敲除aceA和gogat及过表达gltA对谷氨酸棒状杆菌GKGD合成α-酮戊二酸的影响

Effect of aceA and gogat knockout combined gltA overexpression in GKGD on α-ketoglutarate production

α-酮戊二酸(α-ketoglutarate,α-KG)在生命活动中具有重要的作用,被广泛应用于食品、医药等领域。以α-KG生产菌谷氨酸棒状杆菌GKGD为出发菌株,敲除其异柠檬酸裂解酶编码基因ace A以增加异柠檬酸供应,获得GKGD-1,摇瓶发酵条件下其α-KG产量和转化率分别提高14.72%和9.76%;敲除谷氨酸合酶编码基因gogat以降低L-谷氨酸生成量,获得GKGD-2,其α-KG产量和转化率分别提高7.39%和5.43%,L-谷氨酸生成量降低52.87%;过表达柠檬酸合酶编码基因glt A以进一步增加前体物供应,获得GKGD-3,其α-KG产量提高35.9%;于7.5 L发酵罐经30 h发酵,GKGD-3α-KG产量达49.5 g/L,较出发菌株提高36.4%,L-谷氨酸生成量降低50%。敲除ace A和gogat并过表达glt A可显著提高α-KG产量并降低L-谷氨酸生成量。

α-Ketoglutarate （α-KG） plays an important role in life activities and has been widely applied in food and medicine. Isocitrate lyase encoding gene aceA was knocked out to improve isocitrate supply and obtain GKGD-1, by which the α-KG production and yield was increased by 14.72% and 9.76%. In order, glutamate synthase encoding gene gogat was knocked out to reduce L-glutamate accumulation and thus obtain GKGD-2, by which the α-KG production and yield was increased by 7.39% and 5.43% and L-glutamate was decreased by 52.87%. Citrate synthase encoding gene gltA was overexpressed to further increase precursor supply and thus obtain GKGD-3, by which α-KG production was increased by 35.9%. Fermentation assay was performed in 7.5-L fermentor with GKGD-3, α- KG production achieve 49.5 g/L （increased by 36.4% ） and L-glutamate accumulation was decreased by 50%. In summary, aceA and gogat knockout combined with gltA overexpression in GKGD remarkably improved α-ketoglutarate production. Key words α-ketoglutarate; L-glutamate; glutamate synthase; lsocitrate lyase; citrate synthase