关键词: 谷氨酸棒状杆菌, 基因敲除, 磷酸烯醇式丙酮酸-糖磷酸转移酶系统

Abstract:

Corynebacterium glutamicum is a food-grade microorganism widely used to produce amino acids, proteins
and other chemical products in the fields of biological transformation, food additives, animal feed, cosmetics, medicine
and health care. It can not only use glucose and fructose as the carbon sources, but also can use them as the substrates to
produce gluconic acid, mannitol, sorbitol and other products. Blocking sugar metabolism in which the phosphoenolpyruvate
carbohydrate phosphotransferase system (PTS) and phosphokinase are involved through metabolic engineering is an
effective avenue to improve substrate utilization and the accumulation of desired products. In this research, the mutant strains
CGΔptsF lacking the ptsF gene and CGΔptsFΔptsHΔptsI lacking the ptsF, ptsH and ptsI genes were constructed by
homologous recombination and reverse screening. It was shown that both CGΔptsF and CGΔptsFΔptsHΔptsI were grown
in medium with glucose as the only carbon source, while glucose metabolism was not affected by the lack of these three
genes. Compared to the wild-type strain, the growth rates of CGΔptsF and CGΔptsFΔptsHΔptsI were 48.4% and 29.7%
and cell concentrations of CGΔptsF and CGΔptsFΔptsHΔptsI were 61.6% and 34.1%, respectively, when the mutant
strains were grown in medium using sucrose as the sole carbon source. However, when they were grown using fructose
as the sole carbon source, the growth rates of CGΔptsF and CGΔptsFΔptsHΔptsI were 43.2% and 0 compared to the
wild-type strain. It turned out that the fructose PTS system was controlled by combination of the ptsF, ptsH and ptsI genes
encoding proteins associated with PTS. These engineered bacteria capable of blocking fructose metabolism can provide a
genetic resource to construct mannitol or sorbitol-producing strains with fructose as the substrate and also lay the theoretical
basis for the study of carbohydrate metabolism in Corynebacterium glutamicum.

Key words: Corynebacterium glutamicum, gene knockout, phosphoenolpyruvate carbohydrate phosphotransferase system (PTS)