The Entner-Doudoroff Pathway Is an Essential Metabolic Route for Methylotuvimicrobium buryatense 5GB1C.

5GB1C, a fast-growing gammaproteobacterial methanotroph, is equipped with two glycolytic pathways, the Entner-Doudoroff (ED) pathway and the Embden-Meyerhof-Parnas (EMP) pathway. Metabolic flux analysis and C-labeling experiments have shown the EMP pathway is the principal glycolytic route in 5GB1C, while the ED pathway appears to be metabolically and energetically insignificant. However, it has not been possible to obtain a null mutant in the - genes encoding the two unique enzymatic reactions in the ED pathway, suggesting the ED pathway may be essential for 5GB1C growth. In this study, the inducible P promoter was used to manipulate gene expression of , and in addition, the expression of these two genes was separated from that of a downstream gene. The resulting strain shows arabinose-dependent growth that correlates with ED pathway activity, with normal growth achieved in the presence of ∼0.1 g/liter arabinose. Flux balance analysis shows that 5GB1C with a strong ED pathway has a reduced energy budget, thereby limiting the mutant growth at a high concentration of arabinose. Collectively, our study demonstrates that the ED pathway is essential for 5GB1C. However, no known mechanism can directly explain the essentiality of the ED pathway, and thus, it may have a yet unknown regulatory role required for sustaining a healthy and functional metabolism in this bacterium. The gammaproteobacterial methanotrophs possess a unique central metabolic architecture where methane and other reduced C carbon sources are assimilated through the ribulose monophosphate cycle. Although efforts have been made to better understand methanotrophic metabolism in these bacteria via experimental and computational approaches, many questions remain unanswered. One of these is the essentiality of the ED pathway for 5GB1C, as current results appear contradictory. By creating a construct with and genes controlled by P and P , respectively, we demonstrated the essentiality of the ED pathway for this obligate methanotroph. It is also demonstrated that these genetic tools are applicable to 5GB1C and that expression of the target genes can be tightly controlled across an extensive range. Our study adds to the expanding knowledge of methanotrophic metabolism and practical approaches to genetic manipulation for obligate methanotrophs.