Frielingsdorf, S. | Search bioRxiv

bioRxiv2024

The energy metabolism of Cupriavidus necator in different trophic conditions

The knallgas bacterium Cupriavidus necator is attracting interest for biotechnological applications due to its extremely versatile metabolism. C. necator can use hydrogen or formic acid as an energy source, fixes CO2 via the Calvin-Benson-Bassham (CBB) cycle, and also grows well on various other organic acids and sugars. Its tri-partite genome is notable for its size and manifold duplications of key genes (CBB cycle, hydrogenases, nitrate reductases). Comparatively little is known about which of these (iso-) enzymes and their cofactors are actually utilized for growth on different energy sources. Here, we investigated the energy metabolism of C. necator H16 by growing a barcoded transposon knockout library on various substrates including succinate, fructose, hydrogen (H2/CO2) and formic acid. The fitness contribution of each gene was determined from enrichment or depletion of the corresponding mutants. Fitness analysis revealed that 1) some, but not all, molybdenum cofactor biosynthesis genes were essential for growth on formate and nitrate respiration. 2) Soluble formate dehydrogenase (FDH) was the dominant enzyme for formate oxidation, not membrane-bound FDH. 3) For hydrogenases, both soluble and membrane-bound enzymes were beneficial for lithoautotrophic growth. 4) Of the six terminal respiratory complexes in C. necator H16, only some are utilized and utilization depends on the energy source. 5) Deletion of hydrogenase-related genes boosted heterotrophic growth, and we show that the relief from associated protein cost is responsible for this phenomenon. This study evaluates the contribution of each of C. necators genes to fitness in biotechnologically relevant growth regimes. Our results illustrate the genomic redundancy of this generalist bacterium, and may inspire future strategies for strain engineering. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/582058v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1aac357org.highwire.dtl.DTLVardef@1fe970corg.highwire.dtl.DTLVardef@16ea53dorg.highwire.dtl.DTLVardef@122498d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIa barcoded transposon library was used to assess gene fitness for hydrogenase, formate dehydrogenase and electron transport chain complexes C_LIO_LIthe utilization of terminal respiratory complexes is substrate specific C_LIO_LIsoluble formate dehydrogenase is more important than the membrane-bound enzyme C_LIO_LIsoluble hydrogenase and membrane-bound hydrogenase are equally utilized C_LIO_LIinactivation of hydrogenase and its accessory genes leads to faster heterotrophic growth C_LIO_LIwe demonstrate that faster growth is caused by the relief of protein synthesis burden C_LIO_LIfitness data is available in an interactive app at https://m-jahn.shinyapps.io/ShinyLib/ C_LI

microbiology↗

bioRxiv2020

Photorespiration pathways in a chemolithoautotroph

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of 2-phosphoglycolate, an essential process termed photorespiration, was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, but remains uncharacterized in chemolithoautotrophic bacteria. Here, we study photorespiration in the model chemolithoautotroph Cupriavidus necator (Ralstonia eutropha) by characterizing the proxy-process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. We find that the canonical plant-like C2 cycle does not operate in this bacterium and instead the bacterial-like glycerate pathway is the main photorespiratory pathway. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports photorespiration. In this cycle, glyoxylate is condensed with acetyl-CoA to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2. We present bioinformatic data suggesting that the malate cycle may support photorespiration in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so-far unknown photorespiration pathway, highlighting important diversity in microbial carbon fixation metabolism.

microbiology↗