Functions Of Key Enzymes Involved In Drimentines Biosynthesis In Mangrove-derived Streptomyces Sp.OUC6819

Drimentines belong to a class of antibiotics possessinga novel new terpenylateddiketopiperazine structure,which proved to exhibit antibacterial, antifungal,anticancer,and anthelmintic activities.Streptomyces sp.OUC6819, isolated from reedsrhizosphere soil collected from the mangrove conservation area of Guangdongprovince, was previously reported to produce six drimentines.In order to elucidatebiosyntheticpathway of drimentines, the following studies were carried out:Firstly, the NRPS gene clusters in the genome of Sreptomyces sp. OUC6819were systematiclyanalyedusing bioinformatics stategies.The results showedthat therewere seven putativeNRPSs loci in the genome:i, NRPS-1is proposed to encodes aunknown siderophore analogue containing13amino acids, while thesequenceuniqueness (i.e. K517deletion in A1, A4and A8domains) indicates its incompletefunction; ii, NRPS-2~NRPS-6have only one module, and NRPS-4~NRPS-6do nothave acompleteset of domains; iii, NRPS-7is putatively to encode a compoundderived from Gly and Asp. These results suggest that drimentines are probablybiosynthesized via a non-typical NRPS pathway.Secondly, five key genes (driM, driO, driN,driP and driK) involved in thebiosynthesis of drimentines were cloned and expressed. The five genes wereamplified and ligated into different expression vectors, such as pET-22b (+), pET-24a(+), pET-30a (+) and pET-32a (+). The resulting recombinant plasmids weretransformed into different E. coli expression hosts, including BL21(DE3), Rosetta(DE3), Rosetta and Codon plus. DriN, DriP, DriK and DriM were successfullyexpressed in E. coli. The optimized expression conditions are as follows: for DriN andDriK, induced with0.05mM IPTG and incubated at30℃; for DriM,induced with0.05mM IPTG and incubated at16℃; for DriP, induced with0.2mM IPTG and incubated at16℃. However, the expression level of DriK is very low, and no soluble proteinswere obtained for DriO and DriD, no matter what expression vectors and hosts theywere tried in.Thirdly, the acyl carrier protein DriN was characterized. The in vitrobiochemical experimentand HPLC analysis showed that PPTase can convert theapo-DriN into the functional holo-DriN by installing the4’-phosphopantetheineprosthetic group with about48%conversion rate. Moreover, the LC-MS data showedthat the molecular weight difference between apo-form and holo-form is consistentwith the theoretical value, which proved that DriN probably acts as the acyl carrierprotein during the biosynthesis of drimentines.Finally, the catalytic activity of DriM was studied using Pyrophosphate Assay Kit.The result revealed that DriM showed wide substrate flexibility and was able tocatalyze the adenylation of all14amino acidstested.In summary, the systematic bioinformatics analysis of NRPS gene clusters inStreptomyces sp.OUC6819provided an important theoretical guidance for NRPsgenome mining. And the in vitro biochemical studies of the key enzymes laid thenecessary foundation for deciphering the molecular mechanism of drimentinesbiosynthesis.