Impact of growth mode, phase, and rate on the metabolic state of the extremely thermophilic archaeon Pyrococcus furiosus

Abstract

The archaeon Pyrococcus furiosus is emerging as a metabolic engineering platform for production of fuels and chemicals, such that more must be known about this organism's characteristics in bioprocessing contexts. Its ability to grow at temperatures from 70 to greater than 100°C and thereby avoid contamination, offers the opportunity for long duration, continuous bioprocesses as an alternative to batch systems. Toward that end, we analyzed the transcriptome of P. furiosus to reveal its metabolic state during different growth modes that are relevant to bioprocessing. As cells progressed from exponential to stationary phase in batch cultures, genes involved in biosynthetic pathways important to replacing diminishing supplies of key nutrients and genes responsible for the onset of stress responses were up-regulated. In contrast, during continuous culture, the progression to higher dilution rates down-regulated many biosynthetic processes as nutrient supplies were increased. Most interesting was the contrast between batch exponential phase and continuous culture at comparable growth rates (∼0.4 hr^-1 ), where over 200 genes were differentially transcribed, indicating among other things, N-limitation in the chemostat and the onset of oxidative stress. The results here suggest that cellular processes involved in carbon and electron flux in P. furiosus were significantly impacted by growth mode, phase and rate, factors that need to be taken into account when developing successful metabolic engineering strategies.

Keywords: Pyrococcus furiosus; continuous culture; growth phase; growth rate; hyperthermophiles; transcriptome.

Figure 1. Differentially transcribed ORFs in batch and continuous cultures of Pyrococcus furiosus grown at 90°C in SSM supplemented with maltose

(Left) Growth of P. furiosus in batch culture with 1.8L working volume on maltose supplemented SSM medium. Arrows indicate sampling times for RNA extraction (5, 8, 16 hours). EXP: exponential phase. ES: early stationary phase. LS: late stationary phase. (Right) Growth of P. furiosus in continuous culture with 1L working volume on maltose supplemented SSM medium at 0.15 hr⁻¹ (■), 0.25 hr⁻¹ ( ), and 0.45 hr⁻¹ (▲). The arrow indicates the start of steady state (t_o); samples for transcriptional analysis were taken after 5 reactor volume changes. Numbers represent transcribed ORFs (≥ 2-fold) for comparisons between column and corresponding row. For example, 64 ORFs were up-regulated in ES phase (8 hours) compared to E phase (5 hours). Dark gray areas represent comparisons from batch cultures. Light gray areas represent comparisons from continuous cultures. E: exponential phase; ES: early stationary phase;LS: late stationary phase

Figure 2

Heat map of log₂-fold change comparisons in P. furiosus. Inner ring: batch culture at 8 h and 16 h compared to 5 h (inside to outside). Middle ring: chemostat culture at D = 0.25 h⁻¹ and 0.15 h⁻¹ compared to 0.45 h⁻¹(inside to outside). Outer ring: chemostat compared to batch: D = 0.45 h⁻¹ vs. 5 h, D = 0.25 h⁻¹ vs. 8 h, D = 0.15 h⁻¹ vs. 16 h (inside to outside). Only genes with a 3-fold change for at least one contrast are shown.