Structure and Mechanisms of Assembly-Line Polyketide Synthases.

Three decades of studies on the multifunctional 6-deoxyerythronolide B synthase have laid a foundation for understanding the chemistry and evolution of polyketide antibiotic biosynthesis by a large family of versatile enzymatic assembly lines. Recent progress in applying chemical and structural biology tools to this prototypical assembly-line polyketide synthase (PKS) and related systems has highlighted several features of their catalytic cycles and associated protein dynamics. There is compelling evidence that multiple mechanisms have evolved in this enzyme family to channel growing polyketide chains along uniquely defined sequences of 10-100 active sites, each of which is used only once in the overall catalytic cycle of an assembly-line PKS. Looking forward, one anticipates major advances in our understanding of the mechanisms by which the free energy of a repetitive Claisen-like reaction is harnessed to guide the growing polyketide chain along the assembly line in a manner that is kinetically robust yet evolutionarily adaptable.

The Structural Enzymology of Iterative Aromatic Polyketide Synthases: A Critical Comparison with Fatty Acid Synthases.

Polyketides are a large family of structurally complex natural products including compounds with important bioactivities. Polyketides are biosynthesized by polyketide synthases (PKSs), multienzyme complexes derived evolutionarily from fatty acid synthases (FASs). The focus of this review is to critically compare the properties of FASs with iterative aromatic PKSs, including type II PKSs and fungal type I nonreducing PKSs whose chemical logic is distinct from that of modular PKSs. This review focuses on structural and enzymological studies that reveal both similarities and striking differences between FASs and aromatic PKSs. The potential application of FAS and aromatic PKS structures for bioengineering future drugs and biofuels is highlighted.

Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars.

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.

A treasure trove of 1034 actinomycete genomes.

Filamentous Actinobacteria, recently renamed Actinomycetia, are the most prolific source of microbial bioactive natural products. Studies on biosynthetic gene clusters benefit from or require chromosome-level assemblies. Here, we provide DNA sequences from >1000 isolates: 881 complete genomes and 153 near-complete genomes, representing 28 genera and 389 species, including 244 likely novel species. All genomes are from filamentous isolates of the class Actinomycetia from the NBC culture collection. The largest genus is Streptomyces with 886 genomes including 742 complete assemblies. We use this data to show that analysis of complete genomes can bring biological understanding not previously derived from more fragmented sequences or less systematic datasets. We document the central and structured location of core genes and distal location of specialized metabolite biosynthetic gene clusters and duplicate core genes on the linear Streptomyces chromosome, and analyze the content and length of the terminal inverted repeats which are characteristic for Streptomyces. We then analyze the diversity of trans-AT polyketide synthase biosynthetic gene clusters, which encodes the machinery of a biotechnologically highly interesting compound class. These insights have both ecological and biotechnological implications in understanding the importance of high quality genomic resources and the complex role synteny plays in Actinomycetia biology.

Animal FAS-like polyketide synthases produce diverse polypropionates.

Animal cytoplasmic fatty acid synthase (FAS) represents a unique family of enzymes that are classically thought to be most closely related to fungal polyketide synthase (PKS). Recently, a widespread family of animal lipid metabolic enzymes has been described that bridges the gap between these two ubiquitous and important enzyme classes: the animal FAS-like PKSs (AFPKs). Although very similar in sequence to FAS enzymes that produce saturated lipids widely found in animals, AFPKs instead produce structurally diverse compounds that resemble bioactive polyketides. Little is known about the factors that bridge lipid and polyketide synthesis in the animals. Here, we describe the function of EcPKS2 from Elysia chlorotica, which synthesizes a complex polypropionate natural product found in this mollusc. EcPKS2 starter unit promiscuity potentially explains the high diversity of polyketides found in and among molluscan species. Biochemical comparison of EcPKS2 with the previously described EcPKS1 reveals molecular principles governing substrate selectivity that should apply to related enzymes encoded within the genomes of photosynthetic gastropods. Hybridization experiments combining EcPKS1 and EcPKS2 demonstrate the interactions between the ketoreductase and ketosynthase domains in governing the product outcomes. Overall, these findings enable an understanding of the molecular principles of structural diversity underlying the many molluscan polyketides likely produced by the diverse AFPK enzyme family.

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products.

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

A natural single nucleotide mutation in the small regulatory RNA ArcZ of Dickeya solani switches off the antimicrobial activities against yeast and bacteria.

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.

Complexity of fungal polyketide biosynthesis and function.

Where does one draw the line between primary and secondary metabolism? The answer depends on the perspective. Microbial secondary metabolites (SMs) were at first believed not to be very important for the producers because they are dispensable for growth under laboratory conditions. However, such compounds become important in natural niches of the organisms, and some are of prime importance for humanity. Polyketides are an important group of SMs with aflatoxin as a well-known and well-characterized example. In Aspergillus spp., all 34 afl genes encoding the enzymes for aflatoxin biosynthesis are located in close vicinity on chromosome III in a so-called gene cluster. This led to the assumption that most genes required for polyketide biosynthesis are organized in gene clusters. Recent research, however, revealed an enormous complexity of the biosynthesis of different polyketides, ranging from individual polyketide synthases to a gene cluster producing several compounds, or to several clusters with additional genes scattered in the genome for the production of one compound. Research of the last decade furthermore revealed a huge potential for SM biosynthesis hidden in fungal genomes, and methods were developed to wake up such sleeping genes. The analysis of organismic interactions starts to reveal some of the ecological functions of polyketides for the producing fungi.

Synthetic anaplerotic modules for the direct synthesis of complex molecules from CO2.

Anaplerosis is an essential feature of metabolism that allows the continuous operation of natural metabolic networks, such as the citric acid cycle, by constantly replenishing drained intermediates. However, this concept has not been applied to synthetic in vitro metabolic networks, thus far. Here we used anaplerotic strategies to directly access the core sequence of the CETCH cycle, a new-to-nature in vitro CO2-fixation pathway that features several C3-C5 biosynthetic precursors. We drafted four different anaplerotic modules that use CO2 to replenish the CETCH cycle's intermediates and validated our designs by producing 6-deoxyerythronolide B (6-DEB), the C21-macrolide backbone of erythromycin. Our best design allowed the carbon-positive synthesis of 6-DEB via 54 enzymatic reactions in vitro at yields comparable to those with isolated 6-DEB polyketide synthase (DEBS). Our work showcases how new-to-nature anaplerotic modules can be designed and tailored to enhance and expand the synthetic capabilities of complex catalytic in vitro reaction networks.

Enzymology of assembly line synthesis by modular polyketide synthases.

Modular polyketide synthases (PKSs) run catalytic reactions over dozens of steps in a highly orchestrated manner. To accomplish this synthetic feat, they form megadalton multienzyme complexes that are among the most intricate proteins on earth. Polyketide products are of elaborate chemistry with molecular weights of usually several hundred daltons and include clinically important drugs such as erythromycin (antibiotic), rapamycin (immunosuppressant) and epothilone (anticancer drug). The term 'modular' refers to a hierarchical structuring of modules and domains within an overall assembly line arrangement, in which PKS organization is colinearly translated into the polyketide structure. New structural information obtained during the past few years provides substantial direct insight into the orchestration of catalytic events within a PKS module and leads to plausible models for synthetic progress along assembly lines. In light of these structural insights, the PKS engineering field is poised to enter a new era of engineering.

Polyketide synthase positive Escherichia coli one-time measurement in stool is not informative of colorectal cancer risk in a screening setting.

Environmental factors like the pathogenicity island polyketide synthase positive (pks+) Escherichia coli (E. coli) could have potential for risk stratification in colorectal cancer (CRC) screening. The association between pks+ E. coli measured in fecal immunochemical test (FIT) samples and the detection of advanced neoplasia (AN) at colonoscopy was investigated. Biobanked FIT samples were analyzed for both presence of E. coli and pks+ E. coli and correlated with colonoscopy findings; 5020 CRC screening participants were included. Controls were participants in which no relevant lesion was detected because of FIT-negative results (cut-off ≥15 µg Hb/g feces), a negative colonoscopy, or a colonoscopy during which only a nonadvanced polyp was detected. Cases were participants with AN [CRC, advanced adenoma (AA), or advanced serrated polyp (ASP)]. Existing DNA isolation and quantitative polymerase chain reaction (qPCR) procedures were used for the detection of E. coli and pks+ E. coli in stool. A total of 4542 (90.2%) individuals were E. coli positive, and 1322 (26.2%) were pks+ E. coli positive. The prevalence of E. coli in FIT samples from individuals with AN was 92.9% compared to 89.7% in FIT samples of controls (p = 0.010). The prevalence of pks+ E. coli in FIT samples from individuals with AN (28.6%) and controls (25.9%) was not significantly different (p = 0.13). The prevalences of pks+ E. coli in FIT samples from individuals with CRC, AA, or ASP were 29.6%, 28.3%, and 32.1%, respectively. In conclusion, the prevalence of pks+ E. coli in a screening population was 26.2% and did not differ significantly between individuals with AN and controls. These findings disqualify the straightforward option of using a snapshot measurement of pks+ E. coli in FIT samples as a stratification biomarker for CRC risk. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

ClusterCAD 2.0: an updated computational platform for chimeric type I polyketide synthase and nonribosomal peptide synthetase design.

Megasynthase enzymes such as type I modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) play a central role in microbial chemical warfare because they can evolve rapidly by shuffling parts (catalytic domains) to produce novel chemicals. If we can understand the design rules to reshuffle these parts, PKSs and NRPSs will provide a systematic and modular way to synthesize millions of molecules including pharmaceuticals, biomaterials, and biofuels. However, PKS and NRPS engineering remains difficult due to a limited understanding of the determinants of PKS and NRPS fold and function. We developed ClusterCAD to streamline and simplify the process of designing and testing engineered PKS variants. Here, we present the highly improved ClusterCAD 2.0 release, available at https://clustercad.jbei.org. ClusterCAD 2.0 boasts support for PKS-NRPS hybrid and NRPS clusters in addition to PKS clusters; a vastly enlarged database of curated PKS, PKS-NRPS hybrid, and NRPS clusters; a diverse set of chemical 'starters' and loading modules; the new Domain Architecture Cluster Search Tool; and an offline Jupyter Notebook workspace, among other improvements. Together these features massively expand the chemical space that can be accessed by enzymes engineered with ClusterCAD.

Yellow polyketide pigment suppresses premature hatching in social amoeba.

Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoeba Dictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.

In silico prediction of polyketide biosynthetic gene clusters in the genomes of Hypericum-borne endophytic fungi.

BACKGROUND: The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized. RESULTS: The aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein). CONCLUSIONS: The results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.

Discovery and Characterization of the Fully Decorated Nocardiosis-Associated Polyketide Natural Product.

The genomes of 40 strains of Nocardia, most of which were associated with life-threatening human infections, encode a highly conserved assembly line polyketide synthase designated as the NOCAP (NOCardiosis-Associated Polyketide) synthase, whose product structure has been previously described. Here we report the structure and inferred biosynthetic pathway of the fully decorated glycolipid natural product. Its structure reveals a fully substituted benzaldehyde headgroup harboring an unusual polyfunctional tail and an O-linked disaccharide comprising a 3-α-epimycarose and 2-O-methyl-α-rhamnose whose installation requires flavin monooxygenase-dependent hydroxylation of the polyketide product. Production of the fully decorated glycolipid was verified in cultures of two patient-derived Nocardia species. In both E. coli and Nocardia spp., the glycolipid was only detected in culture supernatants, consistent with data from genetic knockout experiments implicating roles for two dedicated proteins in installing the second sugar substituent only after the monoglycosyl intermediate is exported across the bacterial cell membrane. With the NOCAP product in hand, the stage is set for investigating the evolutionary benefit of this polyketide biosynthetic pathway for Nocardia strains capable of infecting human hosts.

Biosynthesis of Cytosporones in Leotiomycetous Filamentous Fungi.

Polyketides with the isochroman-3-one pharmacophore are rare among fungal natural products as their biosynthesis requires an unorthodox S-type aromatic ring cyclization. Genome mining uncovered a conserved gene cluster in select leotiomycetous fungi that encodes the biosynthesis of cytosporones, including isochroman-3-one congeners. Combinatorial biosynthesis in total biosynthetic and biocatalytic formats in Saccharomyces cerevisiae and in vitro reconstitution of key reactions with purified enzymes revealed how cytosporone structural and bioactivity diversity is generated. The S-type acyl dihydroxyphenylacetic acid (ADA) core of cytosporones is assembled by a collaborating polyketide synthase pair. Thioesterase domain-catalyzed transesterification releases ADA esters, some of which are known Nur77 modulators. Alternatively, hydrolytic release allows C6 hydroxylation by a flavin-dependent monooxygenase, yielding a trihydroxybenzene moiety. Reduction of the C9 carbonyl by a short chain dehydrogenase/reductase initiates isochroman-3-one formation, affording cytosporones with cytotoxic and antimicrobial activity. Enoyl di- or trihydroxyphenylacetic acids are generated as shunt products, while isocroman-3,4-diones are formed by autoxidation. The cytosporone pathway offers novel polyketide biosynthetic enzymes for combinatorial synthetic biology to advance the production of "unnatural" natural products for drug discovery.

Antibiotic Skeletal Diversification via Differential Enoylreductase Recruitment and Module Iteration in trans-Acyltransferase Polyketide Synthases.

Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the "blind watchmaker" of evolution creates skeletal diversity is a key question in natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here, we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from Burkholderia gladioli with promising activity against multidrug-resistant Mycobacterium tuberculosis, and etnangien, a structurally related antibiotic produced by Sorangium cellulosum. Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the trans-acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. In vitro reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all the structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a cis-acting enoylreductase (ER) domain in module 1 and in trans recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons.

Tools to make Stachybotrys chartarum genetically amendable: Key to unlocking cryptic biosynthetic gene clusters.

The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.

Biosynthesis of Octacosamicin A: Uncommon Starter/extender Units and Product Releasing via Intermolecular Amidation.

Octacosamicin A is an antifungal metabolite featuring a linear polyene-polyol chain flanked by N-hydroxyguanidine and glycine moieties. We report here that sub-inhibitory concentrations of streptomycin elicited the production of octacosamicin A in Amycolatopsis azurea DSM 43854T . We identified the biosynthetic gene cluster (oca BGC) that encodes a modular polyketide synthase (PKS) system for assembling the polyene-polyol chain of octacosamicin A. Our analysis suggested that the N-hydroxyguanidine unit originates from a 4-guanidinobutyryl-CoA starter unit, while the PKS incorporates an α-hydroxyketone moiety using a (2R)-hydroxymalonyl-CoA extender unit. The modular PKS system contains a non-canonical terminal module that lacks thioesterase (TE) and acyl carrier protein (ACP) domains, indicating the biosynthesis is likely to employ an unconventional and cryptic off-loading mechanism that attaches glycine to the polyene-polyol chain via an intermolecular amidation reaction.

Modifications of Protein-Bound Substrates by Trans-Acting Enzymes in Natural Products Biosynthesis.

Enzymatic modifications of small molecules are a common phenomenon in natural product biosynthesis, leading to the production of diverse bioactive compounds. In polyketide biosynthesis, modifications commonly take place after the completion of the polyketide backbone assembly by the polyketide synthases and the mature products are released from the acyl-carrier protein (ACP). However, exceptions to this rule appear to be widespread, as on-line hydroxylation, methyl transfer, and cyclization during polyketide assembly process are common, particularly in trans-AT PKS systems. Many of these modifications are catalyzed by specific domains within the modular PKS systems. However, several of the on-line modifications are catalyzed by stand-alone proteins. Those include the on-line Baeyer-Villiger oxidation, α-hydroxylation, halogenation, epoxidation, and methyl esterification during polyketide assembly, dehydrogenation of ACP-bound short fatty acids by acyl-CoA dehydrogenase-like enzymes, and glycosylation of ACP-bound intermediates by discrete glycosyltransferase enzymes. This review article highlights some of these trans-acting proteins that catalyze enzymatic modifications of ACP-bound small molecules in natural product biosynthesis.