Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Abstract

To construct a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis, the genome of the industrial microorganism Streptomyces avermitilis was systematically deleted to remove nonessential genes. A region of more than 1.4 Mb was deleted stepwise from the 9.02-Mb S. avermitilis linear chromosome to generate a series of defined deletion mutants, corresponding to 83.12-81.46% of the wild-type chromosome, that did not produce any of the major endogenous secondary metabolites found in the parent strain. The suitability of the mutants as hosts for efficient production of foreign metabolites was shown by heterologous expression of three different exogenous biosynthetic gene clusters encoding the biosynthesis of streptomycin (from S. griseus Institute for Fermentation, Osaka [IFO] 13350), cephamycin C (from S. clavuligerus American type culture collection (ATCC) 27064), and pladienolide (from S. platensis Mer-11107). Both streptomycin and cephamycin C were efficiently produced by individual transformants at levels higher than those of the native-producing species. Although pladienolide was not produced by a deletion mutant transformed with the corresponding intact biosynthetic gene cluster, production of the macrolide was enabled by introduction of an extra copy of the regulatory gene pldR expressed under control of an alternative promoter. Another mutant optimized for terpenoid production efficiently produced the plant terpenoid intermediate, amorpha-4,11-diene, by introduction of a synthetic gene optimized for Streptomyces codon usage. These findings highlight the strength and flexibility of engineered S. avermitilis as a model host for heterologous gene expression, resulting in the production of exogenous natural and unnatural metabolites.

Fig. 1.

The strategy for construction of large-deletion mutants SUKA2 and SUKA3 of S. avermitilis. Detailed procedures are described in SI Materials and Methods.

Fig. 2.

AseI physical maps of S. avermitilis wild type and its large-deletion mutants. The genotype of large-deletion mutants were as follows: SUKA2, Δ(7,7341–1,494,898 nt) ΔolmA (Δ3,557,725–3,594,005 nt); SUKA3, Δ(79,455–1,595,563 nt)::loxP; SUKA4, SUKA2 Δ(olmA4-olmC)::mut-loxP (Δ3,536,700–3,634,730 nt); SUKA5, SUKA3 Δ(olmA4-olmC)::mut-loxP (Δ3,536,700–3,634,730 nt); SUKA6, SUKA2 Δ(8,886,025–8,925,414 nt)::loxP (containing cyp28 and fdxH); SUKA7, SUKA5 Δ(8,886,025–8,925,414 nt)::loxP; SUKA10, SUKA4 Δ(gap1-ptlL)::ermE (Δ3,745,502–3,758,936 nt); SUKA11, SUKA5 Δ(gap1-ptlL)::ermE (Δ3,745,502–3,758,936 nt); SUKA12, SUKA10 Δ(8,886,025–8,925,414 nt)::loxP; SUKA13, SUKA11 Δ(8,886,025–8,925,414 nt)::loxP; SUKA15, SUKA12 ΔgeoA::aadA; SUKA16, SUKA13 ΔgeoA::aadA; and SUKA17, SUKA13 Δ(2,633,682–2,641,994 nt)::mut-loxP. The right column indicates the percentage of the genome size compared with that of the wild type. Shaded boxes [D’; Δ(8,886,025–8,925,414 nt]::loxP, K’; ΔolmA, K”; Δ(olmA4-olmC)::mut-loxP, N’; Δ(gap1-ptlL)::ermE, O’; ΔgeoA::aadA, O” Δ(2,633,682–2,641,994 nt)::mut-loxP, P’; Δ(79,455–1,595,563 nt)::loxP and W’; Δ(7,7341–1,494,898 nt)] on the physical maps indicate the introduction of deletion(s). Thick bars at the top and bottom of the physical maps correspond to the central core region. Open arrows and filled triangles indicate the replication origin and 16S-23S-5S rRNA operon, respectively.

Fig. 3.

Production of streptomycin in S. avermitilis wild type carrying pSM1, its large-deletion mutants SUKA5(pSM1), and the original producer S. griseus. All strains were grown in production medium at 28 °C. Quantitative analysis of streptomycin in the culture broth was carried out by the agar-diffusion method using B. subtilis as an indicator microorganism. The strRp, rpsJp, and aveRp indicate that strR was expressed by its own promoter or the promoters rpsJ and aveR in S. avermitilis, respectively. ΔbdpA indicates disruption mutants of bdpA (sav5261). Construction of pSM1 containing the entire streptomycin biosynthetic gene cluster of S. griseus is described in SI Materials and Methods. SGR, S. griseus IFO13350; SAV, S. avermitilis wild type.

Fig. 4.

Production of cephamycin C in S. avermitilis SUKA17(pCEF2) and in the original producer S. clavuligerus. Microorganisms were grown in the original cephamycin C production medium (A) and avermectin production medium (B) at 28 °C. The titer of cephamycin C was measured by the agar-diffusion method using C. terrigena as an indicator microorganism. SCLA indicates the original producer S. clavuligerus. The ccaRp indicates that the transcriptional regulatory gene ccaR was expressed by its own promoter, and rpsJp is as defined in Fig. 3. ΔrsbV indicates a rsbV-null mutant. Construction of pCEF2 containing the entire cephamycin C biosynthetic gene cluster of S. clavuligerus is described in SI Materials and Methods.