摘要
为提高灰褐链霉菌(Streptomyces griseofuscus LS-6)的ε-聚赖氨酸(ε-poly-lysine,ε-PL)发酵水平,从增加内源性前体赖氨酸角度考虑,将S. griseofuscus LS-6与赖氨酸生产菌株黄色短杆菌(Brebvibacterium flavum S62)进行属间的原生质体融合。以S. griseofuscus LS-6产黑色素作为遗传标记挑选到1株融合子LS-32,其ε-PL摇瓶产量为2.82 g/L,是亲本菌株LS-6的1.73倍;LS-32在补料分批发酵中的ε-PL产量达到56.4 g/L,比亲本提高了38.9%。随机引物扩增多肽DNA技术显示融合子LS-32中确实有来自B. flavum S62的基因;融合子ε-PL代谢途径中关键酶的活力(己糖激酶、天冬氨酸激酶、ε-PL合成酶等)和胞内氨基酸含量(赖氨酸、谷氨酸、精氨酸等)均高于亲本,是LS-32高产ε-PL的重要原因。本研究明确了内源性赖氨酸对于提升ε-PL产量的重要性,且属间融合为ε-PL产生菌的育种研究提供了新的参考途径。
In order to improve the ε-poly-lysine(ε-PL) production of Streptomyces griseofuscus, intergeneric hybridization between S. griseofuscus and Brebvibacterium flavum for enhanced level of the endogenous precursor lysine in the cells was investigated for the first time in this study. One hybrid designated LS-32 was selected by using melanin as genetic marker, which produced 2.82 g/L of ε-PL in a shake flask, 1.73 folds as high as that of S. griseofuscus. In fed-batch fermentation, the ε-PL production of LS-32 was 56.4 g/L, which was increased by 38.9% as compared to that of S. griseofuscus. Randomly amplified polymorphic DNA(RAPD) analysis confirmed the intergeneric hybridization between S. griseofuscus and B. flavum. The activities of several key enzymes in LS-32 were greater than in S. griseofuscus. Furthermore, the concentrations of intracellular amino acids such as Asp, Glu and Arg in LS-32 were much higher than in S. griseofuscus. This research proves the importance of enhancing the endogenous lysine for improving ε-PL production and that intergeneric hybridization is a new breeding strategy for ε-PL production.
引文
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