Identification and application of a different glucose uptake system that functions as an alternative to the phosphotransferase system in Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to uptake and phosphorylate glucose; no other route has yet been identified. Disruption of the ptsH gene in wild-type C. glutamicum resulted, as expected, in a phenotype exhibiting little growth on any of the PTS-sugars: glucose, fructose, and sucrose.However, a suppressor mutant that grew on glucose but not on the other two sugars was spontaneously isolated from the PTS-negative strain WTΔptsH. The suppressor strain SPH2, unlike the wild-type strain, exhibited a phenotype of resistance to 2-deoxyglucose which is known to be a toxic substrate for the glucose-PTS of this microbe, suggesting that strain SPH2 utilizes glucose via a different system involving a permease and native glucokinases. Analysis of the C. glutamicum genome sequence using E. coli galactose permease, which can transport glucose, led to the identification of two candidate genes, iolT1 and iolT2, both of which have been reported as myo-inositol transporters. When cultured on glucose medium supplemented with myo-inositol, strain WTΔptsH was able to consume glucose, suggesting that glucose uptake was mediated by one or more myo-inositol-induced transporters. Overexpression of iolT1 alone and that of iolT2 alone under the gapA promoter in strain WTΔptsH rendered the strain capable of growing on glucose, proving that each transporter played a role in glucose uptake. Disruption of iolT1 in strain SPH2abolished growth on glucose whereas disruption of iolT2 did not, revealing that iolT1 was responsible for glucose uptake in strain SPH2. Sequence analysis of the iol gene cluster and its surrounding region identified a single-base deletion in the putative transcriptional regulator gene Cgl0157 of strain SPH2. Introduction of the frameshift mutation allowed strain WTΔptsH to grow on glucose, and further deletion of iolT1 abolished the growth again, indicating that inactivation of Cgl0157 under a PTS-negative background can be a means by which to express the iolT1-specified glucose uptake bypass instead of the native PTS. When this strategy was applied to a defined lysine producer, the engineered strain displayed increased lysine production from glucose.

A third glucose uptake bypass in Corynebacterium glutamicum ATCC 31833

“…1), unlike the wild-type strain, exhibited a phenotype of resistance to 2-deoxyglucose, which is known to be a toxic substrate for the glucose-PTS of this organism (Mori and Shiio 1987), we predicted that the suppressor strain would utilize glucose via a different system involving a particular permease and the native glucokinases. Our analysis following these observations eventually led to the finding that two myo-inositol transporters encoded by iolT1 (Cgl0181, NCgl0178) and iolT2 (Cgl3058, NCgl2953) mediate glucose uptake in this organism and that glucose phosphorylation depends on glucokinase activity (Ikeda 2012;Ikeda et al 2011). We also showed that inactivation of iolR, a putative transcriptional regulator gene, enables strain WTΔptsH to grow on glucose through the derepression of iolT1, as was the case with the suppressor strain SPH2.…”

“…In our previous report (Ikeda et al 2011), we showed that the myo-inositol transporters IolT1 and IolT2 function as alternatives to the glucose-PTS in C. glutamicum. In this study, we identified a third glucose uptake bypass as the bglF-specified EII component of the β-glucoside-PTS in C. glutamicum ATCC 31833.…”

“…Strains WTΔptsH, WTΔptsI, and WTΔptsHI are WT-96-derived PTS-negative mutants, which have deletions in ptsH, ptsI, and both of them, respectively. Strain SPH2 is one of the suppressor mutants that arose spontaneously from the PTS-negative strain WTΔptsH on an MM agar plate with glucose (Ikeda et al 2011). This strain carries a suppressor mutation leading to inactivation of iolR, a putative transcriptional regulator gene.…”

“…1). Based on these results, we established a strategy for engineering C. glutamicum to express the iolT1-specified glucose uptake bypass instead of the original PTS, and demonstrated the usefulness of this strategy using lysine fermentation as a model (Ikeda 2012;Ikeda et al 2011). In keeping with our results, a German group has independently identified two myo-inositol transporters as alternatives to the PTS in this organism and shown the advantage of expressing the non-PTS route(s) in lysine production (Lindner et al 2011a, b).…”

“…Recently, we happened to find that colonies emerged on glucose agar plates from WTΔptsH, a ptsH-disrupted strain of the C. glutamicum wild-type strain WT-96 (ATCC 31833), at a frequency of 10 −5 (Ikeda et al 2011). Since the suppressor strain SPH2 (Fig.…”