温度和溶菌酶敏感菌株的L-谷氨酸合成

用温度敏感型菌株发酵生产L-谷氨酸不存在生物素亚适量问题,因此该方法在国际上被广泛使用.通常采用对出发菌株进行传统诱变的方法获得温度敏感型菌株.以谷氨酸棒杆菌CICC 10226为出发菌株,先克隆其ItsA基因,然后通过基因敲除的方法构建了突变菌株Corynebacterium glutamicum WT ΔL,该菌株同时具有温度敏感性和溶菌酶敏感性.经透射式电子显微镜观察发现,于38℃培养的突变株细胞与在30℃培养的同一种细胞相比,细胞明显增大,而出发菌株无该现象.发酵试验表明,在生物素过量的情况下,在发酵进入细胞产酸期后通过将发酵温度从原来的30℃提高到38℃,温度敏感突变株的产酸量增加近5倍.如果在发酵培养基巾添加适量的琥珀酸和乙酸,该菌株与不添加的在30℃培养的对照相比,产酸量增加近6.5倍.对于野生型出发菌株而言,在生物素过量的情况下,无论是否采用变温发酵方法都几乎不产酸.说明温度敏感突变株即使在生物素过量的情况下也能通过变温发酵诱导其合成并分泌产生L-谷氨酸.图10表1参13

L-glutamate Production by Engineering Strain with Temperature and Lysozyme Sensitivity

The glutamate excreters obviously possess special properties which allow them to excrete a large quantity of glutamate. These properties are of a nutritional requirement for biotin and the lack, or very low content of a-ketoglutarate dehydrogenase. The biotin requirement is the major controlling factor involved in fermentation. If enough biotin is supplied for optimal growth, the organism produces lactate. With a temperature-sensitive strain, however, glutamate is excreted in spite of enough biotin in the medium. It is a common international practice to produce L-glutamate with temperature-sensitive strains nowadays. Normally, glutamate-overproducing temperature-sensitive strains are obtained from their parent strains by conventional breeding and mutagenesis. Selection of improved organisms is time demanding. In this paper, a glutamate excreter, Corynebacterium glutamicum WT ΔL with temperature and lysozyme sensitivity from Corynebacterium glutamicum CICC 10226 was constructed by recombinant DNA technology. Morphologic change of the mutant strain was analysed by transmission electron microscopy. The fermentation experiments showed that L-glutamate production was induced in the disruptant and its production yield was increased by about 6 folds, more than that of the control, after a temperature shift from 30℃ to 38℃ during the production phase when enough biotin was supplied in the fermentation medium. If a proper amount of succinate and acetate was supplied in the fermentation medium, the yield was increased by about 7.5 folds, more than that of the control. No matter whether a temperature shift was used during the fermentation, less L-glutamate was produced by the wild type strain when enough biotin was supplied in the fermentation medium. The above-mentioned results indicated that L-glutamate production could be induced in the temperature-sensitive mutant by a temperature shift during the fermentation even with excessive biotin in the fermentation broth. Fig 10, Tab 1, Ref 13