Identification of mutation points in Cupriavidus necator NCIMB 11599 and genetic reconstitution of glucose-utilization ability in wild strain H16 for polyhydroxyalkanoate production.

Although the facultative chemolithoautotrophic Cupriavidus necator (formerly Ralstonia eutropha) wild strain H16 is potentially useful as a host for metabolic engineering aimed at polyhydroxyalkanoate production, this organism is deficient in assimilating glucose, a major sugar in non-edible cellulosic resources. Growth properties of C. necator H16 harboring heterologous glf (encoding glucose-facilitated diffusion transporter) and glk (encoding glucokinase) from Zymomonas mobilis strongly suggested that the lack of glucose-utilization ability of C. necator H16 was caused by deficiency of both glucose-uptake and phosphorylation abilities. Next examination focused on previously unknown mutation points in a glucose-utilizing mutant of C. necator NCIMB 11599. Direct sequencing of a region of genes for putative N-acetylglucosamine-specific phosphoenolpyruvate-dependent phosphotransferase system and its upstream region identified a missense mutation in nagE corresponding to Gly265Arg in the EIIC-EIIB component, and a nonsense mutation in nagR encoding a putative GntR-type transcriptional regulator. Further analyses demonstrated that the glucose-utilization ability of C. necator NCIMB 11599 is attributed to extended sugar specificity of the mutated NagE and derepression of nagFE expression by inactivation of NagR. The mutation in nagE and disruption of nagR were then introduced onto chromosome 1 of wild strain H16 by homologous recombination. The resulting engineered strain C. necator nagE_G265R∆nagR exhibited comparable growth and poly(3-hydroxybutyrate) accumulation on glucose to those of the wild strain on fructose, demonstrating successful reconstitution of functional glucose-uptake and phosphorylation system. This recombinant strain is expected to be useful in further engineering for efficient production of PHAs from inexpensive biomass resources.