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The methanotrophic bacterium Methylosinus trichosporium OB3b converts methane to methanol using two distinct forms of methane monooxygenase (MMO) enzyme: a cytoplasmic soluble form (sMMO) and a membrane-bound form (pMMO). The transcription of these two operons is known to proceed in a reciprocal fashion with sMMO expressed at Iow copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of the smmo operon is initiated from a [[sigma].sup.N] promoter 5' of mmoX. In this study the genes encoding [[sigma].sup.N] (rpoN) and a typical [[sigma].sup.N] -dependent transcriptional activator (mmoR) were cloned and sequenced, mmoR, a regulatory gene, and mmoG, a gene encoding a GroEL homologue, lie 5' of the structural genes for the sMMO enzyme. Subsequent mutation of rpoN and mmoR by marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription of mmoX. An rpoN mutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating that [[sigma].sup.N] plays a role in both nitrogen and carbon metabolism in Ms. trichosporium OB3b. The data also indicate that mmoG is transcribed in a [[sigma].sup.N] and MmoR-independent manner. Marker-exchange mutagenesis of mmoG revealed that MmoG is necessary for smmo gene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoR in vivo. The data presented allow the proposal of a more complete model for copper-mediated regulation of smmo gene expression.

Research has been conducted on Bacillus methanolicus MGA3 steady-state methanol-limited cultures. The role of the growth-inhibitory effects of methanol pulses which perturb steady-state methanol concentrations has been investigated and the results are reported.

Research has been conducted on Candida biodinii, a methylotrophic yeast with formaldehyde dehydrogenase activity exhibited during growth on methanol. The cloning of the structural gene from C. biodinii genomic library has been carried out and the results indicate that this gene is responsible for the yeast growth on methanol.

Extracts derived from methanol-grown cells of Amycolatopsis methanolica and Mycobacterium gastri were found to oxidize methanol and ethanol, with concomitant reduction of N, N1 dimethyl-4-nitrosoaniline (NDMA). Anion exchange chromatography proved the existence of an enzyme that catalyzes the activity of oxidation in methanol- or ethanol-grown cells of M. gastri. The methanol:NDMA oxidoreductases (MNO) were distilled to achieve homogeneity from methanol grown cells of M. gastri and A. methanolica.

Methylophaga marina is a Gram-negative methylotroph which can utilize methanol and methylamine as well as fructose as growth substrates. Exploiting the organism's ability to use fructose, mutants affected in methanol utilization were generated and characterized. The experiments produced three classes of methanol oxidation mutants. Class I mutants had disrupted apo-methanol-dehydrogenase, cytochrome C-L and pyrroloquinoline quinone. Class II mutants did not produce active methanol dehydrogenase (MDH) and the class III mutants produced stable MDH. This suggests that other factors are involved in the assembly of MDH.

Research has been conducted on genes involved in methanol oxidation to formaldehyde in methylotroph Methylobacterium extorquens AM1 consisting of several gene clusters. Results demonstrate that these clusters are transcribed as a single operon.

Research has been conducted on Pleurotus sajor-caju gene. The cloning and expressing of this gene into heterologous host Pichia pastoris have been carried out under the control of methanol inducible promoter, and the results deomstrate that P. sajor-caju laccase is effective in directing the gene expressed in P. sajor-caju.