Matsui Daisuke, Asano Yasuhisa
a Biotechnology Research Center and Department of Biotechnology , Toyama Prefectural University , Imizu , Japan.
Biosci Biotechnol Biochem. 2015;79(9):1473-80. doi: 10.1080/09168451.2015.1034654. Epub 2015 Apr 21.
For the heterologous production of l-lysine ε-oxidase (LodA), we constructed a new plasmid carrying LodA gene fused in-frame with an antibiotic (phleomycine) resistant gene. The new plasmid was randomly mutated and the mutated plasmids were transformed into Escherichia coli BL21 (DE3) harboring lodB, which encodes a protein (LodB) acting in posttranslational modification of LodA, and active mutants were selected by phleomycin resistance and oxidase activities. One soluble LodA variant isolated by this method contained six silent mutations and one missense mutation. At these mutation points, the codon adaptations at Lys92, Ala550, and Thr646, and the amino acid substitution at His286 to Arg contributed to the production of its functional form. The active form of LodA variant was induced by post-modification of LodB in the heterologous coexpression, and the activity increased with additional NaCl and heat treatment. This is the first report of heterologous production of LodA by random mutagenesis.
为了实现L-赖氨酸ε-氧化酶（LodA）的异源生产，我们构建了一种新质粒，该质粒携带与抗生素（腐草霉素）抗性基因框内融合的LodA基因。对新质粒进行随机诱变，并将诱变后的质粒转化到携带lodB的大肠杆菌BL21（DE3）中，lodB编码一种参与LodA翻译后修饰的蛋白质（LodB），通过腐草霉素抗性和氧化酶活性筛选出活性突变体。通过这种方法分离得到的一种可溶性LodA变体包含六个沉默突变和一个错义突变。在这些突变位点，Lys92、Ala550和Thr646处的密码子适应性以及His286到Arg的氨基酸取代有助于其功能形式的产生。LodA变体的活性形式在异源共表达中由LodB的后修饰诱导产生，并且通过额外的NaCl和热处理活性增加。这是关于通过随机诱变实现LodA异源生产的首次报道。
