ABSTRACT
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.