Heterologous expression of heterotrophic nitrification genes

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK Author for correspondence: Stephen Spiro. Tel: + 44 1603 592777. Fax: +44 1603 592250. e-mail: s.spiro@uea.ac.uk ABSTRACT Paracoccus denitrificans is a heterotrophic organism capable of oxidizing ammonia to nitrite during growth on an organic carbon and energy source. This pathway, termed heterotrophic nitrification, requires the concerted action of an ammonia monooxygenase (AMO) and hydroxylamine oxidase (HAO). The genes required for heterotrophic nitrification have been isolated by introducing a Pa. denitrificans genomic library into Pseudomonas putida and screening for the accumulation of nitrite. In contrast to the situation in chemolithoautotrophic ammonia oxidizers, the genes encoding AMO and HAO are present in single linked copies in the genome of Pa. denitrificans. AMO from Pa. denitrificans expressed in Ps. putida is capable of oxidizing ethene (ethylene) to epoxyethane (ethylene oxide), which is indicative of a relaxed substrate specificity. Further, when expressed in the methylotroph Methylobacterium extorquens AM1, the AMO endows on this organism the ability to grow on ethene and methane. Thus, the Pa. denitrificans AMO is capable of oxidizing methane to methanol, as is the case for the AMO from Nitrosomonas europaea. The heterotrophic nitrification genes are moderately toxic in M. extorquens, more toxic in Ps. putida, and non-toxic in Escherichia coli. Toxicity is due to the activity of the gene products in M. extorquens, and both expression and activity in Ps. putida. This is the first time that the genes encoding an active AMO have been expressed in a heterologous host. Keywords: heterotrophic nitrification, ammonia monooxygenase, hydroxylamine oxidoreductase, Paracoccus denitrificans, Methylobacterium extorquens Present address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. Present address: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.