Development of a genetic system for combinatorial biosynthesis of lipopeptides in Streptomyces fradiae and heterologous expression of the A54145 biosynthesis gene cluster

Abstract

A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu₁₂), at the expense of factors containing 3-methyl-glutamic acid (3mGlu₁₂). This provided a practical route to produce high levels of pure Glu₁₂-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.

FIG. 1.

Structures of the lipopeptide antibiotics and NRPS protein subunit relationships. (Top) A54145 factors normally produced by S. fradiae. Note that factors A, A₁, D, and F have Glu at position 12, and factors B, B₁, C, and E have 3mGlu at position 12. (Bottom) A21978C factors normally produced by S. rosesosporus and daptomycin.

FIG. 2.

Examples of using λ-Red-mediated recombination in E. coli to engineer BAC pCB01 and derivatives. (A) Insertion of a cassette to drive conjugation from E. coli and site-specific integration in Streptomyces species, generating pDA2002. (B) Integration of the conjugation/insertion promoter cassette and deletion of the genes upstream of lptB to generate pDA2012. (C) Further deletion of genes from pDA2012 and insertion of a terminator cassette to generate pDA2040.

FIG. 3.

Results from HPLC analysis of fermentation broths. The A54145 factors A, A₁, and D (A-core lipopeptides) have Glu₁₂ and Ile₁₃, factors B, B₁, and E (B-core) contain 3mGlu₁₂ and Ile₁₃, and factor F contains Glu₁₂ and Val₁₃. (A) S. fradiae XH25 in DSF roduction medium; (B) XH25 in DSF-Ile; (C) S. fradiae DA613 (ΔlptI::tsr) in DSF-Ile; (D) S. fradiae DA728 (ΔlptD) in DSF-Ile.

FIG. 4.

Ectopic trans-complementation system for engineering A54145 biosynthesis in S. fradiae. (A) The top line shows the organization of A54145 biosynthesis genes cloned in the BAC pCB01. The next series of lines show the extent of deletions (dotted lines) with or without marker insertions (solid boxes) in various S. fradiae mutants. (B) Plasmids derived from pCB01 useful for trans-complementation and genetic engineering of lipopeptide biosynthesis.