Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Nagano, Japan.
Appl Microbiol Biotechnol. 2011 May;90(4):1443-51. doi: 10.1007/s00253-011-3210-x. Epub 2011 Mar 31.
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 Escherichia 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 SPH2 abolished 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.
谷氨酸棒杆菌利用磷酸烯醇式丙酮酸依赖的糖磷酸转移酶系统 (PTS) 摄取和磷酸化葡萄糖；尚未发现其他途径。在野生型谷氨酸棒杆菌中破坏 ptsH 基因，如预期的那样，导致表型在 PTS 糖（葡萄糖、果糖和蔗糖）上生长很少。然而，从 PTS 阴性菌株 WTΔptsH 中自发分离出一种能够在葡萄糖上生长但不能在其他两种糖上生长的抑制突变体。与野生型菌株不同，抑制突变体 SPH2 表现出对 2-脱氧葡萄糖的抗性表型，2-脱氧葡萄糖已知是该微生物葡萄糖-PTS 的有毒底物，这表明菌株 SPH2 通过涉及渗透酶和天然葡萄糖激酶的不同系统利用葡萄糖。使用能够运输葡萄糖的大肠杆菌半乳糖渗透酶对谷氨酸棒杆菌基因组序列进行分析，导致鉴定出两个候选基因，iolT1 和 iolT2，它们都被报道为肌醇转运蛋白。当在补充有肌醇的葡萄糖培养基上培养时，WTΔptsH 菌株能够消耗葡萄糖，表明葡萄糖摄取是由一种或多种肌醇诱导的转运蛋白介导的。iolT1 和 iolT2 分别在 gapA 启动子下在 WTΔptsH 菌株中的单独过表达使该菌株能够在葡萄糖上生长，证明每种转运蛋白都在葡萄糖摄取中发挥作用。在 SPH2 菌株中破坏 iolT1 会使葡萄糖生长能力丧失，而破坏 iolT2 则不会，这表明 iolT1 负责 SPH2 菌株的葡萄糖摄取。iol 基因簇及其周围区域的序列分析确定了 SPH2 菌株中假定的转录调节基因 Cgl0157 存在单个碱基缺失。引入移码突变使 WTΔptsH 菌株能够在葡萄糖上生长，进一步缺失 iolT1 又使生长能力丧失，这表明在 PTS 阴性背景下 Cgl0157 的失活可以作为一种表达 iolT1 指定的葡萄糖摄取旁路而不是天然 PTS 的手段。当将该策略应用于确定的赖氨酸生产菌株时，工程菌株显示出从葡萄糖中增加了赖氨酸的产量。
