欧文氏菌和棒杆菌的属间融合研究

INTERGENERIC CELL FUSION OF CORYNEBACTERIUM AND ERWINA

研究了用原生质体融合技术获得欧文氏菌和棒杆菌的融合细胞。串联发酵Ｄ－葡萄糖产生２－酮基－Ｌ－古龙酸的第一步发酵菌株欧文氏菌ＳＣＢ２４７经０．８ｍｇ／ｍＬ溶菌酶酶解０．５ｈ后，原生质体的形成率和再生率分别为９９．８％和２７．８％。第二步发酵菌株棒杆菌ＳＣＢ３０５８经预处理后由１．３ｍｇ／ｍＬ溶菌酶酶解２ｈ，原生质体的形成率和再生率分别为９９．５％和５６．３％。用携带氨苄青霉素抗性标记的ＳＣＢ２４７和经热灭活的ＳＣＢ３０５８为亲本，在４０％ＰＥＧ６０００和０．２ｍｏｌ／Ｌ新生磷酸钙等适宜条件下融合，融合频率为３．６×１０^(－６)。在非选择和选择培养基上连续传代十几次后，对融合子的单菌形态、染色结果、菌落形态、色泽、总蛋白量、同工酶、发酵性能等方面与亲本进行了比较。结果表明，融合子确系棒杆菌和欧文氏菌的融合细胞。摇瓶发酵结果显示，所得的３８株稳定的融合子中约４０％能转化葡萄糖为维生素Ｃ前体２－酮基－Ｌ－古龙酸。

Given the known information of D-glucose tandem fermentation, this paper did the cell fusion of thetwo strains involved in the tandem fermentation. Lysed in 0.8mg / ml lysozyme for half an hour, Erwinia sp .SCB247 was converted into protoplasts with the formation rate of 99.8% and regeneration rate of 27.8%. Theprotoplasts of Corynebacterium sp. SCB3058 were formed after being treated with 1.3mg/ml lysozyme, formationrate and regeneration rate were 99.5% and 56.3% respectively. Ampicillinresistant strain Erwinia sp. SCB247protoplasts and heat- inactivated Corynebacterium sp. SCB3058 protoplasts were fused in 40% PEG 6000 and 0.2mol/Lnewly-made Ca3(PO4)2 with a fusion rate of 3.6× 10-6. After 15 generations, the fusion cells were comparedwith parents cells in terms of single cell morphology, colony morphology, protein staining, isozymes andfermentation properties. The comparison showed that about 40 percent of the fusants can fermentate D-glucosedirectly and convert it to 2-keto-L-gulonic acid, the precursor of vitamin C.