Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins

Abstract

The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

Keywords: Antibiotic titer improvement; Biosynthesis; Glutarimide-containing polyketides; Regulation; Streptomyces antibiotic regulatory proteins.

Figure 1

(A) Structures of selected members of the glutarimide-containing polyketide family of natural products and (B) genetic organization of the chx, mgs, and ltm biosynthetic gene clusters.

Figure 2

Domain architecture of SARPs and amino acid alignment of MgsA and ChxA with other members of SARPs. (A) Domain architecture of typical SARP and 'PimR-like' SARP. TRC, transcriptional regulatory protein, C terminal; BTAD, bacterial transcriptional activator domain; AAA, ATPase domain, characteristic of ATPases associated with a variety of cellular activities. (B) Alignment of the SARP-like regions of MgsA and ChxA with those of other related SARP family regulators. Numbers indicate amino acid residues from the N-terminus of the protein. Conserved amino acid residues are highlighted. (C) Comparison of the AAA domains of MgsA and ChxA with those of other related proteins. The Walker A and B motifs are underlined. Numbers indicate amino acid residues from the N-terminus of the protein. Conserved amino acid residues are highlighted.

Figure 3

Production of glutarimide-containing polyketides by wild-type and recombinant strains. (A) HPLC traces of CHX (●) and APN (○) production in recombinant strains with the wild-type strain S. sp. YIM56141 as a control. (B) HPLC traces of iso-MGS (▼) production in recombinant strains with the wild-type strain S. platensis NRRL 18993 as a control. (C) HPLC traces of LTM (◆) production in recombinant strains with the wild-type strain S. amphibiosporus ATCC 53964 as a control.

Figure 4

Time course of (A) LTM production in and (B) cell mass of S. amphibiosporus ATCC 53964 wild-type (▲) and recombinant strains SB15008 (○) and SB15009 (□).