Identification, analysis, and manipulation of the Torrubiellone A gene cluster
Fecha
2017Autor
Lazarus, Colin Michael
UNIVERSITY OF BRISTOL
Institución
Resumen
Torrubiellones A-D, extracted from Torrubiella sp. BCC2165, are structurally similar to 2- pyridone compounds. Torrubiellone A is particularly interesting because it has antimalarial activity. Combining knowledge of the gene clusters responsible for the biosynthesis of the structurally similar compounds with in-silico analysis of the Torrubiella genome sequence lead to the identification of the torrubiellone A biosynthetic gene cluster. Torrubiella sp. BCC2165 DNA was extracted, sequenced and analysed to reveal a putative torrubiellone A gene cluster, comprising torS encoding a hybrid polyketide synthase- nonribosomal peptide synthetase, torA and torB encoding two P450 cytochromes and torC encoding an enoyl reductase. Comparison to the tenellin and desmethylbassianin gene clusters identified two additional genes, torD and torE, which could be responsible for structural differences between torrubiellone A and desmethylbassianin. torS was assembled without introns by homologous recombination in yeast and combined with other biosynthetic genes from the putative torrubiellone cluster, on a multigene expression vector. Assembled plasmids were used to transform the filamentous fungus Aspergillus oryzae NSAR1, yielding strongly yellow- pigmented transformants. Analysis of organic extracts from transformants by liquid chromatography-mass spectroscopy indicated that the production of torrubiellone-related compounds has been achieved. torD and torE gene functions were investigated by co- expressing these genes in a tenellin-producing A. oryzae, resulting in the addition of a hydroxyl group to the tenellin compound. A Torrubiella transformation system was developed, initially for promoter analysis of the torD and torE genes because the start codon of torE lies only 17 bp downstream of the torD stop codon in the Torrubiella genome, yet the two genes are transcribed independently, and that torE promoter is encoded within the torD coding region. The potential regulatory function of two transcription factors, ZnTF and C6TF, whose genes flank the torrubiellone biosynthetic genes, was investigated by over-expression in the native host. Overexpression of ZnTF led to the production of novel compounds, but no role, either positive or negative, was found for C6TF. The potential for genetic analysis by gene knockout in Torrubiella was tested with the torS gene. Transformants generated by homologous recombination were detected, as well as ectopic events, but the system requires improvement particularly in the isolation of homokaryons post- transformation.