Hyperthermophilic archaea with unique biochemical and physiological characteristics are important organisms for fundamental research of life science and have great potential for biotechnological applications. However, low transformation efficiency of foreign DNA molecules impedes developments in genetic modification tools and industrial applications. In this study, we applied prolonged overlap extension PCR (POE‐PCR) to generate multimeric DNA molecules and then transformed them into two hyperthermophilic archaea, Thermococcus kodakarensis KOD1 and Pyrococcus yayanosii A1. This study was the first example to demonstrate the enhanced transformation efficiencies of POE‐PCR products by a factor of approximately 100 for T. kodakarensis KOD1 and 8 for P. yayanosii A1, respectively, relative to circular shuttle plasmids. Furthermore, directed evolution of a modestly thermophilic enzyme, Methanothermococcus okinawensis 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR), was conducted to obtain more stable ones due to high transformation efficiency of T. kodakarensis (i.e. ~3 × 10⁴ CFU per μg DNA). T. kodakarensis harbouring the most thermostable MoHMGR mutant can grow in the presence of a thermostable antibiotic simvastatin at 85°C and even higher temperatures. This high transformation efficiency technique could not only help develop more hyperthermophilic enzyme mutants via directed evolution but also simplify genetical modification of archaea, which could be novel hosts for industrial biotechnology.

中文翻译：

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直接PCR产物对古细菌的高效转化及其在热稳定酶的定向进化中的应用

具有独特生化和生理特性的嗜热古细菌是生命科学基础研究的重要生物，在生物技术应用方面具有巨大潜力。然而，外来DNA分子的低转化效率阻碍了基因修饰工具和工业应用的发展。在这项研究中，我们应用了延长的重叠延伸PCR（POE-PCR）来生成多聚体DNA分子，然后将其转化为两个超嗜热古细菌，即柯达卡氏热球菌KOD1和pyyrococcus yayanosii A1。该研究是第一个证明POE-PCR产物转化效率提高的实例，其中T. kodakarensis KOD1的转化效率提高了约100倍，而T. kodakarensis KOD1的转化效率提高了约8倍。相对于圆形穿梭质粒，P。yayanosii A1。此外，由于柯达氏衣原体的高转化效率（〜3 ×10 ⁴  CFU ），对适度嗜热的酶，甲烷氧化甲烷球菌3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）进行了定向进化，以获得更稳定的酶。每微克DNA）。柯达氏衣原体携带最热的MoHMGR突变体的动物可以在85°C甚至更高温度下存在热稳定性抗生素辛伐他汀的情况下生长。这种高转化效率技术不仅可以通过定向进化帮助开发更多的超嗜热酶突变体，而且可以简化古细菌的遗传修饰，这可能是工业生物技术的新型宿主。