Protein allocation and utilization in the versatile chemolithoautotrophCupriavidus necator

Abstract

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy.

Thermodynamic limitations of metabolic strategies for PHB production from formate and fructose in Cupriavidus necator

“…For each EFM we determined the biomass yield, the PHB yield, and the MDF value (Figure 2A-B, Figure S3). While the maximum biomass yield observed for formate (∼0.12 g biomass /g substrate ) corresponded well to experimental results (Grunwald et al, 2015; Jahn et al, 2021), the model overestimated the maximum biomass yield for fructose (∼0.53 g biomass /g substrate , Figure 2A). In vivo flux distributions on this substrate are therefore likely to contain EFMs with lower yields.…”

“…This apparent disagreement arises because the addition of Xfpk allows both high and lower-yield EFMs, so its yield score is not high. On fructose, a functional CBB cycle, represented by PRK and RUBISCO, had a negative score for biomass yield, which agrees with yields determined by proteomics-constrained flux simulations (Jahn et al, 2021). The CBB cycle requires ATP and NADPH, whose production requires decarboxylation in lower glycolysis and the TCA cycle, diverting carbon away from biomass.…”

“…Common reactions with high DFs in the two metabolic strategies were found in the anaplerotic reactions (MAE, PEPC), as well as some CBB cycle reactions (RUBISCO, PRK, GADPH, FBA, FBPase, SBPase), implying lower expression levels to carry flux. However, having a high DF is no guarantee of low expression levels, as is exemplified by the high protein levels of RUBISCO, an enzyme limited by its low rate (Jahn et al, 2021). While it is challenging to precisely determine which metabolic strategy (or combination of EFMs) is used for PHB production in vivo based on stoichiometry and thermodynamics alone, essentiality analysis, such as through use of genetic knockout libraries (Jahn et al, 2021), could help to elucidate the actual in vivo flux distribution to PHB.…”

“…Which EFMs are preferentially used in vivo could be better predicted with additional physiological constraints on the solution space, such as protein abundance, gene essentiality, and enzyme kinetics and biochemical regulations (Gerosa et al, 2015; Jahn et al, 2021; Sánchez et al, 2017). Along those lines, a transposon-library of C. necator identified ACS and PEPC as essential reactions, hinting towards their usage in vivo (Jahn et al, 2021).…”

“…The color of each EFM corresponds to its MDF value. White diamonds indicate measured yields from chemostat experiments of the H16 wild-type at different dilution rates, taken from (Jahn et al, 2021). B) Same as A) but for formate.…”