Characterisation of putative polyketide synthase genes from streptomyces cinnamonensis

Arrowsmith, Teresa Jayne (1990) Characterisation of putative polyketide synthase genes from streptomyces cinnamonensis. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

A clone had been isolated from a lambda library of Streptomyces cinnamonensis A3823.5 genomic DNA by its ability to hybridise to actI and actIII; genes encoding enzymes involved in the early steps of biosynthesis of actinorhodin by S.coelicolor A3(2). A 4.8kb region of DNA from this clone which contains putative polyketide synthase (PKS) genes for the production of the polyether antibiotic, monensin A, was sequenced. Five open reading frames (ORFs) were identified, which are all transcribed in the same direction. The predicted protein products of each of the ORFs show strong homology to the deduced proteins of PKSs from other polyketide-producing streptomycetes, namely those for production of actinorhodin in S.coelicolor, granaticin (S.violaceoruber) and tetracenomycin (S.glaucescens). These proteins include a putative dimeric condensing enzyme, an acyl carrier protein and a ketoreductase, which would constitute part of the PKS multienzyme complex. The high copy number plasmid pWOR120 transformed S.cinnamonensis initially at extremely low frequencies. Stable maintenance of the plasmid in this strain was shown. Transformation frequencies were optimised in S.cinnamonensis using pWOR120 isolated from the organism to eliminate problems of host restriction. Both the conditions used for growth of mycelium, and the medium used for regeneration of protoplasts proved to be important factors in obtaining efficient and reproducible transformation. Frequencies as high as 2.7 x 10⁶ transformants per μg of DNA were obtained. Attempts were made to complement S.cinnamonensis mutants which fail to make monensin A using the cloned DNA which encodes the putative monensin PKS genes. To do this the DNA was subcloned as five fragments into the visual screening vector pWOR125, and the subclones were used to transform the set of mutants. No complementation was observed. The S.cinnamonensis PKS gene cluster was disrupted by use of a temperature-sensitive plasmid containing a fragment of DNA which presumably lies internally to a transcription unit, allowing homologous recombination and subsequent disruption of the gene. This did not, however, result in the loss of monensin production. DNA encoding a resistance gene for the polyether ionophore antibiotic, tetronasin, was used as a probe to isolate DNA encoding a putative monensin resistance gene from a λEMBL4 library of S.cinnamonensis genomic DNA. The predicted protein products of the sequenced S.cinnamonensis DNA show good homology to deduced proteins of PKSs from other streptomycetes, suggesting that this DNA indeed encodes enzymes involved in polyketide biosynthesis. However, the negative results obtained from complementation and gene disruption experiments suggest that this PKS is not involved in the biosynthesis of monensin A.