole function of these domains is to covalently anchor ~20 Å swinging arm that can move from active site to active site shuttling intermediates along the assembly line. The Ppant cofactor is derived from CoA and is attached to the T-domain active site serine through a phosphopantetheinyl transferase most often acting in trans. Condensation domains are composed of ~500 amino acids and catalyze amide bond formation between intermediates on adjacent T-domains. These enzymes carry a signature HHXXXDG motif essential for catalysis. The second H in the strictly conserved motif is proposed to activate the acceptor amino group so that it can act as a nucleophile and attack the donor thioester. The donor T-domain is now available for recharging by its A-domain and the newly formed dipeptide is ready for progression down the assembly line. Peptide bond formation proceeds in this fashion until the end of the last module is reached. Product release can be achieved by hydrolysis of the last thioester with or without macrocyclization [5] as seen in Fig. 2.

PKS core activities of monomer selection (AT), tethering (T, acyl carrier protein (ACP)), and condensation (KS) functions are analogous to the A-, T-, and C- domains of NRPSs and highly related to the AT- ACP- and KS- domains involved in fatty acid synthesis [80]. AT domains are ~45 kDa enzymes that select acyl-CoA starter and extender units for chain initiation and elongation [81]. The AT-domains attack the starter and extender CoA thioester bonds with a conserved serine nucleophile forming a transient covalent intermediate that is the substrate for KSs (for initiation) and ACPs (for elongation) [82]. The T-domains of PKSs are very similar to those of NRPSs and are approximately the same size, 8–10 kDa. The KS is a ~50 kDa enzyme that catalyzes Claisen condensation between a T-domain and KS-domain bound substrates using a conserved cysteine residue [83]. In addition to these minimal functions, PKSs may also include ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER), cyclase (Cyc), thioesterase (TE), C-methyl transferase (C-MT) activities, among others [84]. The diversity in PK structure and activity relies on stereocenters introduced by KRs, degree of reduction, cyclization, aromatization and decoration of the ketide core as opposed to NRPs that owe most of their structural diversity to monomer selection. PKSs can be categorized into three different types, type I, type II and type III, although blurring between the categories has become evident from the growing wealth of DNA sequence [85]. Enzymatic functions are shared between the types; however quaternary structure and chain initiation and termination are different. Type I PKSs are monolithic enzymes comprised of multiple active sites along one polypeptide chain. Type II PKSs are a set of discrete enzymes that act iteratively to produce a polyketide. Type II PKSs utilize stand- alone T-domains. In addition they minimally consist of a KS and chain-length factor (CLF) that are typically encoded by distinct genes. The CLF is an inactive KS enzyme that provides a cavity for the nascent ketide chain to extend into, the depth of which governs ultimate chain length [86]. Type III PKSs use acyl-CoAs but no T-domain and have a single KS which initiates, tethers, extends and terminates the polyketide chain. They function as homodimers and are roughly ~45 kDa in size. Type III PKSs are typically thought of as plant enzymes; however, there are examples of bacterial and fungal type III PKSs [87,88]. For fungi, the most commonly used mode is the type I PKS. In fungi, unlike bacteria, the type I PKSs use a single KS-AT-T module repeatedly and are hence referred to as type I iterative PKSs (IPKSs). These IPKSs can be further subdivided into non-reducing (nr), partially-reducing (pr) and highly-reducing (hr) IPKSs [89]. Theses qualifications reflect the presence or absence of PKS reducing functions, the KR, DH and ER domains.

Footnotes

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