Multicopy integration and expression of heterologous genes in Methylobacterium extorquens ATCC 55366

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 (PmxaF) using the mini-Tn7 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 Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 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 attTn7 site is a noncoding region of the chromosome make the mini-Tn7 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 [beta-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens.

FIG. 1.

Construction of mini-Tn7 delivery plasmids. All genes are transcribed from left to right. Km^r, kanamycin resistance gene; Tn7L and Tn7R, Tn7 left and right ends which are the minimal requirements for transposition; P_mxaF, methanol dehydrogenase promoter; MCS, multiple cloning site; bgl, β-galactosidase gene; estI, esterase gene; gfp, green fluorescent protein gene; M, MluI; A, AflII; N, NotI; K, KpnI; AP, ApaI.

FIG. 2.

(A) Identification of the mini-Tn7 integration site (attTn7) in M. extorquens. The insertion site is indicated by a triangle between nucleotides 24 and 25 downstream of the glmS stop codon in the chromosome of M. extorquens. nt, nucleotides. (B) Identification of mini-Tn7 integration in M. extorquens by colony PCR. Verification of transposition events by colony PCR using the primer pairs indicated by convergent arrows yielded PCR fragments (solid bars) whose sizes are indicated. Lane M, 1-kb DNA ladder; lane 1, wild type; lanes 2 and 5, gfp integrants; lanes 3 and 6, est integrants; lanes 4 and 7, bgl integrants.

FIG. 3.

Specific yields of target proteins produced by recombinant M. extorquens for one-copy gene integration into the chromosome under control of P_mxaF. BGL, β-galactosidase; EST, esterase. Almost identical growth profiles were obtained for each M. extorquens recombinant. All measurements were obtained two or more times, and the deviations are indicated by error bars. OD₆₀₀, optical density at 600 nm.

FIG. 4.

Specific yield and fluorescence profiles for strains containing one (1×), two (2×), three (3×), and five (5×) copies of the GFP expression cassette integrated into the chromosome. The deviations in the specific yield determinations are based on three or more independent experiments with different positive colonies on the same plate.