Diversity of ABBA Prenyltransferases in Marine Streptomyces sp. CNQ-509: Promiscuous Enzymes for the Biosynthesis of Mixed Terpenoid Compounds.

Terpenoids are arguably the largest and most diverse family of natural products, featuring prominently in e.g. signalling, self-defence, UV-protection and electron transfer. Prenyltransferases are essential players in terpenoid and hybrid isoprenoid biosynthesis that install isoprene units on target molecules and thereby often modulate their bioactivity. In our search for new prenyltransferase biocatalysts we focused on the marine-derived Streptomyces sp. CNQ-509, a particularly rich source of meroterpenoid chemistry. Sequencing and analysis of the genome of Streptomyces sp. CNQ-509 revealed seven putative phenol/phenazine-specific ABBA prenyltransferases, and one putative indole-specific ABBA prenyltransferase. To elucidate the substrate specificity of the ABBA prenyltransferases and to learn about their role in secondary metabolism, CnqP1 -CnqP8 were produced in Escherichia coli and incubated with various aromatic and isoprenoid substrates. Five of the eight prenyltransferases displayed enzymatic activity. The efficient conversion of dihydroxynaphthalene derivatives by CnqP3 (encoded by AA958_24325) and the co-location of AA958_24325 with genes characteristic for the biosynthesis of THN (tetrahydroxynaphthalene)-derived natural products indicates that the enzyme is involved in the formation of debromomarinone or other naphthoquinone-derived meroterpenoids. Moreover, CnqP3 showed high flexibility towards a range of aromatic and isoprenoid substrates and thus represents an interesting new tool for biocatalytic applications

Complete Genome Sequence of Streptomyces sp. CNQ-509, a Prolific Producer of Meroterpenoid Chemistry

Rückert C, Leipoldt F, Zeyhle P, et al. Complete Genome Sequence of Streptomyces sp. CNQ-509, a Prolific Producer of Meroterpenoid Chemistry. Journal of Biotechnology. 2015;216:140-141.: Streptomyces sp. CNQ-509 is a marine actinomycete belonging to the MAR4 streptomycete lineage. MAR4 strains have been linked to the production of diverse and otherwise rare meroterpenoid compounds. The genome sequence of Streptomyces sp. CNQ-509 was found to contain 29 putative gene clusters for the biosynthesis of secondary metabolites, some of them potentially involved in the formation of meroterpenoid molecules

Phylogenetic tree of ABBA prenyltransferases of the phenol / phenazine family.

<p>Data include previously biochemically characterised ABBA prenyltransferases and those investigated in this study. The tree was constructed with MEGA6 using default parameter for multiple sequence alignment (CLUSTALW) and neighbour-joining method. Bootstrap values (in percent) calculated from 1000 replications are shown at the respective nodes. The fungal indole prenyltransferase DMATS (shares PT barrel) serves as a root.</p

Genome-Based Discovery of a Novel Membrane-Bound 1,6-Dihydroxyphenazine Prenyltransferase from a Marine Actinomycete

<div><p>Recently, novel prenylated derivatives of 1,6-dihydroxyphenazine have been isolated from the marine sponge-associated <i>Streptomyces</i> sp. SpC080624SC-11. Genome sequencing of this strain now revealed a gene cluster containing all genes necessary for the synthesis of the phenazine and the isoprenoid moieties. Unexpectedly, however, the cluster did not contain a gene with similarity to previously investigated phenazine prenyltransferases, but instead a gene with modest similarity to the membrane-bound prenyltransferases of ubiquinone and menaquinone biosynthesis. Expression of this gene in <i>E. coli</i> and isolation of the membrane fraction proved that the encoded enzyme, Mpz10, catalyzes two successive prenylations of 1,6-dihydroxyphenazine. Mpz10 is the first example of a membrane-bound enzyme catalyzing the prenylation of a phenazine substrate, and one of few examples of membrane-bound enzymes involved in the prenylation of aromatic secondary metabolites in microorganisms.</p></div

Mixed-terpenoid secondary metabolites of <i>Streptomyces</i> sp. CNQ-509.

<p>Mixed-terpenoid secondary metabolites of <i>Streptomyces</i> sp. CNQ-509.</p

HPLC and LC-MS analysis of the reaction products of Mpz10.

<p>(A) and (B): Incubations containing 1,6-dihydroxyphenazine, dimethylallyl diphosphate (DMAPP), and Mg²⁺ with the membrane fractions of <i>E. coli</i> harboring either the empty vector pET-28a(+) (A) or the Mpz10 expression vector pPH23 (B). (C) Authentic DHDMP (structure see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#pone-0099122-g002" target="_blank">Figure 2B</a>). (D) and (E): Authentic JBIR-47 and JBIR-46 (structures see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#pone-0099122-g001" target="_blank">Figure 1B</a>). (F) Incubation containing 1-hydroxyphenazine, DMAPP, and Mg²⁺ with the membrane fraction of <i>E. coli</i> harboring the Mpz10 expression vector pPH23 (for the structure of <b>3</b> see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#pone-0099122-g005" target="_blank">Figure 5</a>). Mass spectra of the three enzymatic products are shown on the right. Detection: A-E, UV 275 nm; F, UV 368 nm.</p

Phenazine biosynthesis in <i>Streptomyces</i> sp. SpC080624SC-11.

<p>(A) Putative biosynthetic gene cluster of JBIR-46, -47, and -48. (B) Proposed pathway for the biosynthesis of JBIR-46, -47, and -48. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#pone-0099122-t001" target="_blank">Table 1</a> for additional information on the genes contained in the depicted gene cluster.</p

Prenylation in the biosynthesis of phenazines.

<p>(A) Reaction catalyzed by the previously discovered prenyltransferases PpzP and EpzP. (B) Structures of the phenazines JBIR-46, -47, and -48 from <i>Streptomyces</i> sp. SpC080624SC-11.</p

Localization of the Mpz10 activity in the membrane fraction.

<p>Comparison of the prenyltransferase activity of different cell fractions. Activity was tested with 1,6-dihydroxyphenazine, DMAPP, and Mg²⁺ as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099122#s4" target="_blank">Experimental Procedures</a>. The membrane fraction was obtained by centrifugation at 100,000×<i>g</i>.</p