A Toolkit for Heterologous Expression of Metabolic Pathways in Aspergillus oryzae

作者：Pahirulzaman Khomaizon A K; Williams Katherine; Lazarus Colin M^*

来源：NATURAL PRODUCT BIOSYNTHESIS BY MICROORGANISMS AND PLANTS, PT C, ELSEVIER ACADEMIC PRESS INC, 525 B STREET, SUITE 1900, SAN DIEGO, CA 92101-4495 USA, 241-260, 2012.

DOI：10.1016/B978-0-12-404634-4.00012-7

Much has been learned about the activities of the key enzymes involved in eukaryotic natural product synthesis by isolating the relevant genes and expressing them in a suitable foreign host. Aspergillus oryzae has proved to be an amenable host for the functional analysis of megasynthases from other fungi, but secondary metabolites are often the products of suites of enzymes, and understanding their biosynthesis requires simultaneous expression of several genes. This chapter describes the development and use of a molecular toolkit that facilitates the rapid assembly of the genes constituting whole biosynthetic pathways in one or a few multiple gene expression plasmids designed to provide high-level expression in A. oryzae. Conventional DNA manipulation by restriction/ligation is replaced by homologous recombination in yeast and Gateway (R)-mediated site-specific recombination in vitro. The toolkit comprises an assembly vector used for the simple construction and modification of large genes from overlapping DNA fragments and three multigene expression vectors. Insertion of three tailoring enzyme genes by homologous recombination and one megasynthase gene by Gateway (R) transfer into each of the expression vectors can be achieved in a little more than 1 week, and alternative selection markers in the expression plasmids permit cotransformation of A. olyzae with up to 12 genes.

• 出版日期2012