Biosynthesis of fungal polyketides by collaborating and trans-acting enzymes

Contents

• 1. Introduction

• 2. PKS activation by a phosphopantetheinyltransferase (PPT)

• 3. PKS loading by collaborating enzymes
  • 3.1 FAS loading NRPKS enzymes with a starter unit
  • 3.2 HRPKS loading NRPKS enzymes with a starter unit
  • 3.3 Structures of SAT domains

• 4. PKS requiring collaborating enzymes for polyketide extension
  • 4.1  Trans-acting ER enzymes
  • 4.2  Trans-acting MT enzymes
  • 4.3  Trans-acting KR enzymes

• 5. PKS requiring collaborating enzymes for polyketide release
  • 5.1  Trans-acting α,β-hydrolases
  • 5.2  Trans-acting acyl transferases
  • 5.3  Trans-acting reductases
  • 5.4  Trans-acting NRPS enzymes
  • 5.5  Trans-acting PLP-dependent enzymes
  • 5.6  Trans-acting cyclases

• 6.  Trans-acting oxidases

• 7. Biosynthetic pathways utilizing multiple trans-acting and collaborating enzymes

• 8. Biosynthetic pathways engineering opportunities

• 9. Conclusions and future prospects

• 10. Conflicts of interest

• Acknowledgements

• References

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Abstract

Covering: 1999 up to 2021

Fungal polyketides encompass a range of structurally diverse molecules with a wide variety of biological activities. The giant multifunctional enzymes that synthesize polyketide backbones remain enigmatic, as do many of the tailoring enzymes involved in functional modifications. Recent advances in elucidating biosynthetic gene clusters (BGCs) have revealed numerous examples of fungal polyketide synthases that require the action of collaborating enzymes to synthesize the carbon backbone. This review will discuss collaborating and trans-acting enzymes involved in loading, extending, and releasing polyketide intermediates from fungal polyketide synthases, and additional modifications introduced by trans-acting enzymes demonstrating the complexity encountered when investigating natural product biosynthesis in fungi.

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Elizabeth Skellam

Elizabeth Skellam received an MChem degree from Swansea University, a Masters in Business Administration from the University of North Carolina at Wilmington, and a PhD in Chemistry from the University of Bristol. She has worked as a Technical Writer at ID Business Solutions and as an Akademischer Ratin at Leibniz University Hannover. She is currently an Assistant Professor at the University of North Texas and her research focuses on elucidating and engineering biosynthetic pathways in microorganisms to discover new enzymes and generate novel natural products.

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1. Introduction

Fungal polyketides possess a diverse range of biological activities, some of which are beneficial to human health and have been utilized in medicine and agriculture e.g. lovastatin 1, strobilurin A 2, and fumagillin 3. In contrast, other fungal polyketides are devastating mycotoxins such as aflatoxin 4, patulin 5, zearalenone 6, and fumonisin B1 7, that contaminate foodstuffs (Fig. 1).

Fig. 1 Examples of important fungal polyketide compounds.

In fungi, the majority of polyketides are synthesized by iterative type I multifunctional polyketide synthases (PKS),^1–4 although some exceptions exist.^5–7 Type I iterative polyketide synthases (PKS) can be subdivided into non-reducing (NR) PKS, partially reducing (PR) PKS and highly reducing (HR) PKS, based on the catalytic domains present and the structure of the resulting polyketide backbone.^8,9 Hybrid PKS enzymes are known such as polyketide synthase/non-ribosomal peptide synthases (PKS–NRPS) (Fig. 2).¹⁰ Although type III PKS also occur in fungi,^11,12 these enzymes are out of scope of this review.

Fig. 2 Domain organization of fungal PKS. Faded domains indicate that these domains are not present in every PKS sub-class. ER⁰ indicates that a domain is present but inactive. Domain abbreviations: SAT = starter unit:ACP-transacylase; KS = ketosynthase; AT = acyltransferase; PT = product template; ACP = acyl carrier protein; MT = C-methyltransferase; TD = terminal domain (involved in release; discussed later); DH = dehydratase (in PRPKS this is a thiolhydrolase (TH) domain¹³); KR = ketoreductase; ER = enoylreductase; C = condensation; A = adenylation; T = thiolation.

NRPKS contain three catalytic domains that are present in all fungal type I PKS: ketosynthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains.¹⁴ However, unlike PRPKS and HRPKS they lack the reductive and dehydrative domains active during β-processing, e.g. ketoreductase (KR), enoyl reductase (ER), and dehydratase (DH)/thiolhydrolase (TH)¹³ domains. Instead, NRPKS contain starter-unit acyl transferase (SAT) and product template (PT) domains.^15,16 Additional domains that may be present include: C-methyltransferase (MT) domains, and terminal domains such as thioesterase (TE),¹⁷ Claisen cyclase (CLC),¹⁸ or reductase (R) domains,¹⁹ that are involved in product release rather than β-processing. Aflatoxin 4 and zearalenone 6 are examples of polyketides synthesized by NRPKS.

PRPKS are relatively rare; in addition to KS, AT, and ACP domains they also contain TH and KR domains.¹³ Only a few PRPKS have been characterized but they typically synthesize 6-methylsalicylic acid,²⁰ the precursor to patulin 5,²¹ or the common fungal metabolite (R)-mellein 8.²² The products of PRPKS are less reduced than the products of HRPKS due to the presence of only a KR domain, however the enzymes themselves form separate phylogenetic clades from HRPKS and PKS–NRPS.²²

HRPKS contain domains required for β-processing e.g. DH, KR, and, usually, ER domains in addition to the standard KS, AT and ACP domains. Often they lack domains involved in product release and consequently are more difficult to study than their NRPKS or PKS–NRPS counterparts. Examples of polyketides synthesized by HRPKS are lovastatin 1, strobilurin A 2, fumagillin 3, and fumonisin B1 7. HRPKS are most similar to fatty acid synthases (FAS) found in mammals in terms of the domains present,¹⁴ however the chemistry differs significantly in that the β-processing domains are used in a programmed manner.

Due to the large size of fungal PKS enzymes (>250 kDa),²³ and the incredible challenges in obtaining soluble protein for structural studies, there is relatively little structural information available for this class of enzymes. Investigations into the biosynthesis of fungal polyketides, therefore, typically require the use of isotopically labelled precursors and/or intermediates,²⁴ targeted gene inactivation, heterologous expression (omitting one or more genes of interest),²⁵ and enzymology of individual catalytic domains.^{14–17,26,27} Often, the structures of mFAS²⁸ (Fig. 3) and structurally characterized bacterial PKS domains are used as homologs for fungal PKS enzymes. Only very recently has the structure of a complete fungal PKS been reported.²³

Fig. 3 The structure of mammalian FAS (PDB = 2VZ8).²⁸ Domain abbreviations as in Fig. 2.

Multifunctional proteins are not unique to secondary metabolism e.g. type I FAS in mammals,²⁹ and the pentafunctional AROM protein required for shikimate production in fungi.³⁰ Multi-domain enzymes are thought to arise from monofunctional enzymes, and be the evolutionary result of smaller ancestral genes encoding the individual enzymes fusing.^31,32 The coordinated use of the different catalytic domains by type I PKS is similar to the coordinated use of discrete type II PKS in bacteria, for example, and it is likely that type I PKS arose from fusion of type II PKS genes. An evolutionary analysis of KS domains from PKS and FAS demonstrate that iterative type I PKS are more closely related to modular PKS than fungal FAS.³³ Similarly, structural analysis of fungal PT domains, DH domains from FAS, and aromatases/cyclases from bacteria (ARO/CYC) indicate that these enzymes have conserved structural features despite sharing little sequence homology.^16,34 These comparisons suggest that the way the individual domains interact with one another may also be conserved.

The recruitment of additional collaborating enzymes is a concept that may be explained by the formation of metabolons,³⁵ where sequential biosynthetic enzymes self-assemble into a multienzyme complex via non-covalent protein–protein interactions.³⁶ These metabolons facilitate efficient substrate transport between the enzymes via channelling and thereby increase the overall flux of the pathway. Metabolons have been reported in primary metabolism, such as the citric acid cycle and fatty acid biosynthesis,^37–41 as well as in secondary metabolism in plants.^42–45

This review will highlight some of the most well-studied fungal polyketide pathways that require trans-acting and collaborating enzymes e.g. lovastatin 1 and aflatoxin 4 biosynthesis, but also discuss the more unexpected collaborations in fungal polyketide biosynthesis that have been proposed based on isolation of intermediates. From a biosynthetic perspective, the simplest classification system can be defined as collaborations that initiate biosynthesis (PKS loading), collaborations that occur during polyketide chain elongation (PKS extension) or collaborations that terminate biosynthesis (PKS release). However, there are additional examples of tailoring reactions by trans-acting enzymes, and, several increasingly more complex pathways where more than one trans-acting enzyme is required. Ultimately, these observations are intended to be useful when engineering fungal polyketide pathways by factoring in the requirement of collaborating enzymes and considering potential protein–protein interactions.

2. PKS activation by a phosphopantetheinyltransferase (PPT)

Although outside of the scope of this review, it is important to mention that PKS require post-translational modification to activate the ACP domains (Scheme 1). This is achieved by a phosphopanthetheinyltransferase (PPT), a class of enzymes that attach 4′-phosphopantetheine derived from coenzyme A 9 (reviewed elsewhere).⁴⁶ 4′-Phosphopantetheine is considered a “prosthetic arm” that is covalently bound to the ACP and essential for the transfer of all substrates and intermediates. NpgA is a PPT identified in Aspergillus nidulans and shown to be involved in both primary (i.e. lysine biosynthesis)^47,48 and secondary metabolism.^49,50 NpgA homologs are found in many fungi including the aflatoxin 4 producer Aspergillus parasiticus.⁵¹In vitro investigations of NpgA from A. parasiticus demonstrated that this PPT could activate an ACP domain from an iterative PKS (PksA) by transferring 4′-phosphopantetheine, but could not activate an ACP domain from the FAS (HexA).⁵¹ Specific ACP/PPT interactions are proposed but not yet elucidated.

Scheme 1 Overview of ACP activation by a trans-acting PPT.

3. PKS loading by collaborating enzymes

Feeding studies with isotopically labelled compounds have demonstrated that the majority of fungal polyketides utilize acetyl-CoA as the starter unit. However, a number of compounds have been identified that are derived from alternative starter units such as: hexanoyl-CoA in norsolorinic acid 10 biosynthesis,⁵² the precursor of the mycotoxin aflatoxin 4; propionyl-CoA e.g. in pseurotin 11;⁵³ nicotinyl-CoA e.g. in pyripyropene A 12;⁵⁴ or malonamoyl-CoA in the biosynthesis of viridicatumtoxin 13 (Fig. 4).⁵⁵ Although there are several examples of HRPKS that utilize non-acetate starter units, such as those involved in the biosynthesis of squalestatin S1 14,⁵⁶ and strobilurin A 2,⁵⁷ the rules governing how certain HRPKS select non-acetate starter units remains unknown, however they are not attributed to collaborating enzymes.

Fig. 4 Examples of fungal polyketides derived from non-acetate starter units (highlighted in red).

A breakthrough in the understanding of fungal NRPKS came from the discovery of the starter unit:ACP-transacylase (SAT) domain.¹⁵ SAT domains select the starter unit substrate and load the ACP, which then transfers the substrate onto the KS domain. Based on experiments with isolated SAT domains there is some flexibility in which substrates are selected in vitro,⁵⁸ however SAT domains exhibit a clear selectivity over the chain length and functionality present in the starter unit.

3.1 FAS loading NRPKS enzymes with a starter unit

The biosynthesis of norsolorinic acid 10, the precursor to aflatoxin 4 and other biosynthetically related mycotoxins, results from the collaboration of two FAS subunits, HexA and HexB, with the NRPKS, PksA.⁵⁹ The two FAS subunits synthesize hexanoyl-CoA that is transferred to the SAT domain of PksA. The N-terminal SAT domain of PksA was cloned, recombinantly expressed, and incubated with holo-ACP and [1-¹⁴C]-hexanoyl-CoA, clearly showing that the N-terminal domain covalently binds radiolabelled hexanoyl-CoA and transfers it to the ACP domain.¹⁵ In later deconstruction experiments PksA was shown to directly accept the hexanoyl starter unit 15 from the ACP domain of HexA (Scheme 2).⁶⁰

Scheme 2 Experimentally confirmed transfer of hexanoyl starter unit 15 from the ACP of the FAS HexA to the SAT domain of the NRPKS PksA.

Besides the fatty acid hexanoyl-CoA, the only other fatty acid to be reported as a starter unit for an SAT domain is octanoyl-CoA in NRPKS from the fungi Coccidioides immitis and C. posadasii.⁶¹ These fungi contain biosynthetic gene clusters (BGC) encoding two FAS subunits and an NRPKS, however, the final structure of the metabolite produced by the cryptic cluster remains unknown.

3.2 HRPKS loading NRPKS enzymes with a starter unit

An emerging class of characterized NRPKS are those that collaborate with a HRPKS lacking a release domain; the HRPKS synthesizes a polyketide starter unit and transfers it directly to the SAT domain of the partnering NRPKS (Scheme 3). To date, di-, tri-, tetra-, penta- and hexaketide starter units have been proposed that are mostly linear and display varying levels of reduction; some of which contain pendant methyl groups (Fig. 5).

Scheme 3 General overview of NRPKS loading by a HRPKS.

Fig. 5 Examples of fungal polyketides derived from collaborating HRPKS and NRPKS. The polyketide transferred by the collaborating HRPKS is highlighted in red.

Zearalenone 6 was the first fungal polyketide recognised as requiring two PKS enzymes for biosynthesis;^62,63 the HRPKS PKS4 is proposed to synthesize a very highly reduced hexaketide that is transferred directly to the NRPKS PKS13 (Fig. 5). Similar biosyntheses are proposed for the anti-fungal agents hypothemycin 16,^64,65 and radicicol 17,^66,67 a HSP90 inhibitor. In contrast, the immune system modulator 10,11-dehydrocurvularin 18 is proposed to arise from an almost fully reduced tetraketide starter unit synthesized by the HRPKS AtCURS1 that primes the NRPKS AtCURS2 (Fig. 5).⁶⁸

The novel metabolite asperfuranone 19 was discovered by genome mining after activation of the gene cluster encoding a unique HRPKS/NRPKS pair.⁶⁹ The HRPKS AN1036.3 synthesizes a dimethylated tetraketide that acts as the starter unit for the NRPKS AN1034.3. Since then, a number of fungal polyketide biosynthetic pathways have been shown to involve the collaborative action of HRPKS and NRPKS pairs including: chaetomugilin A 20, derived from a methylated triketide synthesized by the HRPKS CazF;⁷⁰ the antioxidant sorbicillinoid 21 derived from a triketide diene synthesized by the HRPKS SorA;⁷¹ tricholignan 22 that utilizes the triketide starter unit synthesized by the HRPKS TlnA;⁷² the anti-malarial cladosporin 23, composed of a highly functionalized pentaketide synthesized by the HRPKS Cla2;⁷³ and ascotricholactone A 24, proposed to utilize a reduced oligomeric diketide synthesized by Men1 (Fig. 5).⁷⁴

The corresponding NRPKS subsequently elongates, cyclizes and releases the polyketide product once it reaches the programmed chain length.⁷⁵ Some NRPKS contain methyltransferase domains and introduce methyl groups during chain elongation programmatically, and release can be reductive or thioesterase mediated. The polyketide produced through the collaborative action of the HRPKS and NRPKS may be the final product of the biosynthetic pathway e.g.18 and 21, or it may undergo additional modifications by tailoring enzymes encoded by the same gene cluster e.g.19 and 20.

3.3 Structures of SAT domains

X-ray crystal structures of isolated SAT domains indicate an α,β-hydrolase core and a smaller ferrodoxin-like domain. The structure of the SAT domain from the NRPKS CazM (Fig. 6)²⁶ and the SAT–KS–AT tri-domain from NRPKS CTB1 (Fig. 7)⁷⁶ exhibit hydrophobic residues that line the active site of the SAT domains. These residues are proposed to provide control over the chain length and extent of functional groups of the starter unit entering the cavity.

Fig. 6 Structure of the CazM SAT domain bound with hexanoyl-CoA (PDB = 4RPM).²⁶ The arginine residues proposed to be involved in inter-/intra-molecular docking with ACP domains are labelled.

Fig. 7 The structure of the SAT–KS–AT tri-domain from CTB1 and ACP2 (also from CTB1; circled. PDB = 6FIK).⁷⁶ The arginine residues proposed to be involved in inter-/intra-molecular docking with ACP domains are labelled.

Inter- and intramolecular docking of the SAT domain with ACP domains from the collaborating HRPKS (Fig. 6) and the native NRPKS (Fig. 7) are believed to be facilitated by several basic residues lining the entrance of the SAT domain active site.^26,76 Therefore, the SAT domain has at least three fundamental roles: docking with the downstream ACP domain for starter unit transfer, selecting the preferred starter unit, and docking with the ACP domain from the collaborating enzyme.

To date, SAT domains have been reported to interact with collaborating FAS enzymes or HRPKS enzymes that provide a non-acetate starter unit. It is not yet possible to predict the starter unit selected by the SAT domain; this has to be confirmed experimentally using isotope labelled precursors, isolated domain assays, or via heterologous expression of the NRPKS with its collaborating enzyme partner.

4. PKS requiring collaborating enzymes for polyketide extension

4.1 Trans-acting ER enzymes

The first example of a fungal PKS being reliant on a trans-acting accessory protein for correct polyketide extension was in the biosynthesis of the cholesterol-lowering drug lovastatin 1.⁷⁷ The biosynthetic gene cluster contains two genes encoding PKS enzymes; lovB encodes lovastatin nonaketide synthase (LNKS) and lovF encodes lovastatin diketide synthase (LDKS). LNKS, like many HRPKS, has an inactive ER domains (ER⁰) based on the absence of key active site residues. Also contained in the cluster is lovC, encoding a stand-alone protein with sequency similarity to PKS ER domains.⁷⁷ In heterologous expression experiments in A. nidulans, when LNKS is expressed alone, instead of producing the expected dihydromonacolin L 25, shunt polyene compounds 26 and 27 were detected (Scheme 4A). Furthermore, the shunt products had shorter carbon chain lengths than expected, indicating a loss of LNKS programming fidelity. Co-expression of LNKS and LovC led to synthesis of the expected polyketide 25 (Scheme 4A) demonstrating that the associated trans-acting ER protein, LovC, not only reduces the LNKS olefins selectively but also contributes to overall PKS programming control.⁷⁷

Scheme 4 (A) The trans-acting ER enzyme LovC contributes to the correct programming of LNKS in dihydromonacolin L 25 biosynthesis.⁷⁷ (B) Examples of fungal polyketides biosynthesized through the actions of trans-acting ER enzymes. Chemical modifications introduced by trans-acting ER enzymes are highlighted in light blue.

Trans-acting ER enzymes have been confirmed in a number of other fungal polyketide biosynthetic pathways including tenellin 28 and other pyridones,^78–84 equisetin 29 and other decalins,^85–91 cytochalasin E 30 and other cytochalasans,^92–98 himeic acid 31,⁹⁹ pyrrocidines 32,¹⁰⁰ carlosic acid 33,¹⁰¹ and other acyl tetronic acids,¹⁰² burnettramic acid A 34,¹⁰³ and other lipid-like polyketides¹⁰⁴ (Scheme 4B). It is now widely accepted, even expected, that a gene encoding a HRPKS with an ER⁰ domain, in close proximity to a trans-ER gene in a BGC, must be co-expressed for correct polyketide synthesis.

To date, only a single crystal structure of a fungal trans-acting ER enzyme, LovC, has been reported.¹⁰⁵ LovC contains a medium chain dehydrogenase/reductase (MDR) fold and exists as a monomer. Between the catalytic domains and the NADPH co-factor binding domain are two loops that are highly conserved amongst fungal trans-acting ERs. These extended loops are proposed to prevent dimerization of LovC, since they induce steric clash in computational models.¹⁰⁵ The substrate binding pocket is adjacent to the bound NADP⁺ and contains an electropositive bridge including the residue K54, proposed to dock the phosphopanthetheine arm of the LNKS ACP domain (Fig. 8). The monomeric form of LovC was proposed as being necessary for protein–protein interactions with LNKS and it was hypothesized that LovC forms a heterodimer with LNKS–ER⁰. However, no complex was detected when LovC was incubated with LNKS ER⁰ indicating a different mode of protein–protein interaction.¹⁰⁵

Fig. 8 The structure of LovC in complex with NADP⁺ (PDB ID = 3B70).¹⁰⁵ The putative PPT docking site K54 is labelled.

The structure of LovC reveals important information about substrate specificity and the final structure of the nonaketide synthesized by LNKS. Due to hydrophobic binding, the substrate pocket of LovC accommodates tetra- and pentaketide intermediates more efficiently than the di- and triketide intermediates, and so they undergo enoyl reduction.¹⁰⁵ However, the hexaketide intermediate forms a decalin due to Diels–Alder cyclization being favoured over enoyl reduction.¹⁰⁵

Recently, the cryo-EM structure of the LovC-LNKS complex was reported.²³ LNKS is considered as a HRPKS even though it contains a terminal condensation (C) domain, similar to those observed in non-ribosomal peptide synthetase (NRPS) enzymes. Although the ACP and C domains could not be solved, this is the most complete fungal PKS enzyme structure containing eight connected catalytic domains. The LovC-LNKS complex is an X-shaped dimer and LovC interacts with the AT domain, forming two L-shaped chimeric catalytic chambers (Fig. 9),²³ reminiscent of the structure of mFAS (Fig. 3).

Fig. 9 The structure of LNKS and LovC (PDB ID = 7CPY).²³

Unfortunately, the resolution of the LovC-LNKS interface was not high enough to model protein–protein interactions, but computational docking identified the docking region between residues 695–757 of the LNKS AT domain and the loop region of LovC. Mutating the loop region of LovC and co-expressing with LNKS–AT resulted in the two proteins eluting separately. Furthermore, mutating residues 747–750 within LNKS–AT also causes the two proteins to elute separately, thereby establishing the trans-acting ER interaction between the loop region and a helix of the AT domain.²³

4.2 Trans-acting MT enzymes

Pseurotin A 11 is a PKS–NRPS derived polyketide displaying neuritogenic activities amongst other bioactivities.^106,107 A gene cluster for biosynthesis of 11 was identified in Aspergillus fumigatus as it was the only cluster encoding a PKS–NRPS enzyme.¹⁰⁸ The function of the PKS–NRPS encoding gene, psoA, was confirmed as being essential for biosynthesis of 11 by both knockout and overexpression.¹⁰⁸ The PKS–NRPS product of this gene, PsoA, had all the domains expected for the biosynthesis of 11, including a MT domain, albeit with low homology to other fungal MT domains (Scheme 5).¹⁰⁸

Scheme 5 Biosynthetic pathway of pseurotin A 11.^110,111 Abbreviations: as in Fig. 2; GST = glutathione S-transferase. Only chemical modifications introduced by the bifunctional PsoF are highlighted in colour.

Unexpectedly, the pseurotin gene cluster was later found to be interlinked with the genes required for the biosynthesis of fumagillin 3, within a supercluster.¹⁰⁹ An additional two genes were subsequently identified as being essential for biosynthesis of 11; psoF, encoding a dual function monooxygenase/methyltransferase, and psoG, encoding a hypothetical protein.¹¹⁰ Characterization of the modification enzymes: PsoC, an O-methyltransferase; PsoD, a P450; PsoE, a glutathione S-transferase; and the bifunctional PsoF, established precise functions for PsoC–E and indicated that PsoF participated in trans-C-methylation of the growing polyketide (Scheme 5), despite the MT domain of the PKS–NRPS retaining some methylation activity in in vitro studies.¹¹¹ The role of PsoG remains unknown as it has no homology to any known protein.

The MT domain of PsoF was cloned and expressed in E. coli for structural and mechanistic studies (Fig. 10).¹¹² The structure of PsoF identified a structural fold characteristic of class I S-adenosyl-methionine (SAM) dependent methyltransferases that act on small molecules. Computational docking studies suggested that there is a hydrophobic tunnel, formed by the SAM-binding domain and substrate binding domain, that can accommodate a tetraketide intermediate.¹¹² Mutagenesis of any of the active site residues Tyr687, His799 or Glu825 significantly reduced enzyme activity. Tyr687 is proposed to be essential for substrate binding and catalysis and Tyr833 is proposed to be involved in controlling the chain length of the intermediate that is accepted into the active site. Currently, it is still unknown how exactly the trans-acting PsoF interacts with the PKS–NRPS PsoA.

Fig. 10 The structure of PsoF MT domain in complex with SAH (PDB ID = 6KJI).¹¹² The tyrosine residues proposed to be involved in substrate binding and recognition are labelled.

Compared to trans-acting ER enzymes, reports of trans-acting MT enzymes are relatively rare. A trans-acting MT domain has also been identified in the biosynthesis of the fungal metabolites trichnolignan 22 and calbistrin A 35 (Fig. 11).^72,113 In calbistrin A 35 biosynthesis the function of the trans-acting MT CalH′ was first established through heterologous expression with the HRPKS CalA′ in Aspergillus oryzae.¹¹³ A mixture of compounds 36–39 was detected including 36 that has a carbon backbone identical to the polyene portion of 35 (Scheme 6). Compounds 36 and 38 were also observed in in vitro enzymatic reactions, and assays determined that CalH′ acts on the growing polyketide chain while it is still attached to the PKS.¹¹³

Fig. 11 Structures of tricholignan 22 and calbistrin A 35 indicating the position of a methyl group introduced by a trans-acting MT enzyme. Chemical modifications introduced by the trans-acting MT enzymes are highlighted in green.

Scheme 6 Investigations into the biosynthetic products of CalA′ and the trans-acting MT CalH′.

Similar to the structure of PsoF-MT, CalH′ contains catalytic residues Tyr157, His266 and Glu292 that are well conserved, however the hydrophobic substrate tunnel of CalH′ is larger, to accommodate a longer polyene substrate (Fig. 12).¹¹³ Furthermore, the surface of CalH′, near to the substrate tunnel, also consists of positively charged residues. A computational docking study suggests that the negatively charged region of the CalA′ ACP fully covers the entrance of the active site due to the interaction between the oppositely charged protein surfaces, orchestrating the methylation reaction.¹¹³

Fig. 12 The structure of CalH′ MT domain (PDB ID = 7DMB).¹¹³

The biosynthesis of both tricholignan 22 and calbistrin A 35 contain a number of additional unusual features and so will be discussed further in Section 7.

4.3 Trans-acting KR enzymes

The immunosuppressant agent dalmanol 40 contains a rare 6/6/6/6/6 ring system proposed to derive from 1,3,6,8-tetrahydroxynaphthalene (4HN) 41 and 1-(2,6-dihydroxyphenyl)but-2-en-1-one (PBEO) 42 (Scheme 7).¹¹⁴ The genes required for the biosynthesis of 40 are located in two different gene clusters, physically separated on the genome of Daldinia eschscholzii. One gene cluster encodes a PRPKS, ChrA, adjacent to a ketoreductase enzyme, ChrB, while the second gene cluster encodes an NRPKS, pksTL. Both gene clusters were confirmed as being required for biosynthesis of 40 by gene inactivation and heterologous expression in Aspergillus oryzae.¹¹²

Scheme 7 Biosynthetic pathway of dalmanol A 40. The HRPKS ChrA contains an active KR domain however, the trans-acting KR enzyme ChrB appears to be utilized to diversify the range of polyketide products observed. The chemical modification introduced by the trans-acting KR enzyme ChrB is highlighted in light blue.

ChrA is an unusual PRPKS with an inactive MT domain. Unexpectedly, heterologous expression experiments of ChrA in A. oryzae determined that the KR domain is active during the first round of chain extension due to isolation of 43. However, when the KR enzyme, ChrB, was co-expressed with ChrA, 42 was observed, indicating that ChrB is active in the fourth round of chain extension, specifically reducing the C-3 carbonyl to a hydroxyl group (Scheme 7).¹¹⁴

A trans-acting PKS-like multidomain enzyme, TerB, has been identified as essential for 6-hydroxymellein (6-HM, 44) biosynthesis,¹¹⁵ a precursor to the phytotoxin terrein.¹¹⁶ 6-HM is synthesized through the collaboration of TerA, an NRPKS, and TerB, an unusual enzyme containing an active KR domain and inactive DH and MT domains, in an organization similar to a truncated HRPKS. Heterologous expression of TerA alone in either Aspergillus niger or A. oryzae led to production of triketide 45, tetraketide 46, and pentaketide 47 products indicating an unusual relaxation of the TE domain with respect to both the polyketide length and mode of release i.e. lactonization vs. hydrolysis (Scheme 8).^115,116 Co-expression of TerA and TerB led to a production of 6-HM 44 in a substantially higher titer than 45–47, pointing to a collaborative mode of action between the two enzymes.¹¹⁵

Scheme 8 Biosynthesis of 6-HM 44 requires collaborating NRPKS TerA and the PKS-like enzyme TerB. Site-directed mutations determined that the interaction site is between the PT and DH domains. The chemical modifications introduced by the trans-acting PKS-like enzyme TerB is highlighted in blue.

The interaction between TerA and TerB was investigated via site directed mutations of specific catalytic sites including both ACPs, the TE, the DH and the KR domains. Surprisingly inactivating the catalytic site of the PT domain (H1320A) had the same effect as “activating” the catalytic site of the DH domain (Q46H) during co-expression experiments (Scheme 8). The change in product profile to 48 and 49 indicated that mutating the DH domain within TerB affected the PT domain within TerA, preventing it from functioning as expected and providing insight into where the two enzymes interact.¹¹⁵ 6-HM 44 has also been established as being required for the biosynthesis of cyclohelminthols 50, palmaenones 51, roussoellatide 52 (Fig. 13),^117,118 and is a shunt product in the biosynthesis of 24.⁷⁴

Fig. 13 Structures of 6-HM derived metabolites that require the collaboration of a NRPKS and trans-acting HRPKS-like enzyme.

5. PKS requiring collaborating enzymes for polyketide release

There are relatively few HRPKS that contain a terminal domain for product release,^{104,118–120} instead they are often found fused to an NRPS module generating a PKS–NRPS, or collaborating with NRPKS enzymes, as observed in the biosynthesis of 16–24. HRPKS enzymes lacking a terminal off-loading domain appear to collaborate with trans-acting enzymes for polyketide release and there are various classes of enzymes known to facilitate this polyketide off-loading.

Similarly, NRPKS that belong to group V have no domain for polyketide release,¹²¹ instead a trans-acting TE, or more specifically a metallo-β-lactamase protein, is necessary for product release. Finally, there are trans-acting enzymes with proof-reading roles. Although mechanistically they are not required for polyketide release they greatly facilitate the efficiency of the biosynthesis leading to higher titers of natural product obtained.

5.1 Trans-acting α,β-hydrolases

α,β-Hydrolases are a large class of enzymes with both catalytic and non-catalytic functions that include thioesterases. These enzymes contain a catalytic triad comprised of a nucleophilic residue, an acidic residue (e.g. Glu or Asp) and a catalytic His.¹²² These collaborating α,β-hydrolases will therefore be categorized according to their role in biosynthesis.

5.1.1 Hydolases required for hydrolytic release. The first TE-less NR-PKS to be characterized was atrochrysone carboxylic acid synthase (ACAS) which synthesizes a mixture of products 53–56 in collaboration with atrochrysone carboxyl ACP thiolesterase (ACTE, Scheme 9).¹²³ ACTE belongs to the metallo-β-lactamase (MβL) family. These MβL-TE appear to share no sequence homology with NRPKS TE domains and are more closely related to the β-lactamase superfamily. ACAS and ACTE were reconstituted both in vitro and in vivo, however ACAS did not produce any polyketides when ACTE was not included in the reaction mixture. Further investigations with synthetic substrate analogs confirmed that ACTE specifically catalyzes hydrolysis of a thioester bond to release an octaketide intermediate 57 (Scheme 9).¹²³

Scheme 9 The TE enzyme ACTE releases the octaketide intermediate attached to the NRPKS ACAS by hydrolysis to generate 57. The chemical modification introduced by the trans-acting TE enzyme is highlighted in light pink.

TE-less NRPKS with trans-acting MβL-TE have also been identified in the biosynthesis of viridicatumtoxin 13,⁵⁵ neosartoricin 58,¹²⁴ monodictyphenone 59,¹²⁵ asperthecin 60,¹²⁶ trypacidin 61,¹²⁷ geodin 62,¹²⁸ (Fig. 14) and many other anthraquinone and xanthone products derived from emodin 56.^129–132 A common feature of this class of metabolites is spontaneous decarboxylation of the carboxylic acid group generated by the associated trans-acting MβL-TE.

Fig. 14 Examples of fungal polyketides that utilize a trans-acting TE enzyme for hydrolytic polyketide release from an NRPKS.

Trans-acting hydrolases required for hydrolytic release are also found collaborating with HRPKS, e.g. in lovastatin 1,¹³³ squalestatin S1 14,⁵⁶ byssochalamic acid 63,¹³⁴ fusarielin H 64,¹³⁵ and fusaric acid 65 biosynthesis¹³⁶ (Fig. 15). In lovastatin biosynthesis, LovG, a serine hydrolase, was investigated through gene knock-out experiments where disruption caused a drop in production of 1 by over 95%.¹³³In vitro studies confirmed the role of LovG in releasing polyketide products from LNKS.

Fig. 15 Examples of fungal polyketides that utilize a trans-acting hydrolase enzyme for hydrolytic polyketide release from a HRPKS.

In the biosynthesis of the hexaketide portion of 14, a citrate synthase is proposed to act on the polyketide chain tethered to the HRPKS SQHKS (Clz14) and then the hydrolase (Clz11) releases the modified polyketide product 66 (Scheme 10).⁵⁶ The functions of these enzymes were elucidated in the fungal host A. nidulans; if Clz11 was omitted or replaced with β-lactamase Clz13, there was more than a 99% reduction in titers of 14.⁵⁶ The biosynthesis of byssochalamic acid 63 and other maleic anhydrides have also been shown to require the co-ordination of both a citrate synthase and hydrolase,^134,137 indicating this is another common general release mechanism requiring two collaborating and trans-acting enzymes.¹³⁸

Scheme 10 Biosynthesis of squalestatin hexaketide 66 requires both a citrate synthase and hydrolase. The chemical modifications introduced by the trans-acting enzymes are highlighted in colour.

Often, when HRPKS collaborate with trans-acting hydrolases, lactones or macrolides are formed e.g.67–74 (Fig. 16). The biosynthesis of brefeldin A 67, for example, requires a HRPKS (Bref-PKS) and a thiohydrolase (Bref-TH).¹³⁹In vitro investigation of Bref-PKS led to production of acylic nonaketide products e.g.75 (Scheme 11) which was surprising since 67 is a cyclic octaketide. Including the Bref-TH produced acyclic octaketides e.g.76, revealing a dual role for Bref-TH in hydrolysing the polyketide product as well as controlling the polyketide chain length (Scheme 11).¹³⁹

Fig. 16 Examples of fungal polyketides that utilize a trans-acting TE enzyme for polyketide release from a HRPKS. Chemical modifications introduced by the trans-acting TH/TE enzymes are highlighted in light pink.

Scheme 11 Investigations into the role of Bref-TH.

A remaining question is why macrolactonization of 76 was not observed in the assays and whether Bref-TH is required. It was proposed that the cyclopentane moiety observed in 67 may need to be formed first, suggesting that additional trans-acting enzymes remain to be discovered.¹³⁹ Similarly, the biosynthesis of Sch-642305 68, a bicyclic macrolide similar to 67, also suggests a P450 enzyme acting early in the biosynthesis. Heterologous expression of the PKS (SchPKS) and the corresponding hydrolase (SchR1) did not reveal any linear or cyclic polyketide products, however when the P450 SchR2 was co-expressed with SchPKS and SchR1 both linear polyketides and macrolides were observed for the first time.¹⁴⁰ Knock-out of SchR2 in Phomopsis sp. CMU-LMA, the producer of 68, also abolished biosynthesis and no linear or cyclic polyketides were observed.

Trans-acting hydrolases were revealed in the biosynthesis of 69 by transcriptional factor engineering and by heterologous expression for 70–73 (Fig. 16).^141–146 Recently the thioesterase, DcsB, was shown to collaborate with the HRPKS DcsA during the biosynthesis of the 10-membered lactone decarestrictine C1 74.¹⁴⁵ DcsB was shown to be able to hydrolyse a number of polyketide mimics with varying chain lengths demonstrating its use as a biocatalyst. The crystal structure was obtained showing that DcsB is a homodimer containing an α,β-hydrolase fold (Fig. 17).¹⁴⁵ DcsB was co-crystallized with a HRPKS thioester mimic enabling visualization of the active site, however protein–protein interactions with DcsA were not reported.

Fig. 17 Crystal structure of DcsB (PDB = 6LZH).¹⁴⁵

5.1.2 Hydrolases required for C–C bond formation. Gregatin A 77 is a phytotoxin produced by several fungal species,¹⁴⁶ but until recently had a cryptic biosynthetic pathway. The corresponding gene cluster was identified in Penicillium sp. Sh18 and investigated via heterologous expression in A. oryzae.¹⁴⁷ Expression of the HRPKS (GrgA) and the trans-ER (GrgB) synthesized a linear pentaketide 78, whereas co-expression with the thioesterase (GrgF) led to formation of a heptaketide containing a 6-membered lactone ring 79 (Scheme 12).¹⁴⁷ The interaction between GrgA, GrgB and GrgF was studied in vitro and upon incubation with malonyl-CoA, NADPH and SAM could be observed by LCMS. Further investigation using the GrgA ACP domain, synthetic substrates, a PPT, and GrgF resulted in detection of 77, indicating that GrgA synthesizes two different polyketide chains depending on whether the trans-ER GrgB acts or not.¹⁴⁷

Scheme 12 The biosynthesis of gregatin A 77 revealed that GrgF is a trans-acting TE that fuses two different length polyketides synthesized by the HRPKS GrgA with and without collaboration of trans-acting ER GrgB.

The crystal structure of GrgF demonstrates an α,β-hydrolase fold and contains a typical Cys–His–Asp catalytic triad (Fig. 18).¹⁴⁷ A highly hydrophobic chamber was identified as the substrate binding site and confirmed via docking studies and molecular dynamic simulations. At this time how GrgF interacts with the ACP of GrgA remains unknown but future structural biology studies are proposed.

Fig. 18 Crystal structures of GrgF (PDB = 7D78).¹⁴⁷

α,β-Hydrolases are commonly found in PKS–NRPS gene clusters.^{85,86,88,92–98,110,148,149} The α,β-hydrolase PyiE, involved in the biosynthesis of the cytochalasan pyrichalasin H, was recently proposed to control the formation a specific pyrrolinone tautomer after synthetic and in vivo investigations.¹⁵⁰ Protein–protein interactions are proposed between the PKS–NRPS, PyiS, and hydrolase, PyiE, to enable precise control over the rapid tautomerization, but remain to be confirmed. Similarly, the thioesterase PynI is identified in the biosynthesis of pyranonigrin as being essential for PKS–NRPS product release and tetramic acid formation.¹⁵¹

5.1.3 Hydolases with PKS proof-reading roles. In the biosynthesis of the fungal mycotoxin citrinin an α,β-hydrolase, CitA, was reported to increase titers of the polyketide intermediate resulting from NRPKS PksCT.¹⁵² CitA was investigated in vitro through domain deconstruction experiments of PksCT, and the role of CitA was deduced to be removing acyl intermediates bound to the ACP domain of PksCT (Scheme 13).¹⁵³ Using a homology model two basic residues of CitA, Arg23 and Arg236, were identified as potentially being involved in protein–protein interactions with the ACP domain of PksCT.¹⁵³ Mutation of these residues individually confirmed that R36A was essential for hydrolytic activity and potentially required for interactions with the phosphopantetheine arm of PksCT.

Scheme 13 Overview of the function of the trans-acting hydrolase CitA in the biosynthesis of citrinin.

Trans-acting hydrolases with similar proof-reading activities have been identified in the biosynthesis of lovastatin 1,¹³³ asperfuranone 19,⁶⁹ azanigerone 80,¹⁵⁴ and various Monascus azaphilone-derived pigments.¹⁵⁵ Similar roles for cis-acting TE domains have been characterized in NRPKS such as PksA involved in the biosynthesis of 10.^156,157

5.2 Trans-acting acyl transferases

As mentioned, a distinctive feature of HRPKS is the frequent lack of a domain for polyketide release. The biosynthesis of lovastatin 1 requires two HRPKS, LNKS and LDKS, and a trans-acting hydrolase is required for release of the nonaketide from LNKS. In contrast, the stand alone acyltransferase LovD is required for release of the diketide from LDKS.⁵ The mode of release was studied in vitro by recombinant expression of LDKS in yeast. After incubating with malonyl-CoA, SAM and NADPH; no diketide product was detected, indicating that LDKS is unable to release the polyketide. When LovD and monacolin J acid 81 were included in the reaction mixture 82 could be detected. If LovD was not included, or the LovD S27A active site mutant was used as a replacement, 82 was not detected, indicating that LovD transfers the diketide from LDKS to 81 (Scheme 14A).⁵

Scheme 14 Characterization of the trans-acting acyltransferase LovD in diketide release from the HRPKS LDKS. (A) In vitro investigations of LDKS and LovD. (B) In vitro investigations of the ACP domain of LDKS with LovD. (C) In vitro investigation of LDKS and LovD missing key co-factors.

Further investigation of the diketide transfer from LDKS by LovD through measuring reaction velocities and reaction kinetics demonstrated that LovD and LDKS interact by protein–protein interactions.⁵ The physical interaction was further confirmed by preparing the standalone holo-ACP domain from LDKS and measuring the catalytic efficiency of diketide transfer to LovD (Scheme 14B). The catalytic efficiency of transfer was comparable to that measured for LDKS and LovD, although it cannot be ruled out that LovD interacts with other regions of LDKS. However, when alternative ACP domains from bacterial PKS and FAS systems were substituted for the LDKS ACP no products were detected indicating that LovD is highly selective towards the ACP domain of LDKS.⁵

During investigations of the LDKS and LovD interactions the co-factors SAM and NADPH were not included in the reaction mixture essentially inactivating the MT and KR domains respectively. As expected, 82 was not observed, however analogous products 83–85 were (Scheme 14C).⁵83 contains an unreduced, non-methylated diketide side chain due to the absence of both SAM and NADPH; 84 contains a fully reduced non-methylated diketide side chain due to the absence of SAM; and 85 contains a non-reduced methylated diketide side chain due to the absence of NADPH. Considering that these compounds were not observed under standard cultivation conditions it is speculated that the processing of the diketide occurs more rapidly within LDKS than by the trans-acting LovD. Taken together, these experiments demonstrate that protein–protein interactions are equally as important a consideration as substrate recognition.

The crystal structure of LovD contains an α,β-hydrolase fold, and the Ser76–Lys79–Tyr188 catalytic triad.¹⁵⁸ The interface between the two domains forms a hollow cavity for the substrates with the serine active site at the base of the cleft (Fig. 19).¹⁵⁸ Mutational studies demonstrated that conformational changes could be introduced to LovD that increased catalytic efficiency 11-fold and improved solubility and stability.¹⁵⁸ Further in-depth studies using directed evolution generated a library of 61 779 LovD variants and led to identification of an optimized variant LovD9, 1000 times more active in vitro than the native enzyme.¹⁵⁹

Fig. 19 Structure of LovG (PDB ID = 3HLB).¹⁵⁹ The catalytic serine residue, S76, is labelled and the ACP binding surface is circled.

LovD9 contains 29 mutations at various positions around the enzyme and no longer requires an ACP partner enzyme.¹⁵⁹ Specifically, the width of the active site is reduced, stabilizing catalysis and making it less accessible to partner enzymes i.e., the ACP domain of LDKS. Molecular dynamic simulations indicated that in the native LovD, protein–protein interactions with the ACP domain of LNKS cause conformational changes of the catalytic residues, enhances catalysis, and prevents alternate ACPs from other systems being used as the acyl donor.¹⁵⁹ Furthermore, the ACP and phosphopantetheine binding surface was identified from the crystal structure (Fig. 19).

Subsequently polyketide release from a HRPKS by a collaborating acyltransferase enzyme has been confirmed in the biosynthesis of fumagillin 3,¹⁶⁰ chaetomugilin 20,⁷⁰ and calbistrin A 35 (ref. 113) (Fig. 20). CazE, the acyltransferase required in the biosynthesis of 20, was studied in vitro and shown to release a triketide from the HRPKS CazF.⁷⁰ CazE is proposed to induce a conformational change in CazF to facilitate polyketide release, by protein–protein interactions.⁷⁰

Fig. 20 Examples of fungal polyketides that utilize a trans-acting AT enzyme for polyketide release from a HRPKS. Chemical modifications introduced by the trans-acting AT enzymes are highlighted in brown.

The biosynthesis of azanigerone 80 is also speculated to utilize the acyltransferase AzaD.¹⁵⁴ However, an alternative scenario was proposed where the polyketide product of the HRPKS (AzaB) is released as a carboxylic acid and converted an acyl-CoA, similar to the biosynthesis of the tetraketide portion of 14,¹⁶¹ and various lipopeptides.^162,163 The precise substrate for AzaB-mediated acylation has not yet been determined.

5.3 Trans-acting reductases

Reductive release domains are well known in NRPKS, specifically in group VII,¹²¹ where polyketides are released as an aldehyde. In contrast, the HRPKS required for the biosynthesis of betaenone 85, Bet1, is one of the rare HRPKS that contains a terminal domain for product release; here a reductase (R) domain.^164,165 Collaborating reductases have been proposed in the biosynthesis of sordarial 86,¹⁶⁶ trichoxide 87,¹⁶⁷ and flavoglaucin 88 (ref. 168) biosynthesis (Fig. 21) because the HRPKS required for their biosynthesis lack any release domains, and the resulting PKS products are proposed to arise from aldehyde intermediates. Investigations into the biosynthesis of 87 demonstrated that when the HRPKS VirA was heterologously expressed in A. nidulans an aldehyde product was detected, however when VirA was co-expressed with cupin-domain enzyme VirC relatively higher titers of the aldehyde HRPKS product were detected.¹⁶⁷

Fig. 21 Examples of fungal polyketides that arise from an aldehyde intermediate synthesized via a HRPKS with an R domain or proposed to be the action of a trans-acting reductase.

The biosynthesis of the lanosterol 14a-demethylase (CYP51) inhibitor, restricticin 89, is also proposed to arise from the action of a trans-acting short-chain dehydrogenase/reductase (SDR), Rstn4.¹⁶⁹ Although considering that the biosynthesis of 89, was also investigated in A. nidulans, similar to the investigation of 87, reductases native to the heterologous host cannot be ruled out.

5.4 Trans-acting NRPS enzymes

The initial report on the biosynthesis of the thermolides e.g.90 was attributed to a biosynthetic gene cluster encoding a PKS–NRPS enzyme and several accessory enzymes.¹⁷⁰ However, a number of anomalies were noticed, leading to re-examination of the genome of Talaromyces thermophilus NRRL 2155. A second cluster encoding a PKS/NRPS was identified, however rather than discovering a HRPKS fused to a NRPS, two separate enzymes were predicted.¹⁷¹ Further examination of the NRPS indicated a domain organization that deviates from those typically found in fungal PKS–NRPS. Instead, a C–A–T–C_T domain organization was observed, with an unusual terminal condensation (C_T) domain,¹⁷¹ in contrast to the more common reductase (R) or Dieckmann cyclase (DKC) domains.

The newly discovered Thm cluster was confirmed through heterologous expression in A. nidulans, leading to detection of 90.¹⁷¹ Co-expression of just the HRPKS (ThmA) and the NRPS (ThmB) in A. nidulans produced 91 (Scheme 15). The same compound 91 could also be obtained from recombinant production of ThmA and ThmB in yeast, incubated with acetyl-CoA, malonyl-CoA, SAM, NADPH, MgCl₂, L-Ala/L-Val, indicating that these two enzymes collaborate to synthesize a PKS–NRPS derived product.¹⁷¹ Further investigation determined that the terminal C_T domain of ThmB catalyzes a macrocyclization reaction, rare in fungal PKS–NRPS pathways.

Scheme 15 Biosynthesis of the thermolides requires the collaborative action of a HRPKS and a collaborating NRPS.

Similar collaborating HRPKS and NRPS pairs have also been identified in the biosynthesis of acurin A 92 (Scheme 16).¹⁷²92 was isolated from A. aculeatus and, based on its structural similarities to the fungal mycotoxin fusarin C, was proposed to be synthesized via a PKS–NRPS enzyme. The biosynthetic gene was cluster confirmed as being responsible for the biosynthesis of 92via gene knock out experiments. The cluster encodes separate HRPKS (AcrA) and NRPS (AcrB) enzymes separated by 31 kb of DNA sequence.¹⁷² Individual deletion of AcrA and AcrB by CRISPR/Cas9 led to loss of 92, and no intermediates were reported. Unlike the NRPS ThmB, AcrB contains a common terminal R domain, and homologs of the genes encoding AcrA and AcrB are found in nine other Aspergillus spp. indicating that the separation of these genes is not a result of errors in genome sequencing or annotation.¹⁷² The BGC encoding the biosynthesis of 92 also contains an α,β-hydrolase, AcrC, and disruption of the encoding gene did not reveal accumulation of intermediates, although this was observed for several other tailoring enzymes, therefore potential physical interactions between two or more tailoring enzymes are suggested.¹⁷²

Scheme 16 The biosynthesis of acurin A requires a trans-acting NRPS enzyme, AcrB.

5.5 Trans-acting PLP-dependent enzymes

The infamous fumonisin mycotoxins derive from a polyketide fused to alanine, however not through amide bond formation nor by a PKS–NRPS enzyme.¹⁷³ The biosynthesis requires a HRPKS enzyme, Fum1p, that lacks an obvious domain for polyketide release, similar to many other HRPKS (Scheme 17).¹⁷⁴ Also contained within the fumonisin gene cluster is a gene encoding a protein that is a member of the 2-oxoamine synthase family, Fum8p. Fum8p is a pyridoxal 5′-phosphate (PLP)-dependent enzyme and, due to the diverse range of reactions catalyzed by PLP-dependent enzymes, was a candidate for being involved in decarboxylative condensation, and therefore polyketide release. Fum8p was heterologously expressed in yeast microsomes, incubated with recombinant Fum1p-ACP, PLP, C18-CoA, L-alanine, and shown to catalyze the PLP-dependent release of the C-18 chain from Fum1p-ACP to form 93.¹⁷⁵ The assay also showed some tolerance of Fum8p towards C-16 and C-14 polyketide chains, although Fum8p clearly preferred the C-18 substrate.

Scheme 17 The biosynthesis of fumonisins requires a trans-acting PLP-dependent enzyme for polyketide extension and release.

Similar to fumonisins, AAL-toxins e.g.94 (Fig. 22) also derive from a HRPKS condensed with an amino acid, however AAL-toxins comprise of a C-16 polyketide chain, synthesized by Alt1p, fused with glycine. The AAL-toxin gene cluster reportedly also contains an α-oxoamine synthase type enzyme, Alt4p, proposed to function in a similar mechanism to Fum1p.¹⁷⁶ The addition of an amino acid to a highly reduced polyketide chain shares similarities with the coupling of serine with palmitic acid in sphingosin biosynthesis.¹⁷⁷

Fig. 22 Examples of natural products that require a trans-acting PLP-dependent enzyme for polyketide release from a HRPKS.

The biosynthesis of the indolizidine alkaloid curvulamine 95 (Fig. 22) also requires a trans-acting PLP-dependent enzyme, CuaB, for condensation with alanine, resulting in decarboxylative condensation.¹⁷⁸ However CuaB is a bi-functional aminotransferase and also catalyzes α-hydroxlation of alanine. Using CuaB, fungal genome databases were mined identifying 10 BGCs suspected of encoding indolizidine alkaloids.¹⁷⁸ Over-expression of a transcription factor within one of these cryptic clusters in Bipolaris maydis led to the identification of nine new indolizidine alkaloids, named bipolamines A–I.¹⁷⁸

5.6 Trans-acting cyclases

The aurovertins e.g.96 (Fig. 23) are ATP inhibitors derived from a polyketide polyene α-pyrone. A BGC was discovered in Calcarisporium arbuscula that encodes the HRPKS (AurA) and a number of tailoring enzymes, including a protein, AurE, with sequence homology to bacterial aromatic polyketide cyclases.¹⁷⁹ The cluster was investigated through gene knockout and heterologous expression in yeast. Inactivating AurE reduced the titers of polyketides obtained, whilst including AurE in heterologous expression experiments increased the rate of product release via pyrone formation. Although an exact function of AurE was not yet determined it seems likely that it collaborates with AurA to facilitate polyketide release.¹⁷⁹

Fig. 23 Structure of the ATP inhibitor aurovertin E 96 proposed to require an unusual collaborating cyclase.

6. Trans-acting oxidases

In addition to trans-acting enzymes that appear to introduce functionality reminiscent of missing or inactive PKS domains, are trans-acting tailoring enzymes that introduce specific functionality that PKS enzymes are unable to. For example, in the biosynthesis of TAN-1612 97, a tetracycline-like compound synthesized by the NRPKS AdaA, two trans-acting enzymes are required; the trans-acting TE, AdaB, and the flavin-dependent monooxygenase (FMO), AdaC.¹⁸⁰ When AdaA and AdaB were co-expressed without AdaC, a tricyclic anthracenone 98 was observed (Scheme 18). 98 is an off-pathway shunt intermediate arising from hydrolysis that is not converted to the final metabolite 97. Similarly, when AdaA and AdaC were co-expressed only trace amounts of intermediate 98 was detected. The biosynthesis of 97 requires FMO AdaC to hydroxylate the C-2 position of the polyketide intermediate while it is still tethered to AdaA and this site-specific hydroxylation enables the trans-acting TE, AdaB, to function correctly to release 99.¹⁸⁰ Finally, the O-methyltransferase AdaD acts on the released polyketide product 99 to complete the biosynthesis of 97 (Scheme 18).

Scheme 18 The biosynthesis of TAN-1612 97 requires the collaborative action of the trans-acting FMO, AdaC, followed by release via the trans-acting TE, AdaB. Only chemical modifications introduced by the trans-acting enzymes are highlighted in colour.

The three gene cassette encoding an NRPKS, FMO and TE has also been identified in other fungi that have been confirmed as producing polycyclic polyketide products with a C-2 hydroxylation including asperthecin 60 and viridicatumtoxin 13 (Fig. 24).¹⁸⁰ Currently no structural information is available to understand the potential protein–protein interactions between these three enzymes.

Fig. 24 Examples of fungal polyketides that utilize a trans-acting FMO for correct polyketide release from the NRPKS.

The biosynthesis of oxaleimide 100 requires the action of the trans-acting cytochrome P450 (PoxM) to hydroxylate the polyketide intermediate tethered to the HRPKS (PoxF) prior to release of the carboxylic acid product 101.¹⁸¹ This was confirmed via heterologous expression in A. nidulans since expression of PoxF alone did not yield any detectable product. PoxM is an iterative P450 and further oxidizes 101 to 102 after release from PoxF (Scheme 19).¹⁸¹

Scheme 19 The biosynthesis of oxaleimide 100 requires the action of the trans-acting P450 PoxM. Chemical modifications introduced by the trans-acting P450 are highlighted in light green.

A trans-acting P450 has recently been proposed in the biosynthesis of the C4-alkylated dihydroisocoumarin 103.¹⁶⁷ The gene cluster responsible for the biosynthesis of 103 encodes: an NRPKS (AcreC), a P450 (AcreB), an O-methyltransferase (AcreA), and a hydrolase (AcreD). Heterologous expression of AcreA–D in A. nidulans resulted in formation of 103, as expected (Scheme 20).¹⁸² Investigating AcreA, B and D in vitro revealed that AcreB did not hydroxylate the NRPKS product 104. To exclude the possibly that AcreB was inactive/misfolded, 104 was fed to an A. nidulans strains expressing only AcreB, however no conversion to 105 was detected. As a control, AcreA expressed in A. nidulans could convert 104 to 106, confirming that 104 can penetrate the cell wall, but is not a substrate for AcreB. Therefore, the P450, AcreB, appears to act in-trans hydroxylating the polyketide chain still tethered to AcreC.¹⁸² Genome mining identified gene clusters encoding homologous NRPKS, P450, and hydrolase in other fungi, however the polyketide products are unknown.¹⁸²

Scheme 20 The biosynthesis of 103 requires the action of the trans-acting P450 AcreB. Chemical modifications introduced by the trans-acting P450 is highlighted in light green.

These examples of oxidases hydroxylating intermediates tethered to the PKS are similar to the proposal of the role of the P450 in the biosynthesis of brefeldin 67. Therefore, this type of collaboration between a PKS and oxidases may be more frequent than anticipated. Although the oxidase is not involved in chain extension or chain release, it introduces a functional group that is essential for biosynthesis to proceed as expected and demonstrates the ever-evolving nature of these pathways.

7. Biosynthetic pathways utilizing multiple trans-acting and collaborating enzymes

With an increasing number of fungal biosynthetic pathways being fully interrogated, using both in vivo and in vitro approaches, it is clear that trans-acting and collaborating enzymes are a recurrent feature and often more than one trans-acting enzyme can be found in a specific pathway. For example, the biosynthesis of TAN-1612 97 (Scheme 18) requires hydroxylation by the trans-acting FMO (AdaC) to facilitate release of the correct polyketide intermediate by the trans-acting TE (AdaB).¹⁸⁰ As might be expected, where biosynthetic pathways require more than one PKS megasynthase that collaborate with trans-acting partners the number of protein–protein interactions increase significantly.

The biosynthesis of lovastatin 1 requires two HRPKS, LNKS and LDKS, and three trans-acting enzymes, LovC, LovG and LovD (Scheme 21A).^5,77,133,183 Without the trans-ER, LovC, LNKS is unable to synthesize the required polyketide intermediates and without the trans-acting thiolesterase, LovG, LNKS is unable to release the polyketide product 25. Similarly, LDKS requires the trans-acting AT to transfer its diketide intermediate to 81 resulting in formation of lovastatin acid 82.

Scheme 21 Biosynthetic pathways using multiple trans-acting or collaborating enzymes (A) lovastatin acid 82, (B) chaetomugilin A 20 and (C) calbistrin A 35. Chemical modifications introduced by the trans-acting enzymes are highlighted in colour.

Likewise, the biosynthesis of chaetomugilin A 20 requires the collaborating HRPKS and NRPKS enzymes, CazF and CazM, in addition to a trans-acting AT (CazE).^70,184 However, the main reason for the complexity in this pathway is that CazF is capable of synthesizing two different triketides 108 and 109; 108 is transferred to CazM, and 109 is transferred by CazE to the modified product of CazF/CazM cazisochromene 110 (Scheme 21B).¹⁶⁹ This is a revealing example of collaborating enzymes competing for substrates and altering the product profile of a megasynthase.

The biosynthesis of calbistrin A 35 also demonstrates how competing trans-acting enzymes can alter the product profile of a megasynthase and generate highly complex polyketide products.¹¹³ Unlike chaetomugilin A 20, calbistrin A 35 requires only a single HRPKS CalA′. If CalA′ collaborates with the trans-acting ER CalK′ a decalin product 111 is formed. However, if CalA′ collaborates with the trans-acting MT CalH′ this abolishes the collaboration with CalK′ and a shorter polyketide with a different methylation 112 pattern is synthesized (Scheme 21C).¹¹³ Intriguingly the reductase (R) domain of CalA′ that is clearly active, no longer functions when CalH′ acts and instead the trans-acting AT CalD′ releases the polyene polyketide product, coupling it with the modified polyketide product 113 of CalA′/CalK′.¹¹³

In the biosynthesis of oxaleimide 100 a HRPKS (PoxF) collaborates with the trans-acting P450 (PoxM) to synthesize the precursor 102 of the amino acid substrate 114 utilized by the PKS–NRPS (PoxE) (Scheme 22A).¹⁸¹ As is common for PKS–NRPS PoxE collaborates with the trans-acting ER (PoxP) and it appears that the Diels-Alderase PoxQ, is able to act on the polyketide chain 115 still tethered to PoxE.¹⁸¹

Scheme 22 Biosynthetic pathways using multiple trans-acting or collaborating enzymes (A) oxaleimide A 100 and (B) tricholognan A 22. Chemical modifications introduced by the trans-acting enzymes are highlighted in colour.

Finally, the biosynthesis of tricholignan A 22 requires a HRPKS and NRPKS pair, TlnA and TlnB, that collaborate with trans-acting MT and TE domains, TlnC and TlnC respectively, in a highly unusual biosynthetic pathway (Scheme 22B).⁷² TlnC is a didomain protein consisting of an inactive ACP (ψACP) fused to a MT domain, reminiscent of the organization of a truncated NRPKS. Investigating the stand alone TlnC-MT domain in vitro indicated that the TlnC-ψACP is required to increase the efficiency of the methylation step.⁷² When TlnC-ψACP is activated by introducing the active site Ser residue a non-methylated pentaketide 116 is observed, instead of 117, indicating that an active TlnC-ACP inhibits TlnC-MT. Together these findings indicate protein–protein interactions between TlnC and TlnB to facilitate methylation during the third round of chain extension i.e. on the tetraketide intermediate.⁷² Domain deconstruction experiments using TlnB–SAT–KS–AT⁰, TlnB–ACP, TlnC, and a tetraketide mimic demonstrated that higher concentrations of TlnC inhibited chain elongation. The TlnC-ψACP domain contains a number of negatively charged residues throughout the first half of its sequence, similar to other ACPs.⁷²

TlnC is therefore proposed to form electrostatic protein–protein interactions with the KS domain of TlnB enabling TlnC to intercept the tetraketide and methylate before the tetraketide is shuttled to the KS domain for elongation.⁷²

These examples reveal the high complexity of these pathways and the precise co-ordination between multiple enzymes. They also demonstrate how thoroughly fungal biosynthetic pathways may need to be interrogated in order to fully understand how some of the first enzyme-free intermediates are produced. Although the pathway genes encoding the biosynthetic enzymes are clustered, the genes encoding trans-acting enzymes are not always adjacent to each other within the BGC. This review is intended to categorize the many examples of trans-acting enzymes and act as a guide when considering pathway engineering.

8. Biosynthetic pathways engineering opportunities

Due to the importance of natural products in the pharmaceutical industry, and the declining rate of discovery of novel antibiotics entering the market, methods to engineer analogues of bioactive molecules with improved properties may be desirable. Without a thorough understanding of the elaborate network of collaborating megasynthases and trans-acting accessory enzymes, this may be difficult to achieve. Furthermore, the process will be neither quick nor easy as firstly BGCs have to be fully dissected/reassembled, potential collaborations identified and interrogated, and eventually novel collaborations engineered.

Because there are so few structures of fungal PKS and collaborating enzymes reported, compared to the number of fungal polyketides discovered, the majority of engineering attempts have been curiosity driven in trying to understand the enzymes' tolerance rather than the nature of the collaboration specifically. For example, when studying how HRPKS collaborate with NRPKS during benzenediol lactone biosynthesis using phylogenetically diverse donors, it was determined that generally the NRPKS enzyme as a whole could tolerate unnatural starter units leading to product diversity (Scheme 23A).¹⁸⁵ However, in many cases the SAT domain of the NRPKS acceptor needed to be swapped to ensure efficient substrate transfer,¹⁸⁶ indicating that protein–protein interactions are just as important a consideration as the substrate tolerance of the enzyme being investigated.

Scheme 23 Summary of results of combinations of trans-acting enzymes with non-native PKS enzymes. (A) The investigation of HRPKS and NRPKS shuffling; (B) trans-ER swaps in closely related PKS–NRPS; (C) swaps between PKS-like enzymes TerB and RslB; (D) substituting trans-acting thiolhydrolase BrefTH with trans-acting acyltransferases; (E) shuffling trans-acting TE and FMOs with NRPKS that produce related polyketides but with different chain lengths.

Similarly, when investigating the programming of PKS–NRPS and their trans-ER partners, it was demonstrated that trans-ER enzymes could be swapped between closely related BGCs (∼90% sequence identity).¹⁸⁷ The swaps had no effect on the structure of the final molecules 118 and 119 as both trans-ER catalyzed the same chemistry on the same substrate (Scheme 23B). However, in the absence of the trans-ER a shunt product was observed confirming the essential role of this trans-acting enzyme in PKS programming.⁷⁶

Likewise, when investigating the trans-acting KR enzyme involved biosynthesis of 44, homologous enzymes from the terrein and roussoellatide pathways could be swapped without adverse effects on titers 44 (Scheme 23C).¹¹⁸ Although not necessarily surprising due to these enzymes catalyzing identical chemistry, the enzymes are quite different in that RslB is 300aa shorter that TerB and does not contain the MT⁰ domain.¹¹⁸

To understand Bref-TH in the biosynthesis of 75 and determine its precise role in chain length control, the acyltransferases CazE and Fma-AT, from the chaetomugilin A 20 and fumagillin 3 pathways respectively, were substituted for Bref-TH (Scheme 23D).¹³⁹ Both of these acyltransferases are trans-acting and confirmed as being involved in polyketide chain release from their partner HRPKS.^70,160 However, the major products remained nonaketides, similar to Bref-PKS alone. Considering that only Bref-TH is able to compete with the KS domain demonstrates highly specific protein–protein interactions with Bref-PKS.

The roles of the trans-acting oxidase (AdaC) and thioesterase (AdaB) in the biosynthesis of 97 were studied by combinatorial biosynthesis with the homologous enzymes required for the biosynthesis of asperthecin 60, AptC and AptB, respectively (Scheme 23E).¹⁸⁰ The precursors to 97 and 60, 99 and 120 respectively, differ in that 99 is a decaketide and 120 is a nonaketide, however both molecules are hydroxylated at position C-2. Combination of AptA with AdaB and AdaC led to formation of the expected product 120 in similar titers to the natural combination of AptABC. In contrast combination of AdaA with AptB and AptC also formed the expected product 120 but with significantly lower titers, indicating a less efficient collaboration.¹⁸⁰

Finally, there are several reports of deconstructed NRPKS domains that are able to interact with type II PKS enzymes from bacteria (Scheme 24).¹⁸⁸ For example, PKS4 from Gibberella fujikuroi synthesizes the aromatic polyketide SMA76a 121 in E. coli (Scheme 24A) yet when the TE domain is removed the product profile changes to SMA93 122 (Scheme 24B).¹⁸⁹ Constructing an artificial PKS (PKS_WJ) consisting of unnatural fused KS–AT–ACP domains enabled the synthesis of napthopyrone 123 (Scheme 24C) which was the identical product of the three domains individually expressed in E. coli.¹⁸⁸

Scheme 24 Summary of experiments investigating the potential collaborations between fungal PKS and PKS modification enzymes from bacteria.

The addition of bacterial cyclases (CYC) e.g. ZhuI or MtmQ, caused a significant change in product profile to 124 and 125 (Scheme 24D) indicating that these cyclases can collaborate with the fungal enzymes and introduce programmed cyclization and re-direct the biosynthesis to compounds typically found in bacteria.¹⁸⁹ The study was expanded to include cyclase enzymes from different bacteria that introduce different modes of cyclization, and a trans-acting KR. Again, these bacterial enzymes collaborated with PKS-WJ, this time synthesizing SEK26 126 (Scheme 24E).¹⁸⁹ Furthermore, by studying the complete PKS4 with various combinations of bacterial accessory enzymes, it was demonstrated that the bacterial enzymes are capable of competing with the functional PT domain of PKS4 and diverting the product profile to generate mutactin 127, SEK34 128, DMAC 129 and 126, polyketides typically found in bacteria (Scheme 24F).¹⁸⁹

Although these PKS enzyme are very distantly related, the range of substrate flexibility tolerated by the PKS enables successful engineering. However, in all examples given, the driving factor appears to be appropriate protein–protein interactions that orientate both enzymes close enough that they have access to the polyketide intermediates. A similar observation has been made when engineering chimeric modular PKS enzymes; protein–protein interactions between domains is a more significant factor to consider than enzyme–substrate recognition.¹⁹⁰

9. Conclusions and future prospects

For the future investigation and engineering of fungal PKS enzymes, including combinatorial biosynthesis, researchers must consider that these multifaceted systems may require considerable forethought into the variables. This review collates the various classes of collaborating and trans-acting enzymes that have been identified in fungal polyketide biosynthesis demonstrating some common themes and also some rare examples. As more pathways are interrogated we will gain an understanding of which collaborations are the “rule” and which are the exceptions.

Of the collaborations discovered, still very little is known about how these enzymes interact. For example, it is unknown whether all interactions are a result of protein–protein interactions or mere physical proximity of enzymes. As more information is gained from pathway elucidation combined with structural biology approaches it may be possible to rationally engineer these complex systems to generate novel bioactive molecules with improved properties.

10. Conflicts of interest

There are no conflicts to declare.

11. Acknowledgements

Protein structures were visualized and manipulated using the Mol* Viewer.¹⁹¹

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