Molecular Ecology Study On Methanotrophs And Fundermantal Research On The Applications Of Methanotrophs

Methanotrophs are capable of utilizing methane as the sole carbon and energysource, and contain the key enzymes of methane monooxygenases (MMO) whichcatalyze the oxidation of methane and short-chain alkanes and alkenes.Methanotrophs and MMOs play significant roles in biotransformation,bioremediation and greenhouse gas assimilation. Whereas many problems ofmethanotrophs have limited their applications in industrial biotechnology, includinglack of efficient NADH regeneration system, low growth rate and low cell density,lack of strains which are suitable for applications under various conditions, and lackof genetic engineering tools for MMOs. In order to overcome these deficiencies,molecular ecology, gene engineering and biotechnology methods were used toinvestigate the diversity of methanotrophs in coal mine, hetero-expression of MMO,high cell density culture of methanotroph and optimization of oxidation of propenecatalyzedbyMMO.We successfully elucidated the methanotroph community and activity from atypical Chinese alkaline coal mine soil using several complementary molecularecologytechniques, including microarrayand stable isotope probing. Both type I andtype II methanotrophs were present and active. To our knowledge this is the firstdetailed molecular analysis of methanotrophs in the coal mine environment and thedata obtained from the molecular ecology study will provide essential information toisolate and establish biofilters using the most appropriate methanotrophs to mitigateexplosionsandreleaseofmethanegasfromcoalmines.Heterogenous expression of particulate methane monooxygenase (pMMO) wassystemicallystudiedby"TrialandError"approach.Byconstructionofdifferentkindsof promoters and vectors combined with different host cells, hetero-expressionsystem of pMMO was established. Expression plasmid pCompmo was constructed inwhich pmoCAB gene was under the promoter of alkane monooxygenase. It wasproved that the recombinant Enterobacter aerogenes pCompmo showed transcription and translation activityunder certain cultivation conditions. Unfortunately, due to theimproperpMMOrefoldingorassembly,theenzymeactivityinrecombinant cells wasnot as stabled as that in wild strain. Furtherly, another plasmid pCHP containing thewhole pmo gene cluster including the native promoter was constructed andtransformed into Escherichia coli. This was the first report on successful cloning ofpmo genecluster,althoughpreviousresearchersbelievedpMMOwastoxicto E.coli.Paraffin as the methane vector, which can shorten the lag phase and helpMethylosinus trichosporium OB3b to reach high cell density, was selected to enhancethe growth rate of OB3b. Pilot study was carried out in 5L fermenter. With theaddition of 5% (v/v) paraffin, the highest densityof cultivated cells was 9.89gdrywtL-1,whichwas4.5timesofthecontrol.Optimization of epoxypropane biosynthesis from propene using the whole cellsof M. trichosporium OB3b was investigated. The cells suspended in phosphate buffer(pH 7) (OD660=8) accumulated 12.2 mM epoxypropane over 22 h. Meanwhile it wasfound that the cell culture broth could directlycatalyze the biotransformation withoutany pretreatments, which benefit for simplifying the traditional two-stagegrowth-catalysisprocess,andshortenthewholeprocesstime.