Development of a codon-based mutagenesis method enabling deletion, substitution, and insertion of codons

2021 Fiscal Year Final Research Report

• Project/Area Number: 19K05163
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 27040:Biofunction and bioprocess engineering-related
• Research Institution: Osaka University
• Principal Investigator: Okano Kenji 大阪大学, 工学研究科, 助教 (40623335)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: 進化分子工学 / コドン / 置換 / 欠失 / 挿入

Outline of Final Research Achievements

Directed evolution of enzyme, which is based on introduction of mutation and following selection of functional mutant enzyme, is a powerful technique for improving the function of enzyme. To expand the variety of mutant, in this study, I developed a mutagenesis method allowing deletion, substitution, and insertion of codons for the target codon of the target gene.
Specifically, inverse PCR was performed using a plasmid containing the target gene as a template, and adapter sequences were added to both termini of the target codon. The adapter sequence contains recognition sequences for three types of Type IIS restriction enzymes. By using different types of restriction enzymes and following self-ligation, deletion, substitution, and insertion of codons into target site were successfully achieved. The method was also successfully applied to all codons of the target gene to create a mutant enzyme library.

Free Research Field

生物化学工学

Academic Significance and Societal Importance of the Research Achievements

バイオ市場はかつてない活況を呈しており、酵素や多数の酵素が協奏的に働く微生物細胞による物質生産も多分に漏れず、各国が技術開発に鎬を削っている。望みの機能を有する酵素の開発は、未だランダム変異に頼るところが大きいものの、エラープローンPCRに代表される既往の変異導入法は「変異体のバラエティ」と「発生頻度の均一性」に乏しい。したがって、変異体の解析数とラウンド数は自ずと増加し、多大な時間を有する。一方、本研究で開発した作成した変異酵素群は変異のバラエティに富み、同一の変異を含む確率は従来法より低い。従って、高機能な変異体を短時間で取得できるものと期待できる。