Development of a system for enhancing protein thermostability in vivo using hyperthermophiles

我们一直在研究来自其他古细菌菌株的同源基因是否可以补充T.kodakaraensis的内源性基因破坏，以及热稳定性较差的蛋白质是否可以在增加热稳定性方面受到定向进化的影响。takakaraensis KUW1是一株pyrF和trpE基因缺失的菌株。通过同源重组，从嗜热古细菌furiosus（最适生长温度为100℃）和嗜热古细菌Thermoplasma volcanium（最适生长温度为60℃）中分别引入了pyrF基因。结果发现，重组菌株携带的卟啉卟啉pyrF基因表现出尿嘧啶原生化，表明基因互补已经发生。火山T的基因也获得了类似的结果，但是，只有在75℃或以下的温度下才能观察到持续的生长。有趣的是，在75℃时，我们在该菌株中发现了CpkB的细胞内积累，而在野生型T.kodakaraensis KOD1和携带P.furiosus pyrF的菌株中则没有。由于75℃的温度远低于红枣的最佳生长温度，因此CpkB的积累不可能是热胁迫的结果。这就提出了CpkB表达可能是对T火山中部分变性的PyrF蛋白的细胞内存在作出反应，这一机制与在细菌和真核生物中观察到的未折叠蛋白反应相对应。

We have been examining whether homologous genes from other archaeal strains could complement endogenous gene disruption in T.kodakaraensis and whether proteins with less thermostability could be subject to directed evolution in terms of increasing thermostability. T.kodakaraensis KUW1 is a strain whose pyrF and trpE genes are deleted. To this strain, we introduced pyrF genes from the hyperthermophilic archaeon Pyrococcus furiosus (optimum growth temperature 100℃) or the thermophilic archaeon Thermoplasma volcanium (optimum 60℃) via homologous recombination. We found that the recombinant strain with the P. furiosus pyrF gene exhibited uracil prototrophy, indicating that gene complementation had occurred. Similar results were obtained with the gene from T volcanium, however, consistent growth was observed only at temperatures at or below 75℃. Intriguingly, at 75℃, we found an intracellular accumulation of CpkB in this strain, but not in the wild type T.kodakaraensis KOD1 or in the strain harboring P.furiosus pyrF. As the 75℃ temperature is well below the optimal growth temperature of T.kodakaraensis, the accumulation of CpkB cannot be due to thermal stress. This raise the possibilities that CpkB expression is responding to the intracellular presence of partially denatured PyrF protein from T volcanium, a mechanism which corresponds to the unfolded protein response observed in bacteria and eukaryotes.