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.

Synthetic biology often employs enzymes in the biosynthesis of compounds for purposeful function. Here, we define synthetic enzymology as the application of enzymological principles in synthetic biology and describe its use as an enabling platform in synthetic biology for the purposeful production of compounds of biomedical and commercial importance. In particular, we demonstrated the use of synthetic polyketide enzymology as a means to develop lead polyketide based compounds for antimicrobial therapeutics, as exemplified by the modular coupling of acid:CoA ligases to type III polyketide synthases in the biosynthesis and development of polyketide-based biochemicals. Using wild-type and rationally designed mutants of a type III polyketide synthase isolated from Oryza sativa (OsPKS), we produced a chemically diverse library of novel polyketides and identified two bioactive antimicrobials, 4-hydroxy-6-[(1E)-2-(4-hydroxyphenyl)­ethenyl]-2H-pyran-2-one (bisnoryangonin) and 3,6,7-trihydroxy-2-(4-methoxybenzyl)-4H-1-benzopyran-4,5,8-trione (26OH), respectively, from a screen against a collection of Gram-positive and Gram-negative bacteria. The purification, crystallization, and structural resolution of recombinant OsPKS at 1.93 Å resolution are also reported. Using the described route of synthetic polyketide enzymology, a library of OsPKS mutants was generated as an additional means to increase the diversity of the polyketide product library. We expect the utility of synthetic enzymology to be extended to other classes of biomolecules and translated to various purposeful functions as the field of synthetic biology progresses.

Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the “drug of last resort”. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.

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.

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