Multicopy Integration and Expression of Heterologous Genes in Methylobacterium extorquens ATCC 55366

Abstract

ABSTRACT High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (P mxaF ) using the mini-Tn 7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein ( gfp ) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn 7 integration locus in the chromosome of M. extorquens , known as the Tn 7 attachment site ( att Tn 7 site), was identified. This single att Tn 7 site was identified in an intergenic region between glmS , which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT , which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the att Tn 7 site is a noncoding region of the chromosome make the mini-Tn 7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes ( bgl [β-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens .