Transcriptome and growth efficiency comparisons of recombinant thermophiles that produce thermolabile and...

Geobacillus kaustophilus is a thermophilic bacterium that grows at temperatures ranging between 42 and 74 °C. Here, we modified this organism to produce the thermolabile protein (PyrF_A) or its thermostable variant (PyrF_V) and analyzed the transcriptome and growth efficiency profiles of the resultant strains. In the producer of PyrF_A, the transcriptome profile was changed to facilitate ATP synthesis from NADH without pooling reduced quinones. This change implies that PyrF_A production at elevated temperatures places an energy burden on cells potentially to maintain protein homeostasis. This was consistent with the observation that the PyrF_A producer grew slower than the PyrF_V producer at > 45 °C and had a lower cellular fitness. Similar growth profiles were also observed in the PyrF_A and PyrF_V producers derived from another thermophile (Geobacillus thermodenitrificans) but not in those from Escherichia coli at 30 °C. Thus, we suggest that the production of thermolabile proteins impairs host survival at higher temperatures; therefore, thermophiles are under evolutionary selection for thermostable proteins regardless of whether their functions are associated with survival advantages. This hypothesis provides new insights into evolutionary protein selection in thermophiles and suggests an engineering approach to select thermostable protein variants generated via random gene mutagenesis.