Department of Biological Engineering, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310025, China.
Appl Biochem Biotechnol. 2013 Jul;170(6):1533-45. doi: 10.1007/s12010-013-0297-9. Epub 2013 May 23.
Genome shuffling is a powerful approach for efficiently engineering industrial microbial strains with interested phenotypes. Here we reported a high producer of nuclease P1, Penicillium citrinum G-16, that was bred by the classical physics-mutagenesis and genome shuffling process. The starting populations were generated by (60)Co γ-irradiation mutagenesis. The derived two protoplast fractions were inactivated by heat-treatment and ultraviolet radiation respectively, then mixed together and subjected to recursive protoplast fusion. Three recombinants, E-16, F-71, and G-16, were roughly obtained from six cycles of genome shuffling. The activity of nuclease P1 by recombinant G-16 was improved up to 1,980.22 U4/ml in a 5-l fermentor, which was 4.7-fold higher than that of the starting strain. The sporulation of recombinant G-16 was distinguished from the starting strain. Random amplified polymorphic DNA assay revealed genotypic differences between the shuffled strains and the wild type strain. The close similarity among the high producers suggested that the genetic basis of high-yield strains was achieved by genome shuffling.
基因组改组是一种高效的工程工业微生物菌株与感兴趣的表型的方法。在这里，我们报道了一个高产核酸酶 P1 的菌株，桔青霉 G-16，是通过经典的物理诱变和基因组改组过程选育的。起始种群是通过（60）Co γ-射线诱变产生的。所得的两个原生质体部分分别通过热处理和紫外线辐射失活，然后混合在一起，并进行递归原生质体融合。从六轮基因组改组中得到了三个重组子，E-16、F-71 和 G-16。重组体 G-16 的核酸酶 P1 的活性在 5 升发酵罐中提高到 1980.22 U4/ml，比起始菌株高 4.7 倍。重组体 G-16 的产孢能力与起始菌株不同。随机扩增多态性 DNA 分析显示改组菌株与野生型菌株之间存在基因型差异。高产菌株之间的高度相似性表明，高产菌株的遗传基础是通过基因组改组实现的。
