Identification and heterologous expression of the kocurin biosynthetic gene cluster

Abstract

The antibiotically bioactive thiopeptide compound kocurin was identified in extracts from a newly isolated Kocuria rosea strain. The axenic strain was retrieved from a soil sample of the intertidal area at the Paracas National Park, Peru. The genetic basis of this promising natural product with activity against methicillin-resistant Staphylococcus aureus (MRSA) strains was revealed by comparative genome analysis of this new isolate and other reported thiopeptide producer strains. The functionality of the predicted gene locus was experimentally proven by heterologous expression in Streptomyces coelicolor M1146. Expression of the gene cluster under the control of a constitutive promoter enabled the transgenic strain to produce kocurin in selected media. The kocurin biosynthetic gene cluster comprises nine open reading frames and spans around 12 kbp of the genome.

The “Three Cs” of Novel Antibiotic Discovery and Production through Synthetic Biology: Biosynthetic Gene Clusters, Heterologous Chassis, and Synthetic Microbial Consortia

“…Additionally, the kocurin gene cluster was recently introduced into S. coelicolor through traditional conjugation methods—however, the yield of kocurin was extremely low, attributed to the difficulty previously seen when expressing thiopeptides in multiple Streptomyces spp. due to very strong influence of media components on production . For example, in a previously reported study, thiopeptide GE2270 was expressed by S. coelicolor in only one media condition out of 35 tested .…”

Recent Advances in the Heterologous Biosynthesis of Natural Products from Streptomyces

“…In vitro cloning includes DNA assembly methods, such as Gibson assembly, DiPAC, PfAgo-based AREs, SSRTA, SIRA, and CATCH. Gibson assembly was applied to clone DNA fragments with a size up to 14 kb, such as bicyclomycin [147], kocurin [23], and lasso peptides [148]. Gibson assembly needs DNA polymerases, exonucleases, and DNA ligases to assemble BGCs with vectors.…”

“…The BGCs vary in size from a small size to large sizes, such as 12 kb for the kocurin BGC [23], 60 kb for the pikromycin BGC [22], 65 kb for the daptomycin BGC [24], 90 kb for the meridamycin BGC [25], and 141 kb for the vancoresmycin BGC [26]. Depending on the size of the BGC, several cloning methods and vector systems have been developed for the heterologous expression of BGCs.…”

Novel bioactive natural products from bacteria via bioprospecting, genome mining and metabolic engineering

“…Further analysis confirmed not only the heterologous expression of both clusters, but also the elevated, in comparison with the native strains, level of production of chloramphenicol and congocidine. Two of these engineered S. coelicolor hosts, M1152 and M1154, have later been also found useful for heterologous expression of a wide spectrum of BGCs, such as the ones for indolocarbazole, aminocoumarines, liponucleoside and thiopeptides (Flinspach et al ., , ; Li et al ., ; Linares‐Otoya et al ., ). Additional engineering steps have been performed in order to make S. coelicolor M1152 to efficiently produce compounds biosynthesized by type III PKS (Thanapipatsiri et al ., ).…”