Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

Abstract

Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster.

Keywords: Fusarium; marine fungi; non-ribosomal peptide synthetases; polyketide synthases; regulation; secondary metabolites; transcription factor.

Figure 1

Structure of scopularide A and the related compounds emericellamide A and W493-B.

Figure 2

Overview of the scopularide and emericellamide biosynthetic gene clusters in S. brevicaulis and A. nidulans.

Figure 3

(A) Chromatogram of a scopularide A of the lowest concentration of standard (0.02 µg/mL) used in the experiments; (B) Standard curve for scopularide A.

Figure 4

Specific and volumetric scopularide A production by S. brevicaulis LF580 and transformants TF1-5-1 and TF5-2, which contain the gene encoding the putative transcription factor for the NRPS1-PKS2 gene cluster under a constitutive promoter, when grown in (a) defined medium and (b) complex medium. Error bars represent ± sem (n = 2 for TF1-5-1 and n = 2 for LF580 in defined medium, n = 3 for LF580 in complex medium [16]).

Figure 5

Proposed biosynthetic pathway for scopularide A using emericellamide and W493-B as models [14,15].

Figure 6

Generation of vectors for targeted deletion of NRPS1 and for overexpression of the cluster transcription factor. The deletion vector pHU2-NRPS1KO was generated by cloning 707–717 bp from the border regions of NRPS1 into a vector carrying a hygromycin resistance cassette [28]. To generate the transcription factor expression vector, the gene was cloned into a vector carrying a geneticin (G418) resistance cassette [29] and a promoter from the A. nidulans translation elongation factor 1α (pTEF-1α). This vector also contained border sites from the F. graminearum β-tubulin [29]. Both vectors were introduced into S. brevicaulis by Agrobacterium tumefaciens mediated transformation.