Abstract
The unique capability of acetogens to ferment a broad range of substrates renders them ideal candidates for the biotechnological production of commodity chemicals. In particular the ability to grow with H2:CO2 or syngas (a mixture of H2/CO/CO2) makes these microorganisms ideal chassis for sustainable bioproduction. However, advanced design strategies for acetogens are currently hampered by incomplete knowledge about their physiology and our inability to accurately predict phenotypes. Here we describe the reconstruction of a novel genome-scale model of metabolism and macromolecular synthesis (ME-model) to gain new insights into the biology of the model acetogen Clostridium ljungdahlii. The model represents the first ME-model of a Gram-positive bacterium and captures all major central metabolic, amino acid, nucleotide, lipid, major cofactors, and vitamin synthesis pathways as well as pathways to synthesis RNA and protein molecules necessary to catalyze these reactions, thus significantly broadens the scope and predictability. Use of the model revealed how protein allocation and media composition influence metabolic pathways and energy conservation in acetogens and accurately predicted secretion of multiple fermentation products. Predicting overflow metabolism is of particular interest since it enables new design strategies, e.g. the formation of glycerol, a novel product for C. ljungdahlii, thus broadening the metabolic capability for this model microbe. Furthermore, prediction and experimental validation of changing secretion rates based on different metal availability opens the window into fermentation optimization and provides new knowledge about the proteome utilization and carbon flux in acetogens.
Highlights
Acetogens have been investigated as promising alternative to convert waste gases containing CO2, H2, and CO into multi-carbon commodities [1,2]
We describe a novel genome-scale model of metabolism and gene expression (ME-model) to gain insights into this model acetogen
A gene expression network (i.e., E-matrix) was reconstructed [23,24,25,26]. This reconstruction included an additional 196 protein-coding open reading frames (ORFs), 89 RNA genes, 576 transcription units (415 of which were rho-dependent and 29 were RNA-stable), 19 types of ribosomal RNA (rRNA) modifications, 17 types of tRNA modifications, 735 protein complexes with updated stoichiometry, 219 modified protein complexes, and 134 translocated proteins (Tables 1–15 in S2 File)
Summary
Acetogens have been investigated as promising alternative to convert waste gases containing CO2, H2, and CO (i.e., syngas) into multi-carbon commodities [1,2]. The Wood-Ljungdahl pathway (WLP) enables acetogens to use either H2 or CO as an electron donor with accompanied reduction of CO2, thereby making WLP the only known CO2-fixing pathway coupled to energy conservation [3]. Energetics of autotrophic growth was poorly understood for a long time as no ATP was gained at the substrate level, and not all acetogens contain cytochromeencoding genes to maintain the proton motive force. It was recently discovered that proton exportation could be coupled to ferredoxin oxidation and NAD+ reduction by the Rnf complex [4]. Models like constraint-based genome-scale models of metabolism (i.e., M-models) have been useful for gaining insight to possible routes of energy flux [5,6,7,8]. While M-models have enabled much progress in elucidating cofactor fluxes, other critical components of the cell (e.g., production of macromolecules and mechanistic utilization of metals, vitamins, and cofactors) are usually absent in these models, thereby limiting in-depth understanding of cellular life
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