Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120

Abstract

Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-alpha-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-alpha-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.

FIG. 1.

Structures, common names (in bold), and chemical names in the Chemical Abstracts Service registry for the most relevant sesquiterpenes discussed in the text.

FIG. 2.

Unrooted phylogenetic trees of sesquiterpene synthase homologs identified in completed bacterial sequences available in the NCBI database. (A) Unrooted phylogenetic tree of single-domain sesquiterpene synthase homologs. (B) Unrooted phylogenetic tree of germacradienol/geosmin synthase-like homologs composed of two fused terpene synthase domains. Sequences were identified by BLAST using known bacterial sesquiterpene synthases. In the case in which sequences of several strains of a bacterial species were available, the sesquiterpene synthase sequence(s) of only one representative strain was selected for sequence analysis. Alignments of sequences used for tree building are shown in Fig. S1 in the supplemental material. Phylogenetic tree branches are labeled with accession numbers followed by species names for each sesquiterpene synthase sequence. Nostoc terpene synthase described in this study are indicated in brackets. Note that some species have several terpene synthase paralogs.

FIG. 3.

Clustering of cyanobacterial sesquiterpene synthase ORFs from Nostoc and Anabaena with ORFs encoding a cytochrome P450 and a putative hybrid two-component protein. ORFs flanking the terpene synthase mini-gene cluster differ in the three cyanobacterial genomes, suggesting functional significance of the gene cluster. The lower panel shows the different domains identified in the putative hybrid two-component protein by searching the NCBI conserved domain database. The GAF domain acronym derives from the names of the first three protein classes identified to contain this domain: mammalian cGMP-stimulated phosphodiesterases, Anabaena adenylyl cyclases, and E. coli transcription factor FhlA.

FIG. 4.

GC-MS analysis of volatile organic compounds produced by E. coli transformants expressing single-domain sesquiterpene synthases from Nostoc. (A) E. coli cells expressing terpene synthase NS1 from Nostoc sp. produced germacrene A which undergoes a Cope rearrangement to form β-elemene under the high temperatures of the injection port. (B) E. coli cells expressing terpene synthase NP1 from N. punctiforme produced a germacrene-like compound that was structurally identified by NMR spectroscopy as 8a-epi-α-selinene.

FIG. 5.

Two structural representations (with atom numbering) of 8a-epi-α-selinene (structure 1). Coupling constant (J) values on the right support the all-cis orientation of the dominant substituents at C-2, C-4a, and C-8a (i.e., their relative configuration). Me, methyl.

FIG. 6.

GC-MS analysis of oxidized sesquiterpenes produced from germacrene A by Nostoc sp. P450. Culture supernatant of E. coli cells coexpressing single-domain sesquiterpene synthase NS1 and P450NS from Nostoc sp. along with the ferredoxin/ferredoxin:NADPH reductase system from Nostoc sp. were analyzed for the accumulation of new oxygenated sesquiterpenes. Three new peaks (labeled 1 to 3; top trace), each with a molecular ion of an m/z of 220, appeared in the chromatogram compared to control cells (bottom trace) that coexpress only terpene synthase NS1 and reductase proteins. β-Elemene (the rearrangement product of germacrene A) present in the supernatant of the control culture is not present in the culture expressing terpene synthase and P450. Mass spectra for the two larger peaks are shown and suggest the addition of one hydroxyl group to germacrene A.

FIG. 7.

GC-MS analysis of sesquiterpenoids produced by fusion-type terpene synthase NP2 from N. punctiforme. Culture supernatant of E. coli cells expressing fusion-type sesquiterpene synthase NP2 for N. punctiforme was analyzed for the accumulation of sesquiterpene products. One major peak (peak 1) with an m/z of 222 and a typical sesquiterpene alcohol fragmentation pattern was identified as (E,E)-germacradienol. Two other peaks were identified as germacrene D (peak 2) and β-elemene (peak 3; Cope rearrangement product of germacrene A). Mass spectra for peaks 1 and 2 are shown.

FIG. 8.

Proposed mechanism for the formation of 8a-epi-α-selinene. Shown are alternative routes and possible intermediate species 8 and 9.