Novel, versatile, and tightly regulated expression system for Escherichia coli strains.

A novel tightly regulated gene expression system was developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-isopropylbenzoate (cumate) as an inducer. This novel expression system, referred to as the cumate gene switch, includes a specific expression vector, pNEW, that contains a partial T5 phage promoter combined with the Pseudomonas-based synthetic operator and the cymR repressor protein-encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer (cumate), which is nontoxic to the culture, water soluble, and inexpensive. The characteristics and potential of the expression system were determined. Using flow cytometry and fed-batch fermentations, we have shown that, with the newly developed cumate-regulated system, (i) higher recombinant product yields can be obtained than with the pET (isopropyl-beta-D-thiogalactopyranoside [IPTG])-induced expression system, (ii) expression is tightly regulated, (iii) addition of cumate quickly results in a fully induced and homogenous protein-expressing population in contrast to the bimodal expression profile of an IPTG-induced population, (iv) expression can be modulated by varying the cumate concentration, and (v) the cumate-induced population remains induced and fully expressing even at 8 h following induction, resulting in high yields of the target protein Furthermore, the cumate gene switch described in this article is applicable to a wide range of E. coli strains.

Production of an insecticidal crystal protein from Bacillus thuringiensis by the methylotroph Methylobacterium extorquens.

The Cry1Aa protein from Bacillus thuringiensis is an insecticidal protein that is highly active against several species of Lepidoptera. We cloned and expressed the cry1Aa gene in a plant-colonizing methylotroph, Methylobacterium extorquens, under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter, P(mxaF). Transmission electron microscopy revealed characteristic bipyramidal intracellular delta-endotoxin crystals similar to the crystalline inclusions formed by B. thuringiensis. Both the protoxin protein and the activated toxin were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis. In single-dose assays of the recombinant against the silkworm, Bombyx mori, both whole cells and cell lysates caused rapid feeding inhibition followed by mortality. The biomass and growth rate of recombinant cells in shake flask culture were similar to those of the wild-type strain, indicating a lack of fitness cost to the recombinant under controlled culture conditions. Recombinant Cry1Aa was expressed at a level of 4.5% of total M. extorquens cell protein. The potential benefits of modifying M. extorquens to deliver insecticidal Cry proteins for crop and forest protection are discussed.

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

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.

Bestowing inducibility on the cloned methanol dehydrogenase promoter (PmxaF) of Methylobacterium extorquens by applying regulatory elements of Pseudomonas putida F1.

PmxaF is a strong methanol-inducible promoter in Methylobacterium extorquens. When this promoter is cloned in expression vectors and used to drive heterologous gene expression, methanol inducibility is either greatly reduced or entirely lost. In order to bestow inducibility upon the cloned PmxaF promoter in expression vectors, we adopted combinational methods (regulatory elements of the Pseudomonas putida F1 cym and cmt operons and Tn7 transposon system) to control reporter gene expression at the transcriptional level in M. extorquens. An operator fragment (26 nucleotides) of the cmt operon was inserted downstream of the cloned PmxaF promoter in the broad-host-range expression vector (pCHOI3). The repressor gene (cymR) located upstream of the cym operon in P. putida F1 was amplified by PCR. To avoid cellular toxicity for M. extorquens caused by the overexpression of CymR, single and/or double copies of cymR were integrated into the chromosome of M. extorquens using the mini-Tn7 transposon system. Cultures containing the chromosomally integrated cymR gene were subsequently transformed with pCHOI3 containing modified PmxaF (i.e., PmxaF plus operator). In this construct, inducibility is afforded by cumate (p-isopropylbenzoate). In this report, we describe the inducible and tightly regulated expression of heterologous genes (bgl [for beta-galactosidase], est [for esterase], and gfp [for green fluorescent protein]) in M. extorquens. This is the first documented example of an inducible/regulated heterologous gene expression system in M. extorquens.