Customizable Cloning of Whole Polysaccharide Gene Clusters by Yeast Homologous Recombination

Abstract

Cloning of whole polysaccharide biosynthesis gene clusters for expression in a common Escherichia coli tester strain has the major advantage of enabling direct functional comparisons between gene clusters that are normally found in different strains, where their expression is potentially under differential regulatory control. However, due to the large size of many of these gene clusters, classical cloning methods are highly inefficient, time-consuming, and/or labor-intensive. Here we describe a recently developed system, called the operon assembly protocol (OAP), in which yeast homologous recombination pathways are used to assemble overlapping PCR fragments onto a specially engineered yeast E. coli shuttle vector, resulting in full-length customizable gene cluster clones on single-copy plasmids. Multiple versions of the same gene cluster can also be assembled in parallel with genes deleted, replaced, or rearranged, allowing the function and/or specificity of individual genes to be examined. Since the vector can be easily modified to include other bacterial replicons, it can also be broadly applied to the functional analysis of a wide range of bacterial gene clusters and operons.