In order to understand metabolic functions essential for methane assimilation, we investigate dribulose monophosphate pathway and adjacent pathways in gammaproteobacterial Methylomicrobium alcaliphilum 20Z by using combined approaches of RNA-seq, LC-MS, and 13C-labeled techniques. The absolute quantification of metabolome showed that the concentrations of intermediates, such as glucose-6-phosphate and 2-dehydro-3-deoxy-phosphogluconate, involved in Entner-Doudoroff (EDD) pathway were (150.95 +/- 28.75) micromol/L and below the limit of detection of mass spectrometry. In contrast, fructose-1, 6-bisphosphate, glyceraldehyde-3-phosphate/dihydroxyacetone and phosphoenolpyruvate in Embden-Meyerhof-Parnas (EMP) pathway had significantly higher concentrations with (1 142.02 +/- 302.88) micromol/L, (1 866.76 +/- 388.55) micromol/L and (3 067.57 +/- 898.13) micromol/L, respectively. 13C-labeling experiment further indicated that the enrichment of [3-13C1]-pyruvate involved in EMP pathway was 4-6 fold higher than [1,13C1]-pyruvate in EDD pathway in a dynamic course. Moreover, gene expression profile showed that the expression levels of genes in EMP pathway (e.g. fbaA, tpiA, gap and pykA) were 2 479.2, 2 493.9, 2 274.6 and 1 846.0, respectively, but gene expressionlevels in EDD pathway (e.g. pgi, eda and edd) were only 263.8, 341.2 and 225.4, respectively. Overall our current results demonstrated that EMP pathway was the main route for methane assimilation in M. alcaliphilum 20Z. This discovery challenged our understanding of methane assimilation pathway in gammaproteobacterial methanotrophic bacteria, and further provided an important insight for efficient methane biocatalysis in the future.
Glycolysis ; Industrial Microbiology ; Methane ; metabolism ; Methylococcaceae ; metabolism ; Pyruvic Acid ; metabolism

Methanotrophs are a group of bacteria capable of utilizing methane as the sole carbon and energy source for their anabolism and catabolism. Since methanotrophs contain the unique enzymes of methane monooxygenases (MMOs), which can catalyze the oxidation of methane and short-chain alkanes and alkenes, they have potential applications in carbon recycle of nature and industrial biotechnology. Therefore, methanotrophs have been paid much more attention by the researchers in recent 20 years. In this paper, the latest progresses in studies of methanotrophs and MMOs were reviewed, including taxonomy, function and distribution of methanotrophs, and structure, function and genetic engineering of MMOs. The future research directions of methanotrophs and MMOs as well as their applications were also discussed.
Methane ; metabolism ; Methylococcaceae ; enzymology ; genetics ; Oxidation-Reduction ; Oxygenases ; metabolism

Aerobic methane oxidizing bacteria (methanotrophs) can use methane as carbon source and energy source, eliminating 10%-20% of global methane. Methanotrophs can also effectively synthesize valuable methane-derived products. This article introduced the methane oxidizing mechanism of methanotrophs, and summarized the practical application and research hotspots of methanotrophs in the field of methane emission reduction in the landfill, ventilation air methane mitigation in coal mines, valuable chemicals biosynthesis, as well as oil and gas reservoir exploration. Main factors influencing the pollutant removal and the biosynthesis efficiency in various applications were also discussed. Based on the study of large-scale cultivation of methanotrophs, some measures to benefit the application and promotion of aerobic methane oxidizing biotechnology were proposed. This includes investigating the effect of intermediate metabolites on methanotrophs activity and population structure, and exploiting economical and efficient alternative culture media and culture techniques.
Biotechnology ; Carbon ; Culture Media/chemistry* ; Methane/metabolism* ; Methylococcaceae/metabolism* ; Oxidation-Reduction

Using a fluidized bed as immobilization system, mixed culture methanotrophic attached-films were developed on diatomite particles. The Methane Monooxygenase (MMO) activity was found to increase obviously as soon as the lag phase ended. Greater than 90% of the MMO activity in the bed was attached. Biofilm concentration of 3.3-3.7 mg dry weight cell/g DS was observed. Batch experiments were performed to explore the possibility of producing epoxypropane by a cooxidation process. The effect of methane on the oxidation of propene to epoxypropane and the effect of propene on the growth of methanotroph were also studied. In continuous experiments, optimum mixed gaseous substrates (methane: 35%; propene: 20%; oxygen: 45%) were continuously circulated through the fluidized bed reactor to remove product. Initial epoxypropane productivity was 110-150 mumol/d. The bioreactor operated continuously for 25 d without obvious loss of epoxypropane productivity.
Adhesins, Bacterial ; physiology ; Biofilms ; growth & development ; Bioreactors ; microbiology ; Cells, Immobilized ; drug effects ; enzymology ; microbiology ; Epoxy Compounds ; metabolism ; Methane ; metabolism ; pharmacology ; Methylococcaceae ; drug effects ; enzymology ; growth & development ; Methylosinus ; drug effects ; enzymology ; growth & development ; Oxidation-Reduction ; Oxygenases ; metabolism ; Propane ; metabolism ; pharmacology