Cloning and Biochemical Characterization of the Hectochlorin Biosynthetic Gene Cluster from the Marine Cyanobacterium Lyngbya majuscula

Abstract

Cyanobacteria, or blue-green algae, are a rich source of novel bioactive secondary metabolites that have potential applications as antimicrobial or anticancer agents or useful probes in cell biology studies. A Jamaican collection of the cyanobacterium Lyngbya majuscula has yielded several unique compounds including hectochlorin ( 1) and the jamaicamides A-C ( 5- 7). Hectochlorin has remarkable antifungal and cytotoxic properties. In this study, we have isolated the hectochlorin biosynthetic gene cluster ( hct) from L. majuscula to obtain details regarding its biosynthesis at the molecular genetic level. The genetic architecture and domain organization appear to be colinear with respect to its biosynthesis and consists of eight open reading frames (ORFs) spanning 38 kb. An unusual feature of the cluster is the presence of ketoreductase (KR) domains in two peptide synthetase modules, which are predicted to be involved in the formation of the two 2,3-dihydroxyisovaleric acid (DHIV) units. This biosynthetic motif has only recently been described in cereulide, valinomycin, and cryptophycin biosynthesis, and hence, this is only the second such report of an embedded ketoreductase in a cyanobacterial secondary metabolite gene cluster. Also present at the downstream end of the cluster are two cytochrome P450 monooxygenases, which are likely involved in the formation of the DHIV units. A putative halogenase, at the beginning of the gene cluster, is predicted to form 5,5-dichlorohexanoic acid.

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…A similar process is believed to be at play during the biosynthesis of hectochlorin (mixed NRPS/PKS) where the PCP-bound substrates are believed to be 2-oxo-isovaleric acid. 229 An example of a two-step transformation of a PCP-bound amino acid has been suggested to occur in the biosynthesis of the cyclic peptides JBIR-34 and JBIR-35. 230 In this pathway, the di-domain NRPS module FmoA1 presents a tryptophan residue (or chlorinated derivative) to the P450 FmoC, which performs two oxidation reactions; hydroxylation of the indole ring as well as b-hydroxylation.…”

More than just a Halogenase: Modification of Fatty Acyl Moieties by a Trifunctional Metal Enzyme

“…[8] Because unbranched, hydrophobic fatty acyl chains (presumably housed in "slippery" hydrophobic channels) are expected to show more flexibility than the small amino acid and 3-hydroxy-3-acyl-glutaryl building blocks of amino-acyl and keto-acyl halogenases, we were particularly interested in the chemo-and regioselectivities of fatty acyl halogenases. The first documented example of these for which the encoding gene cluster and natural product are known is HctB, [9] the target enzyme of this work.…”

Fungal cyclooligomerdepsipeptides: From classical biochemistry to combinatorial biosynthesis

“…98,108,109,133 Instead of utilizing preformed 2-hydroxycarboxylic acids as substrates, the respective bacterial NRPS A domains activate and load the corresponding 2-ketocarboxylic acids onto the multienzymes. Here, the ketocarboxylic acyl-thioesters undergo stereospecific reduction in cis by a ketoreductase (KR) domain, yielding the D-or L-hydroxycarboxylic acyl-thioesters ready for condensation.…”