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 CO ₂ 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 (H ₂ /CO ₂ ) 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. necator ’s 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

Highlights

• a barcoded transposon library was used to assess gene fitness for hydrogenase, formate dehydrogenase and electron transport chain complexes

• the utilization of terminal respiratory complexes is substrate specific

• soluble formate dehydrogenase is more important than the membrane-bound enzyme

• soluble hydrogenase and membrane-bound hydrogenase are equally utilized

• inactivation of hydrogenase and its accessory genes leads to faster heterotrophic growth

• we demonstrate that faster growth is caused by the relief of protein synthesis burden

• fitness data is available in an interactive app at https://m-jahn.shinyapps.io/ShinyLib/