Investigation of a biosynthetic genecluster from the proteaseinhibitor bestatin, isolated from Streptomyces olivoreticuli MD976-C7

Bestatin is a natural product isolated from Streptomyces olivoreticuli MD976-C7 that acts as a protease inhibitor for aminopeptidase B, N or leucine aminopeptidase (Umezawa et al. 1976a; Umezawa et al. 1976b; Suda et al. 1976a; Suda et al. 1976b; Nishizawa et al. 1977). The natural product has been studied in numerous clinical trials and is already being used pharmaceutically. It has been shown to be an approved therapeutic agent for cancer and bacterial infections, by enhancing the immune response (Fontijn et al. 2006; Ishii et al. 2001; Shang et al. 2018; Abe et al. 1985; Aozuka et al. 2004; Li et al. 2020).

The aim of this work was the identification of a biosynthetic gene cluster and the establishment of heterologous expression of bestatin. In silico analysis using antiSMASH (Medema et al. 2011) identified a putative gene cluster in the Streptomyces olivoreticuli MD976-C7 genome sequence (Zhang et al. 2019). Following this, a fosmid library (pCC1FOS-based) was generated. Furthermore, positive clones containing the putative gene cluster were genetically modified by integrating the int_neo cassette (Bierman et al. 1992; Kaysser et al. 2012) to enable heterologous expression in Streptomyces coelicolor M512. The gene cluster was introduced into the expression strain using λ-Red- mediated recombination (Gust et al. 2003). After cultivation of the heterologous host and extraction of the natural product, analytical measurement (LC-MS) indicated the presence of a mass for bestatin. These results suggest that a gene cluster for bestatin has been identified. Further verification, such as the generation of knockouts, is required.