利用Genome shuffling技术对1株融合重组菌Streptomyces sp.Feel-1产ε-聚赖氨酸(ε-PL)进行了育种研究。首先对Feel-1进行了甲基磺酸乙酯(EMS)诱变,以甘氨酸(Gly)和S-2-氨乙基-L-半胱氨酸(AEC)作为抗性指标进行筛选,得到4株正向突变株,ε-PL摇瓶产量分别为1.78 g/L、1.89 g/L、1.85 g/L、1.86 g/L,比原始菌株Feel-1(1.60 g/L)分别提高了11.3%、18.1%、15.6%、16.3%;然后将这4株产量提高株作为出发菌库,采用双亲灭活法进行了3轮递推式原生质体融合(Genome shuffling),最终得到了3株产量大幅提高的重组菌,摇瓶产量分别为2.42 g/L、2.35 g/L和2.37 g/L,比原始菌株分别提高了51.3%、46.9%和48.1%;选择其中1株(G3-67)进行补料分批发酵,192 h时ε-PL产量达到32.2 g/L。

Genome shuffling was adopted to breed a recombinant strain named Streptomyces sp.Feel-1 for producing ε-poly-L-lysine(ε-PL) in this research.First Feel-1 was mutated by mutagen EMS and spread on agar plate supplemented with Gly and AEC(S-2-ammonia ethyl-L-homocysteine) as resistance markers.Four improved mutants were obtained in shake-flask fermentation with ε-PL production as 1.78 g/L,1.89 g/L,1.85 g/L,1.86 g/L,respectively,which was increased by 11.3%,18.1%,15.6% and 16.3% compared with Feel-1(1.6 g/L).Then these four positive mutants were used as starting population and subjected for three rounds of genome shuffling by fusing the inactivated protoplasts.Finally three recombinants were proved to give a high yield of ε-PL production as 2.42 g/L,2.35 g/L and 2.37 g/L,respectively,which was over Feel-1 by 51.3%,46.9% and 48.1%,respectively.Fed-batch fermentation by one of the recombinants G3-67 was carried out and the ε-PL production accumulated to 35 g/L,which was the highest reported level in China at present.