Exploration of useful thermostable enzymes using Geobacillus spp. and their genetic engineering

2015 Fiscal Year Final Research Report

• Project/Area Number: 25450105
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Applied microbiology
• Research Institution: Tottori University (2014-2015); Kyushu University (2013)
• Principal Investigator: Suzuki Hirokazu 鳥取大学, 工学(系)研究科(研究院), 准教授 (80462696)
• Co-Investigator(Renkei-kenkyūsha): Morono Yuki 海洋研究開発機構, 高知コア研究所, サブリーダー (30421845)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: ジオバチラス / 活性スクリーニング / 好熱菌 / ゲノムライブラリー / 高温機能性 / 宿主ベクター系

Outline of Final Research Achievements

This study aimed at facile construction of thermophile DNA libraries in thermophiles and efficient identification of thermostable enzyme genes via in vivo functional screening of the libraries. We established an efficient method to construct DNA libraries in the thermophile Geobacillus kaustophilus MK244, which lacks DNA restriction-modification systems. This method can be used to construct DNA libraries of some thermophiles and to identify the genes for orotidine-5′-phosphate decarboxylase via in vivo functional screening. We also examined functional screening for other enzyme genes but obtained false positives, probably due to instability of the cloning vector pUCG18T. Thus, we developed new host-vector systems and successfully identified Geobacillus spp. OS27, which can maintain the plasmid pUCG18T with higher copy numbers and higher stabilities than G. kaustophilus. This system may be further practical in constructing DNA libraries and identifying thermophile genes.

Free Research Field

応用微生物学，酵素工学，進化工学