Elucidation of an intercalation structure of the gene cluster for nonribosomal peptide Surugamides biosynthesis

Abstract:

[Objective] The Surugamides (sgm) biosynthetic gene cluster has been reported to contain four NRPS genes surA-D which give two unrelated NRPS pathways. It was experimentally confirmed that surA and surB were essential for Surugamide A and Surugamide F production respectively. However, the functions of surC and surD genes are not verified with experimental evidence. This work is designed to confirm if surA and surD genes are responsible for Surugamide A biosynthesis so as to pave the way to either genetically engineering of Surugamides biosynthetic pathways or study the recognition mechanism of their NRPS proteins. [Methods] The Actinomycetes isolated from marine sponge were identified by analyzing their 16S rRNA gene sequences. The natural product biosynthetic gene clusters were analyzed by submitting genomic sequences to the online database antiSMASH. The chemical structures of isolated compounds were elucidated by UPLC-Q-TOF-MS and ¹³C NMR. To generate the mutation of gene deletion and replacement, a plasmid was constructed with two fragments used for target homologous recombination. The plasmid was then transformed into the target strain for screening of double-crossover mutants. [Results] We discovered Surugamides gene cluster from the genome of Streptomyces albidoflavus LHW3101 isolated from marine sponge Dactylospongia elegans. The compound Surugamide A and Surugamide F were then identified from the fermentation extract of the strain. The surB and surC gene in Surugamides gene cluster were replaced with a constitutive and strong promoter ermEp^*, which was located just before the transcription frame of surD gene. The resulted mutant RJ9 lost the production of Surugamide F but kept on producing Surugamide A with around two-fold yield of the wide type strain.[Conclusion] Gene surB and surC are verified to be not related to sgm A production. It is reported that surA is essential for Surugamide A production. And a remarkable yield improvement for Surugamide A was achieved after introducing a strong promoter before the open reading frame of surD in this work. Therefore we concluded that surA and surD take charge of the biosynthesis of Surugamide A.

Key words:microbial natural products;nonribosomal peptide synthase;biosynthetic gene cluster