Pooled CRISPRi screening of the cyanobacterium<i>Synechocystis</i>sp. PCC 6803 for enhanced growth, tolerance, and chemical production

Yao, Lun; Shabestary, Kiyan; Björk, Sara; Asplund-Samuelsson, Johannes; Joensson, Haakan N.; Jahn, Michael; Hudson, Elton P.

doi:10.1101/823534

Cited by 1 publication

(1 citation statement)

References 81 publications

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“…For chemostat experiments, the C. necator H16 transposon mutant library was grown as described previously (Jahn et al, 2021). Briefly, an 8-tube MC-1000-OD bioreactor (Photon System Instruments, Drasov, CZ) was customized to perform chemostat cultivation (Jahn et al, 2018, Yao et al, 2020). Bioreactors (65 mL) were filled with minimal medium supplemented with the respective carbon and nitrogen source, and inoculated with an overnight preculture to a target OD 720nm of 0.05.…”

Section: Methodsmentioning

confidence: 99%

The energy metabolism ofCupriavidus necatorin different trophic conditions

Jahn,

Crang,

Gynnå

et al. 2024

Preprint

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SummaryThe ‘knallgas’ bacteriumCupriavidus necatoris attracting interest of the biotech industry due to its extremely versatile metabolism.C. necatorcan use hydrogen or formic acid as an energy source, fixes CO2viathe Calvin–Benson–Bassham (CBB) cycle, and grows well on various other organic acids and sugars. Its three 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 enzymes and their cofactors are actually utilized for growth on different energy sources.Here, we investigated the energy metabolism ofC. necatorH16 by growing a barcoded transposon knockout library on various substrates including hydrogen and formic acid. The fitness contribution of each gene was calculated as the degree of enrichment or depletion of its mutants. Clustering of gene fitness revealed several distinct pathways: 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 means of formate oxidation, not the membrane-bound FDH. 3) For hydrogenases, both soluble and membrane-bound enzymes were beneficial for lithoautotrophic growth. 4) Of the six terminal respiratory complexes inC. necatorH16, only some are used and utilization depends on the energy source. 5) Deletion of hydrogenase-related genes boosted growth, and we show that the relief from associated protein cost is responsible for this phenomenon.This study evaluates the contribution of each ofC. necator’s genes to fitness in industrially relevant growth regimes. Our results illustrate the genomic redundancy of this generalist bacterium, and may inspire future strategies for strain engineering.Graphical AbstractHighlightsa barcoded transposon library was used to assess gene fitness for hydrogenase, formate dehydrogenase and ETC complexesthe utilization of terminal respiratory complexes is substrate specificsoluble formate dehydrogenase is more important than membrane-bound FDHsoluble hydrogenase and membrane-bound hydrogenase are equally utilizedinactivation of hydrogenase and its accessory genes leads to faster growthfurther experiments demonstrate that this is caused by the relief of protein burden

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Section: Methodsmentioning

confidence: 99%