Efficient expression system for production of natural products in Aspergillus oryzae

Khomaizon Abdul Kadir Pahirulzaman, Zahida Wasil, Andrew M Bailey, Thomas J Simpson, Russell J Cox, Colin M Lazarus

Author address: 

University of Bristol, UK

Abstract: 

pTAex3 vector has previously been modified by insertion of a GATEWAY destination module into the amyB expression cassette to produce pTAex3GS. This facilitates directional transfer of genes such as fungal polyketide synthases (PKS) and hybrid polyketide synthase8208;non8208;ribosomal peptide synthases (PKS8208;NRPS) into the expression site. To simplify plasmid construction for whole8208;pathway expression pTAex3GS was first converted to a yeast8208;E. coli shuttle vector, pTAYA.GS. An EST database was used to identify genes expressed at a high level under the culture conditions we use for heterologous gene expression in A. oryzae, and the promoters of three of them, Padh, Peno and Pthia, were evaluated. A. oryzae transformants expressing eGFP from Padh and Peno exhibited intense green fluorescence. We used homologous recombination in yeast to combine Padh and Peno together with the strong constitutive A. nidulans promoter PgpdA in pTAYA.GS8208;Page, a novel multiple gene expression vector which has AscI sites downstream of each promoter. The system was tested by reconstructing and expressing the Beauveria bassiana tenellin and Aspergillus nidulans aspyridone synthesis pathways, each of which comprises a hybrid PKS8208;NRPS together with an enoyl reductase and one or more cytochrome P450s, in A. oryzae. Yeast recombination between the AscI8208;cut vector and three PCR products simultaneously placed the tailoring genes downstream of the promoters, creating pTAYA.GSargTen and pTAYA.GSargAsp. Subsequent introduction of the PKS8208;NRPS gene by GATEWAY recombination created pTAYAargTenellin and pTAYAargAspyridone. Reconstruction of the tenellin and aspyridone biosynthetic pathways proved the multiple gene assembly concept, and chemical analysis showed that 5 of the 11 pTAYAargTenellin transformants analysed produced tenellin, pretenellin B and prototenellin A. Similarly 13 of 14 pTAYAargAspyridone transformants analysed produced aspyridone A and preaspyridone. The results show that our system allows the rapid and simple reconstruction of whole (small) biosynthetic pathways for heterologous expression from a single plasmid in A. oryzae. Further development of the system has included replacement of the arginine selectable marker with basta8208;  and phleomycin8208;resistance genes to allow expression of biosynthetic pathways of up to 12 genes by co8208;transformation of A. oryzae with just 3 plasmids
2012

abstract No: 

PR8.20

Full conference title: 

11 th European Conference on Fungal Genetics
    • ECFG 11th (2012)