Longkuan Xiang

Enzymatic total synthesis of enterocin polyketides

Polyketides are clinically important natural products that often require elaborate organic syntheses owing to their complex chemical structures. Here we report the multienzyme total synthesis of the Streptomyces maritimus enterocin and wailupemycin bacteriostatic agents in a single reaction vessel from simple benzoate and malonate substrates. To our knowledge, our results represent the first in vitro assembly of a complete type II polyketide synthase enzymatic pathway to natural products.

Inactivation, complementation, and heterologous expression of encP, a novel bacterial phenylalanine ammonia-lyase gene.

The enzyme phenylalanine ammonia-lyase, which catalyzes the nonoxidative deamination ofl-phenylalanine to trans-cinnamic acid, is ubiquitously distributed in plants. We now report its characterization for the first time in a bacterium. The phenylalanine ammonia-lyase homologous gene encP from the “Streptomyces maritimus” enterocin biosynthetic gene cluster was functionally characterized and shown to encode the first enzyme in the pathway to the enterocin polyketide synthase starter unit benzoyl-coenzyme A. The disruption of the encP gene completely inhibited the production of cinnamate and enterocin, whereas complementation of the mutant with benzoyl-coenzyme A pathway intermediates or with the wild-type gene encP restored the formation of the benzoate-primed polyketide antibiotic enterocin. Heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor furthermore led to the production of cinnamic acid in the fermented cultures, confirming that the encP gene indeed encodes a novel bacterial phenylalanine ammonia-lyase.

Exploiting marine actinomycete biosynthetic pathways for drug discovery.

Drug discovery relies on the generation of large numbers of structurally diverse compounds from which a potential candidate can be identified. To this end, actinomycetes have often been exploited because of their ability to biosynthesize an impressive array of novel metabolites particularly polyketides. The genetic organization of polyketide synthases (PKSs) makes them readily amenable to manipulation, and thus re-engineering artificial or hybrid PKSs to produce unnatural natural products is a reality. This review highlights two approaches we have used to generate novel polyketides by manipulating genes responsible for starter unit biosynthesis in the ‘Streptomyces maritimus’ enterocin type II PKS. Our preliminary investigation into the biosynthesis of neomarinone, a rare marine actinomycete-derived meroterpenoid, is also presented.

A mechanism of benzoic acid biosynthesis in plants and bacteria that mirrors fatty acid beta-oxidation

The aromatic metabolite benzoic acid (1) is a biosynthetic building block of numerous benzoyl and benzyl groups that serve as important structural elements in a large number of natural products. In eukaryotes, for example, benzoate is a component of the zaragozic acids (2), paclitaxel (taxol ; 3), salicylic acid (4), and cocaine (5) (Figure 1). Although rarer in prokaryotic organisms, benzoyl-coenzyme A (benzoyl-CoA) is a starter unit for a few bacterial polyketides, such as enterocin (6) and the wailupemycins. Despite its simple structure and widespread occurrence, the biosynthesis of 1 and its thioester 1-CoA are only partially understood. In the field of plant secondary metabolism, two major routes from L-phenylalanine (7) to benzoic acid have been reported: the b-oxidation-type pathway (Scheme 1, route a) ; and the so-called nonoxidative pathway, via benzaldehyde (11) (Scheme 1, route b). 6, 7] Both routes possess common intermediates like cinnamoyl-CoA (8) and 3-hydroxy-3-phenylpropionyl-CoA (9-CoA) before branching to involve either oxidation and thiolation (route a) or retro-aldol cleavage followed by oxidation (route b). Until recently, the only known pathway from 7 to 1 in bacteria proceeded through two oxidative decarboxylation reactions involving the intermediates phenylpyruvate and phenylglyoxylate. We recently reported that the biosynthesis of 1 in the terrestrial plants Nicotiana attenuata and Cucumis sativus, and the marine actinomycete aStreptomyces maritimuso proceeds by a similar pathway involving the intermediate 3-hydroxy-3-phenylpropionate. Figure 1. Structures of some important natural products containing benzoatederived residues.

Exploiting Enzymatic Promiscuity to Engineer a Focused Library of Highly Selective Antifungal and Antiproliferative Aureothin Analogues

Aureothin is a shikimate-polyketide hybrid metabolite from Streptomyces thioluteus with a rare nitroaryl moiety, a chiral tetrahydrofuran ring, and an O-methylated pyrone ring. The antimicrobial and antitumor activities of aureothin have caught our interest in modulating its structure as well as its bioactivity profile. In an integrated approach using mutasynthesis, biotransformation, and combinatorial biosynthesis, a defined library of aureothin analogues was generated. The promiscuity of the polyketide synthase assembly line toward different starter units and the plasticity of the pyrone and tetrahydrofuran ring formation were exploited. A selection of 15 new aureothin analogues with modifications at the aryl residue, the pyrone ring, and the oxygenated backbone was produced on a preparative scale and fully characterized. Remarkably, various new aureothin derivatives are less cytotoxic than aureothin but have improved antiproliferative activities. Furthermore, we found that the THF ring is crucial for the remarkably selective activity of aureothin analogues against certain pathogenic fungi.

Engineered Biosynthesis of Phenyl-Substituted Polyketides

Benzoic acid, activated as its coenzyme A thioester (benzoylCoA) serves as a building block in the biosynthetic pathways to a number of important natural products, including zaragozic acid A (squalestatin S1) from fungi and paclitaxel (taxol) and cocaine from plants. It also provides the starter unit for the polyketides enterocin (1) and soraphen (2) from prokaryotic micro-organisms (Scheme 1). We have previously