Precursor-directed Biosynthesis Research Articles

Metabolic Engineering to Create New Class of Nature-Inspired Corrosion Inhibitors

For decades, natural products have indispensable role on drug discovery. But, if natural products applications are limited to therapeutic uses? To explore new horizons, we have applied our expertise on metabolic engineering to heterologously overproduce a siderophore titled Yersiniabactin (Ybt) in E.coli . E . coli host offers advantages of rapid growth rate, easy and cheap large-scale cultivation and well developed genetic manipulation and characterization techniques. Siderophores naturally show great affinity toward metal ions and surfaces. We have tested directing this innate ability toward corrosion inhibition. Preliminary data demonstrates adding reduced quantity of Ybt leads to 50% decrease in corrosion rate of stainless steel in acidic solution, mainly due to preventive layer formation. Moreover, precursor-directed biosynthesis has been applied to generate new Ybt analogues with the potential for improved or altered metal surface binding capabilities. Novel analogues with higher binding constant and those with higher spatial surface area may enhanced corrosion inhibitory. Small molecules were characterized using HPLC and LC/MS. Corrosion study was performed using weight loss experiment and direct measurement by potentiostat . Our findings confirms nature can be a sustainable source for discovering new anti-corrosion small molecules which in contrast to common corrosion inhibitors are not environmental hazardous or human health risks. Figure 1

Precursor directed biosynthesis of odd-numbered fatty acids by different yeasts

Precursor-directed biosynthesis was used for directed preparation of positional isomers of heptadecanoic acid (17:1), which have convenient pharmacological properties. Cultivation of Candida sp., Kluyveromyces polysporus, Rhodotorula glutinis, Saccharomyces cerevisiae, Torulaspora delbrueckii, Trichosporon cutaneum, and Yarrowia lipolytica on 20 g/L glucose, 4 g/L acetic, or 4 g/L propionic acids yielded different proportions of 17:1. Cultivation on carbon sources with even numbers of carbon atoms (glucose and acetic acid) produced preferentially 8Z- and 10Z-heptadecenoic acids in about equal amounts, in agreement with the proposed biosynthesis of fatty acids, whereas cultivation on propionic acid as the only carbon source produced over 90 % of total fatty acids of 9-17:1 out of all possible positional isomers. The structures of positional isomers of 17:1 acid were determined using dimethyl disulfides of fatty acid methyl esters. In cultivation of Candida sp. on propionic acid, the yield of heptadecenoic acid reached 111 mg/L cultivation medium. Principal component analysis was used for identifying the effect of cultivation conditions on the production of the 17:1 acid by individual yeast strains.

Recent advances in combinatorial biosynthesis for drug discovery.

Because of extraordinary structural diversity and broad biological activities, natural products have played a significant role in drug discovery. These therapeutically important secondary metabolites are assembled and modified by dedicated biosynthetic pathways in their host living organisms. Traditionally, chemists have attempted to synthesize natural product analogs that are important sources of new drugs. However, the extraordinary structural complexity of natural products sometimes makes it challenging for traditional chemical synthesis, which usually involves multiple steps, harsh conditions, toxic organic solvents, and byproduct wastes. In contrast, combinatorial biosynthesis exploits substrate promiscuity and employs engineered enzymes and pathways to produce novel “unnatural” natural products, substantially expanding the structural diversity of natural products with potential pharmaceutical value. Thus, combinatorial biosynthesis provides an environmentally friendly way to produce natural product analogs. Efficient expression of the combinatorial biosynthetic pathway in genetically tractable heterologous hosts can increase the titer of the compound, eventually resulting in less expensive drugs. In this review, we will discuss three major strategies for combinatorial biosynthesis: 1) precursor-directed biosynthesis; 2) enzyme-level modification, which includes swapping of the entire domains, modules and subunits, site-specific mutagenesis, and directed evolution; 3) pathway-level recombination. Recent examples of combinatorial biosynthesis employing these strategies will also be highlighted in this review.

Enzymatic formation of a resorcylic acid by creating a structure-guided single-point mutation in stilbene synthase.

A novel C17 resorcylic acid was synthesized by a structure-guided Vitis vinifera stilbene synthase (STS) mutant, in which threonine 197 was replaced with glycine (T197G). Altering the architecture of the coumaroyl binding and cyclization pocket of the enzyme led to the attachment of an extra acetyl unit, derived from malonyl-CoA, to p-coumaroyl-CoA. The resulting novel pentaketide can be produced strictly by STS-like enzymes and not by Chalcone synthase-like type III polyketide synthases; due to the unique thioesterase like activity of STS-like enzymes. We utilized a liquid chromatography mass spectrometry-based data analysis approach to directly compare the reaction products of the mutant and wild type STS. The findings suggest an easy to employ platform for precursor-directed biosynthesis and identification of unnatural polyketides by structure-guided mutation of STS-like enzymes.

Preparation of thermocleavable conjugates based on ansamitocin and superparamagnetic nanostructured particles by a chemobiosynthetic approach.

A combination of mutasynthesis, precursor-directed biosynthesis and semisynthesis provides access to new ansamitocin derivatives including new nanostructured particle-drug conjugates. These conjugates are based on the toxin ansamitocin and superparamagnetic iron oxide-silica core shell particles. New ansamitocin derivatives that are functionalized either with alkynyl- or azido groups in the ester side chain at C-3 are attached to nanostructured iron oxide core-silica shell particles. Upon exposure to an oscillating electromagnetic field these conjugates heat up and the ansamitocin derivatives are released by a retro-Diels-Alder reaction. For example, one ansamitocin derivative exerts strong antiproliferative activity against various cancer cell lines in the lower nanomolar range while the corresponding nanostructured particle-drug conjugate is not toxic. Therefore, these new conjugates can serve as dormant toxins that can be employed simultaneously in hyperthermia and chemotherapy when external inductive heating is applied.

Precursor-directed biosynthesis of 5-hydroxytryptophan using metabolically engineered E. coli.

A novel biosynthetic pathway was designed and verified reversely leading to the production of 5-hydroxytryptophan (5-HTP) from glucose. This pathway takes advantage of the relaxed substrate selectivities of relevant enzymes without employing the unstable tryptophan 5-hydroxylase. First, high-titer of 5-HTP was produced from 5-hydroxyanthranilate (5-HI) by the catalysis of E. coli TrpDCBA. Then, a novel salicylate 5-hydroxylase was used to convert the non-natural substrate anthranilate to 5-HI. After that, the production of 5-HI from glucose was achieved and optimized with modular optimization. In the end, we combined the full pathway and adopted a two-stage strategy to realize the de novo production of 5-HTP. This work demonstrated the application of enzyme promiscuity in non-natural pathway design.

Novel fluorinated lipopeptides from Bacillus sp. CS93 via precursor-directed biosynthesis.

While attempting to improve production of fluoro-iturin A in Bacillus sp. CS93 new mono- and di-fluorinated fengycins were detected in culture supernatants by (19)F NMR and tandem mass spectrometry, after incubation of the bacterium with 3-fluoro-L-tyrosine. The fluorinated amino acid was presumably incorporated in place of one or both of the tyrosyl residues in fengycin. Investigations to generate additional new fluorinated derivatives were undertaken using commercially available fluorinated phenylalanines and 2-fluoro- and 2,3-difluoro-tyrosine that were synthesised by Negishi cross-coupling of iodoalanine and fluorinated bromo-phenols. The anti-fungal activity of the fluorinated lipopeptides was assayed against Trichophyton rubrum and found to be similar to that of the non-fluorinated metabolites.

Direct MALDI-TOF/TOF analyses of unnatural beauvericins produced by the endophytic fungus Fusarium oxysporum SS46

The best time of production of the cyclohexadepsipeptide beauvericin by the endophytic fungus Fusarium oxysporum SS46 in Czapek medium was evaluated. The highest level of beauvericin production was found on day 21 of fermentative culture, as assessed by quantitative analysis by high performance liquid chromatography coupled with a photodiode array detector. Precursor-directed biosynthesis experiments were carried out to produce new analogues of beauvericin by feeding F. oxysporum with ten analogues of L-phenylalanine. In order to evaluate which precursor analogues were incorporated by the microorganism, the obtained extracts were analyzed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF). The precursor-directed biosynthesis studies led to the biosynthesis of novel beauvericin derivatives by replacement of one, two, or all three L-phenylalanine residues in beauvericin with DL-3-fluorophenylalanine, L-3-fluoro-phenylalanine, L-4-fluorophenylalanine, or L-tyrosine. Beyond these precursor analogues, one unit of L-4-aminophenylalanine, L-4-chlorophenylalanine, DL-4-bromophenylalanine, or L-4-bromophenylalanine was also incorporated by the endophyte F. oxysporum SS46. Units of L-4-nitrophenylalanine and L-histidine were not incorporated by the microorganism to produce unnatural beauvericins.

Structural characterization of cyclosporin A, C and microbial bio-transformed cyclosporin A analog AM6 using HPLC-ESI-ion trap-mass spectrometry.

Cyclosporin A (CyA), a cyclic undecapeptide produced by a number of fungi, contains 11 unusual amino acids, and has been one of the most commonly prescribed immunosuppressive drugs. To date, there are over sixty different analogs reported as congeners and analogs resulting from precursor-directed biosynthesis, human CYP-mediated metabolites, or microbial bio-transformed analogs. However, there is still a need for more structurally diverse CyA analogs in order to discover new biological potentials and/or improve the physicochemical properties of the existing cyclosporins. As a result of the complexity of the resulting mass spectrometric (MS) data caused by its unusual amino acid composition and its cyclic nature, structural characterization of these cyclic peptides based on fragmentation patterns using multiple tandem MS analyses is challenging task. Here, we describe, an efficient HPLC-ESI-ion trap MS(n) (up to MS(8)) was developed for the identification of CyA and CyC, a (Thr(2))CyA congener in which L-aminobutyric acid (Abu) is replaced by L-threonine (Thr). In addition, we examined the fragmentation patterns of a CyA analog obtained from the cultivation of a recombinant Streptomyces venezuelae strain fed with CyA, assigning this analog as (γ-hydroxy-MeLeu(6))CyA (otherwise, known as an human CYP metabolite AM6). This is the first report on both the MS(n)-aided identification of CyC and the structural characterization of a CyA analog by employing HPLC-ESI-ion trap MS(n) analysis.

Unsaturated Macrocyclic Dihydroxamic Acid Siderophores Produced byShewanella putrefaciensUsing Precursor-Directed Biosynthesis

To acquire iron essential for growth, the bacterium Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (pbH2; [M + H(+)](+), m/zcalc 373.2) as its native siderophore. The assembly of pbH2 requires endogenous 1,4-diaminobutane (DB), which is produced from the ornithine decarboxylase (ODC)-catalyzed decarboxylation of l-ornithine. In this work, levels of endogenous DB were attenuated in S. putrefaciens cultures by augmenting the medium with the ODC inhibitor 1,4-diamino-2-butanone (DBO). The presence in the medium of DBO together with alternative exogenous non-native diamine substrates, (15)N2-1,4-diaminobutane ((15)N2-DB) or 1,4-diamino-2(E)-butene (E-DBE), resulted in the respective biosynthesis of (15)N-labeled pbH2 ((15)N4-pbH2; [M + H(+)](+), m/zcalc 377.2, m/zobs 377.2) or the unsaturated pbH2 variant, named here: E,E-putrebactene (E,E-pbeH2; [M + H(+)](+), m/zcalc 369.2, m/zobs 369.2). In the latter system, remaining endogenous DB resulted in the parallel biosynthesis of the monounsaturated DB-E-DBE hybrid, E-putrebactene (E-pbxH2; [M + H(+)](+), m/zcalc 371.2, m/zobs 371.2). These are the first identified unsaturated macrocyclic dihydroxamic acid siderophores. LC-MS measurements showed 1:1 complexes formed between Fe(III) and pbH2 ([Fe(pb)](+); [M](+), m/zcalc 426.1, m/zobs 426.2), (15)N4-pbH2 ([Fe((15)N4-pb)](+); [M](+), m/zcalc 430.1, m/zobs 430.1), E,E-pbeH2 ([Fe(E,E-pbe)](+); [M](+), m/zcalc 422.1, m/zobs 422.0), or E-pbxH2 ([Fe(E-pbx)](+); [M](+), m/zcalc 424.1, m/zobs 424.2). The order of the gain in siderophore-mediated Fe(III) solubility, as defined by the difference in retention time between the free ligand and the Fe(III)-loaded complex, was pbH2 (ΔtR = 8.77 min) > E-pbxH2 (ΔtR = 6.95 min) > E,E-pbeH2 (ΔtR = 6.16 min), which suggests one possible reason why nature has selected for saturated rather than unsaturated siderophores as Fe(III) solubilization agents. The potential to conduct multiple types of ex situ chemical conversions across the double bond(s) of the unsaturated macrocycles provides a new route to increased molecular diversity in this class of siderophore.

Precursor-directed biosynthesis of micacocidin derivatives with activity against Mycoplasma pneumoniae

Micacocidin is a promising natural product for the treatment of Mycoplasma pneumoniae infections. In the biosynthesis of this antibiotic, a fatty acid-AMP ligase (FAAL) activates the starter unit hexanoic acid as acyl-adenylate and forwards it to an iteratively acting polyketide synthase. Biochemical analysis of the FAAL revealed an extended substrate tolerance, thereby opening the door for the modification of a micacocidin residue that is barely accessible via semisynthesis. A total of six new analogues were generated by precursor-directed biosynthesis in this study and profiled against M. pneumoniae.

Production of the novel lipopeptide antibiotic trifluorosurfactin via precursor-directed biosynthesis

Incorporation of fluorine into antibiotics can moderate their biological activity, lipophilicity and metabolic stability. The introduction of fluorine into an antimicrobial lipopeptide produced by Bacillus sp. CS93 via precursor-directed biosynthesis is described. The lipopeptide surfactin is synthesised non-ribosomally by various Bacillus species and is known for its biological activity. Administering 4,4,4-trifluoro-dl-valine to cultures of Bacillus sp. CS93 results in the formation of trifluorosurfactin in quantities sufficient for detection by LC–MS/MS. 19F NMR analysis of the culture supernatant revealed that the bulk of the fluorinated amino acid was transformed and thus was unavailable for incorporation into surfactin. Detection of ammonia, and MS analysis indicated that the transformation proceeds with deamination and reduction of the keto acid, yielding 4,4,4-trifluoro-2-hydroxy-3-methylbutanoic acid.

Precursor Directed Biosynthesis of an Orthogonally Functional Erythromycin Analogue: Selectivity in the Ribosome Macrolide Binding Pocket

The macrolide antibiotic erythromycin A and its semisynthetic analogues have been among the most useful antibacterial agents for the treatment of infectious diseases. Using a recently developed chemical genetic strategy for precursor-directed biosynthesis and colony bioassay of 6-deoxyerythromycin D analogues, we identified a new class of alkynyl- and alkenyl-substituted macrolides with activities comparable to that of the natural product. Further analysis revealed a marked and unexpected dependence of antibiotic activity on the size and degree of unsaturation of the precursor. Based on these leads, we also report the precursor-directed biosynthesis of 15-propargyl erythromycin A, a novel antibiotic that not only is as potent as erythromycin A with respect to its ability to inhibit bacterial growth and cell-free ribosomal protein biosynthesis but also harbors an orthogonal functional group that is capable of facile chemical modification.

Small molecule perimeter defense in entomopathogenic bacteria

Two gram-negative insect pathogens, Xenorhabdus nematophila and Photorhabdus luminescens, produce rhabduscin, an amidoglycosyl- and vinyl-isonitrile-functionalized tyrosine derivative. Heterologous expression of the rhabduscin pathway in Escherichia coli, precursor-directed biosynthesis of rhabduscin analogs, biochemical assays, and visualization using both stimulated Raman scattering and confocal fluorescence microscopy established rhabduscin's role as a potent nanomolar-level inhibitor of phenoloxidase, a key component of the insect's innate immune system, as well as rhabduscin's localization at the bacterial cell surface. Stimulated Raman scattering microscopy visualized rhabduscin at the periphery of wild-type X. nematophila cells and E. coli cells heterologously expressing the rhabduscin pathway. Precursor-directed biosynthesis created rhabduscin mimics in X. nematophila pathway mutants that could be accessed at the bacterial cell surface by an extracellular bioorthogonal probe, as judged by confocal fluorescence microscopy. Biochemical assays using both wild-type and mutant X. nematophila cells showed that rhabduscin was necessary and sufficient for potent inhibition (low nM) of phenoloxidases, the enzymes responsible for producing melanin (the hard black polymer insects generate to seal off microbial pathogens). These observations suggest a model in which rhabduscin's physical association at the bacterial cell surface provides a highly effective inhibitor concentration directly at the site of phenoloxidase contact. This class of molecules is not limited to insect pathogens, as the human pathogen Vibrio cholerae also encodes rhabduscin's aglycone, and bacterial cell-coated immunosuppressants could be a general strategy to combat host defenses.

Establishing a Toolkit for Precursor-Directed Polyketide Biosynthesis: Exploring Substrate Promiscuities of Acid-CoA Ligases

Polyketides are chemically diverse and medicinally important biochemicals that are biosynthesized from acyl-CoA precursors by polyketide synthases. One of the limitations to combinatorial biosynthesis of polyketides has been the lack of a toolkit that describes the means of delivering novel acyl-CoA precursors necessary for polyketide biosynthesis. Using five acid-CoA ligases obtained from various plants and microorganisms, we biosynthesized an initial library of 79 acyl-CoA thioesters by screening each of the acid-CoA ligases against a library of 123 carboxylic acids. The library of acyl-CoA thioesters includes derivatives of cinnamyl-CoA, 3-phenylpropanoyl-CoA, benzoyl-CoA, phenylacetyl-CoA, malonyl-CoA, saturated and unsaturated aliphatic CoA thioesters, and bicyclic aromatic CoA thioesters. In our search for the biosynthetic routes of novel acyl-CoA precursors, we discovered two previously unreported malonyl-CoA derivatives (3-thiophenemalonyl-CoA and phenylmalonyl-CoA) that cannot be produced by canonical malonyl-CoA synthetases. This report highlights the utility and importance of determining substrate promiscuities beyond conventional substrate pools and describes novel enzymatic routes for the establishment of precursor-directed combinatorial polyketide biosynthesis.

Assembly and Absolute Configuration of Short‐Lived Polyketides from Burkholderia thailandensis

Complex polyketides from bacteria comprise a broad range of macrolides, polyenes, and polyethers, many of which play eminent roles for the development of therapeutics. Typically, these compounds feature multiple chiral centers that render them challenging benchmarks for organic synthesis. In light of this, it is impressive to learn how swiftly complex polyketides are assembled in microorganisms, utilizing only simple building blocks such as activated acyl and malonyl units. The types, number, and processing of these basic blocks are programmed by the architecture of modular polyketide synthases (PKS) which are encoded in the microbial genomes. One most remarkable discovery was that the order of catalytic domains involved in chain elongation and side-chain processing is typically colinear with the final product. This principle has not only opened the door to rationally engineering polyketides, but also enabled forecasting PKS architectures from polyketide structures and vice versa. However, through recent full genome sequencing projects it has become more and more obvious that most of the genes coding for biosynthetic assembly lines remain silent under standard laboratory conditions. Furthermore, PKS expression and polyketide formation may only occur transiently, thus suggesting that the metabolites function as temporary signals. This remarkable case has been observed for Burkholderia thailandensis, a bacterium that serves as a model for the pathogenic relatives B. mallei and B. pseudomallei. Mining the genome of B. thailandensis revealed a giant trans-acyltransferase (trans-AT) PKS gene cluster, and a bioinformatic structure prediction aided in the discovery of a hitherto unknown polyketide, thailandamide A (1, Scheme 1A). However, the absolute configuration of the labile polyene 1 and the structure of a short-lived congener (thailandamide B; 2 ; see Figure 2 for structure), have remained elusive. Yet this very structural information would serve as a stepping-stone to clarify the biological function of this enigmatic, quorum-sensing-regulated metabolite. Herein we report a multidisciplinary approach involving bioinformatics, chemical degradation, and precursor-directed biosynthesis to rigorously determine the absolute configuration of thailandamide A. On the basis of the stereochemical assignments, in silico predictions, and mutational pathway dissection, we also unveil the structure of the immediate, thermolabile PKS product thailandamide B. Thailandamide features six chiral centers, at C2, C3, C13, C22, C24, and C29. We initiated the stereochemical analysis by chemical degradation and derivatization. For the isolation of preparative amounts of thailandamide A (1) we employed a genetically engineered strain, the quorum sensing mutant B. thailandensis DPthaA, which produces thailandamide constitutively. From an upscaled fermentation (10 L) of the mutant we succeeded in isolating sufficient amounts of Scheme 1. Structure of thailandamide A (1) and experiments revealing the absolute configuration of 1. A) Degradation and derivatization. a) O3, 78 8C; b) 35% H2O2, AcOH, 60 8C; c) 6m HCl, 105 8C; d) S-PGME, PyBOP; e) l-FDAA; f) (S,R)-PGME, PyBOP. B) Synthesis of deoxythailandamide. g) B. thailandensis DPthaA, precursor-directed biosynthesis; h) (S,R)-MTPA chloride, pyridine. B. Dd values in ppm.

Combinatorial biosynthesis of unnatural polyketides using a type III polyketide synthase from Oryza sativa

Polyketides are a class of secondary metabolites found in many bacteria and fungi that display broad range biological and pharmacological activities. Biological syntheses of these molecules by type III polyketides synthases are particularly interesting, due to their ability to catalyze the complete synthesis of the polyketide molecule within the active site of the enzyme. Studies have also shown that these enzymes are promiscuous, allowing them to accept various unnatural substrates as starter molecules to generate novel polyketides. In this study, a recombinant chalcone synthase from Oryza sativa (OsCHS) was purified and characterized. Like most other chalcone synthases, OsCHS is able to utilize ρ‐coumaroyl‐CoA as the starter molecule and catalyze the addition of three acetate units to make naringenin. We also further demonstrate that precursor‐directed combinatorial biosynthesis with unnatural substrates and rational engineering of mutant enzymes can be adopted as strategies to alter the product specificity of OsCHS. Coupling these approaches to highthroughput screening methods will allow us to generate and identify novel polyketides with interesting biological activities.

Developing polyketide‐based anti‐microbial therapeutics using synthetic enzymology

Enterococcus faecalis is an example of a bacterium that has gained multi‐drug resistance and is a major cause of nosocomial infections. In this project, we are targeting the enzymes present in the shikimate pathway within E. faecalis, as this pathway is absent in humans and crucial to the microbe, making the pathway an attractive target for drug design. A preliminary screen using a limited compounds library was performed on two enzymes present within the shikimate pathway and several potential inhibitors bearing the polyketide scaffold were identified. Polyketides are a large class of natural biomolecules biosynthesized from acyl‐CoA precursors using polyketide synthases (PKSs) that includes clinically important antibiotics such as tetracycline. Our project focuses on an uncharacterized Type III PKS, a putative chalcone synthase, from Oryza sativa (OsCHS). In order to produce a chemically diverse library of polyketides, a precursor‐directed biosynthesis approach was employed whereby different acyl‐CoA esters were tested as possible substrates for OsCHS and the enzyme was found to be promiscuous. The results suggested that the synthesis of novel compounds using synthetic enzymology was feasible and these polyketides could be used to screen for inhibitory activity against E. faecalis shikimate pathway enzymes to identify lead compounds for future antibiotic drug development.

Novel applications of plant polyketide synthases

The structurally and mechanistically simple type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkably diverse structures and biological activities. By exploiting the enzymes, we combined precursor-directed biosynthesis with nitrogen-containing substrates and structure-based enzyme engineering and generated unnatural, novel polyketide-alkaloid scaffolds with promising biological activities. The nucleophilic nitrogen atom and the engineered enzymes thus facilitated the formation of additional CC and CN bonds during the enzymatic transformations. The methodology will contribute to the further production of chemically and structurally divergent, unnatural natural products, as well as the rational design of novel biocatalysts with unprecedented catalytic functions.

Benzalacetone Synthase

Benzalacetone synthase, from the medicinal plant Rheum palmatum (RpBAS), is a plant-specific chalcone synthase (CHS) superfamily of type III polyketide synthase (PKS). RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6–C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site “gatekeeper” Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.