Methanotrophs, Methylosinus trichosporium OB3b, sMMO, and their application to bioremediation

One of the most problematic groups of the USEPA and EU priority pollutants are the halogenated organic compounds. These substances have a wide range of industrial applications, such as solvents and cleaners. Inadequate disposal techniques and accidental spillages have led to their detection in soil, groundwater, and river sediments. Persistence of these compounds in the environment has resulted from low levels of biodegradation due to chemical structural features that preclude or retard biological attack. Research has indicated the idea that treatment systems based on methanotrophic co-metabolic transformation may be a cost-effective and efficient alternative to physical methods because of the potential for high transformation rates, the possibility of complete compound degradation without the formation of toxic metabolites, applicability to a broad spectrum of compounds, and the use of a widely available and inexpensive growth substrate. A substantial amount of work concerning methanotrophic cometabolic transformations has been carried out using the soluble form of methane monooxygenase (sMMO) from the obligate methanotroph Methylosinus trichosporium OB3b. This NADH-dependent monooxygenase is derepressed when cells are grown under copper stress. sMMO has a wider specificity than the particulate form. sMMO has been shown to degrade trichloroethylene (TCE) at a rate of at least one order of magnitude faster than obtained with other mixed and pure cultures, suggesting it has a wider application to bioremediation. Furthermore, sMMO catalyzes an unusually wide range of oxidation reactions, including the hydroxylation of alkanes, epoxidation of alkenes, ethers, halogenated methanes, cyclic and aromatic compounds including compounds, that are resistant to degradation in the environment. However, the practical application of methantrophs and Methylosinus trichosporium OB3b to the treatment of chlorinated organics has met with mixed success. Although oxidation rates are rapid, compound oxidation with M. trichosporium OB3b is difficult. This fastidious organism grows relatively slowly, which limits the speed with which sMMO expressing biomass can be generated. Furthermore, product toxicity toward the cell, affecting the stability of the enzyme when transforming certain compounds has been observed, for example, by the products of 1,2,3 trichlorobenzene hydroxylation (2,3,4- and 3,4,5-trichlorophenol) and of TCE degradation (chloral hydrate). Because of this toxicity and the inability of sMMO to further oxidize its own hydroxylation products, the ability of methane monoxygenase to carry out the monooxygenation of a wide variety of substituted aromatics and polyaromatics cannot be fully exploited in M. trichosporium OB3b. Many of these problems could be overcome by the use of either a mixed downstream heterotrophic population of organisms that could accommodate the products of hydroxylation or to express sMMO in an organism that could metabolize the products of hydroxylation. The latter of these two approaches would have several advantages. The main benefit would be the removal of the need for methane, which is required to induce sMMO in M. trichosporium OB3b, and supply carbon and energy to the cells that continuously oxidise the target compound, but also acts as a competitive inhibitor of sMMO. Instead, the recombinant could utilize the products of sMMO-mediated hydroxylation as a carbon source.