Applications of Gene Replacement Technology to Streptomyces clavuligerus Strain Development for Clavulanic Acid Production

Abstract

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine -aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2-to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.

Genetic engineering of an industrial strain of Streptomyces clavuligerusfor further enhancement of clavulanic acid production

“…Increased dosage of the pleiotropic regulator AdpA enhanced CA production by almost 2-fold (López-García et al, 2010). In another approach, inactivation of the lat gene in the cephamycin C biosynthesis pathway in a commercial S. clavuligerus strain resulted in a 2.5-fold increase in CA production (Paradkar et al, 2001). On the other hand, deletion of the glyceraldehyde 3-phosphate dehydrogenase (gap1) gene in S. clavuligerus channeled G-3-P flux, a limiting factor in CA biosynthesis, to the CA pathway rather than to the glycolytic one and resulted in a 2-fold enhancement in CA production (Li and Townsend, 2006).…”

“…In general, the amount of secondary metabolite produced by standard strains is much lower than what is required for industrial-scale production; therefore development of a super-producer strain by manipulating high-titer industrial ones is a very rational option (Nielsen et al, 2009;Pickens et al, 2011). In the literature, there is only one relevant report in which a double disruption of the lat and cvm genes (lat::apr-cvm::apr) involved in cephamycin C and clavam biosynthesis, respectively, resulted in a 10% increment in CA yield in commercial S. clavuligerus (Paradkar et al, 2001). …”

In silico aided metabolic engineering of Streptomyces roseosporus for daptomycin yield improvement

“…Although the daptomycin production has been improved by decanoic acid feeding during fermentation (Huber et al 1988), it still lags far behind the requirement of the economical large-scale production. Since metabolic engineering is an effective method for strain improvement and production enhancement (Li and Townsend 2006;Paradkar et al 2001;Okamoto et al 2003;Tamehiro et al 2003), it raises our interest to investigate whether daptomycin production could be improved by employing an engineered strain with goal-oriented genetic modification, which directs the metabolic flux toward the target and reduces the by-products. For instance, Li and Townsend (2006) disrupted the gap1 gene encoding the 3-phosphate glyceraldehydes dehydrogenase and kept down the gap2 gene which was related to glycolysis pathway, thus leading to the accumulation of 3-phosphate glyceraldehydes.…”

Induction of Holomycin Production and Complex Metabolic Changes by the argR Mutation in Streptomyces clavuligerus NP1

“…The complexity of CA-clavam biosynthesis is also reflected at the level of regulation, and at least 6 regulatory genes (ccaR [45,52], claR [43], cvm7P [59], and orf21 to orf23 [25,55]) were identified among the three gene clusters. Intricate cross-regulation between the arginine and CA (12, 51), cephamycin C and CA (44,45), and CA and holomycin (11) pathways were also reported. In the case of arginine-CA cross-regulation, the oat genes (homologous to the arginine biosynthetic gene argJ, encoding ornithine acetyltransferase) were found in both the CA (oat2) and the paralog (oat1) clusters, and the oat2 mutant showed interesting changes in CA production levels, depending on the arginine concentrations, indicating it plays a role in controlling the flux between arginine and CA (12).…”