Abstract
Sucrose accumulation has been observed in some methylotrophic bacteria utilizing methane, methanol, or methylated amines as a carbon and energy source. In this work, we have investigated the biochemical pathways for sucrose metabolism in the model halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. The genes encoding sucrose-phosphate synthase (Sps), sucrose-phosphate phosphatase (Spp), fructokinase (FruK), and amylosucrase (Ams) were co-transcribed and displayed similar expression levels. Functional Spp and Ams were purified after heterologous expression in Escherichia coli. Recombinant Spp exhibited high affinity for sucrose-6-phosphate and stayed active at very high levels of sucrose (K i =1.0±0.6M). The recombinant amylosucrase obeyed the classical Michaelis-Menten kinetics in the reactions of sucrose hydrolysis and transglycosylation. As a result, the complete metabolic network for sucrose biosynthesis and re-utilization in the non-phototrophic organism was reconstructed for the first time. Comparative genomic studies revealed analogous gene clusters in various Proteobacteria, thus indicating that the ability to produce and metabolize sucrose is widespread among prokaryotes.
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