Heterologous production of the antifungal polyketide antibiotic soraphen A of Sorangium cellulosum So ce26 in Streptomyces lividans

Abstract

The antifungal polyketide soraphen A is produced by the myxobacterium Sorangium cellulosum So ce26. The slow growth, swarming motility and general intransigence of the strain for genetic manipulations make industrial strain development, large-scale fermentation and combinatorial biosynthetic manipulation of the soraphen producer very challenging. To provide a better host for soraphen A production and molecular engineering, the biosynthetic gene cluster for this secondary metabolite was integrated into the chromosome of Streptomyces lividans ZX7. The upstream border of the gene cluster in Sor. cellulosum was defined by disrupting sorC, which is proposed to take part in the biosynthesis of methoxymalonyl-coenzyme A, to yield a Sor. cellulosum strain with abolished soraphen A production. Insertional inactivation of orf2 further upstream of sorC had no effect on soraphen A production. The genes sorR, C, D, F and E thus implicated in soraphen biosynthesis were then introduced into an engineered Str. lividans strain that carried the polyketide synthase genes sorA and sorB, and the methyltransferase gene sorM integrated into its chromosome. A benzoate-coenzyme A ligase from Rhodopseudomonas palustris was also included in some constructs. Fermentations with the engineered Str. lividans strains in the presence of benzoate and/or cinnamate yielded soraphen A. Further feeding experiments were used to delineate the biosynthesis of the benzoyl-coenzyme A starter unit of soraphen A in the heterologous host.

Utilization of the Methoxymalonyl-Acyl Carrier Protein Biosynthesis Locus for Cloning the Oxazolomycin Biosynthetic Gene Cluster from Streptomyces albus JA3453

“…For example, OzmG (287 amino acids) showed significant homology to members of the HADH family of enzymes, such as Asm13 (61% identity) (28), GdmK (61% identity) (20), FkbK (62% identity) (27), SorD (40% identity) (10,29), and TtmE (64% identity) (9). Similarly, OzmF (221 amino acids) is a probable O-methyltransferase, OzmE is a probable ACP, OzmD is an ADH, and OzmB is an activating enzyme to channel a glycolytic pathway intermediate to methoxymalonyl-ACP biosynthesis, all of which have homologs in known methoxymalonyl-ACP biosynthetic loci (9,10,20,(27)(28)(29). Unique to the ozm locus is the presence of ozmC, whose deduced product showed significant sequence homology to DpsC (26% identity) from Streptomyces peucetius for doxorubicin biosynthesis.…”

“…were similarly designed according to two conserved regions (QGMVVWTV and CVGILRAC) of ADHs from known methoxymalonyl-ACP biosynthesis loci (9,10,20,(27)(28)(29) and used to examine the four HADH-positive cosmids by PCR for the presence of an ADH gene. A distinct product with the predicted size of 0.5 kb was readily amplified from pJTU1059 and cloned (pJTU1052), whose identity as an ADH gene was confirmed by DNA sequencing.…”

“…Although the isotope-labeling experiments had failed to establish the biosynthetic origin of the C 3 OC 4 unit of OZM, similar moieties are present in several other polyketide natural products, such as ansamitocins P-3 (28), FK520 (27), geldanamycin (20), soraphen (10,29), and tautomycin (9) (Fig. 1B).…”

“…Chemical, biochemical, and genetical investigations of the biosynthesis of these metabolites have established that this unit is derived from an intermediate of the glycolytic pathway, which is used to form methoxymalonyl-ACP as an extender unit. Five genes typically are involved (9,10,20,(27)(28)(29), and the structure of FkbI, the acyl-ACP dehydrogenase for methoxymalonyl-ACP biosynthesis from the FK520 biosynthetic pathway, supports the preferred substrate specificity for an acyl-ACP instead of an acyl CoA (26).…”

“…Degenerate primers (HADH-FP [5Ј-GTC CTG GGC GCC GGS GTS ATG GG-3Ј] and HADH-RP [5Ј-GTT GTC CAG GCC GAT SAG GTC NGC-3Ј]) were designed according to two conserved regions (VLGAGVMG and ADLIG LDN) of 3-hydroxyacyl-CoA dehydrogenases (HADHs) known for methoxymalonyl-ACP biosynthesis (9,10,20,(27)(28)(29) and used to amplify putative HADH genes by PCR from S. albus JA3453 total DNA using conditions suitable for the GC-rich DNA (8). A distinct product with the predicted size of 0.73 kb was amplified, cloned into pGEM-EZ (Promega, Madison, WI) as pJTU1051, and sequenced.…”