Biology subjects

Nwaokorie, U. J.

Publications and source records attributed to Nwaokorie, U. J..

3 recordsLinked to original sources

bioRxiv2026

Targeted Deletions Uncover Previously Uncharacterised C1-fixing Cluster Genes as Essential for Autotrophy in Clostridium autoethanogenum

Recycling carbon oxides from industrial off-gases and gasified organic waste using gas fermentation offers a sustainable route for biomanufacturing of fuels and chemicals. Acetogens are attractive biocatalysts here and fix CO and CO2 via the Wood-Ljungdahl pathway (WLP). However, the incomplete functional annotation of the associated C1-fixing gene cluster limits rational engineering of acetogen cell factories. Here, we investigated five highly conserved but previously uncharacterised C1-fixing cluster genes to quantify their contributions to the growth of the model-acetogen Clostridium autoethanogenum using targeted CRISPR/Cas9-mediated single-gene deletions, physiological characterisation, comparative computational structural analyses, CRISPRi, and functional genomics. Importantly, we identified acsV, lpdA, cooC, and cooC2 as essential for autotrophy but not heterotrophy, whereas deletion of gcvH reduced autotrophic growth and shifted carbon flux from acetate towards 2,3-butanediol. Amino acid supplementation experiments and computational analyses indicate that lpdA and gcvH do not primarily function in glycine or serine biosynthesis but instead fulfil specialised roles in WLP-associated C1 and redox metabolism. Furthermore, genetic and physiological analyses showed that the two CooC-family ATPases, CooC and CooC2, have non-redundant roles in growth, with only cooC2 deletion impairing heterotrophy. Acetate supplementation tests revealed that {Delta}acsV cells retained metabolic activity despite abolished autotrophy, and computational analyses suggest a likely role for acsV in ATP-dependent corrinoid activation. Surprisingly, CRISPRi-mediated repression of acsV was translationally buffered, resulting in altered carbon partitioning without impairing autotrophy. Together, our findings provide insights into the physiological importance of the C1-fixing cluster genes in acetogens and support their metabolic engineering as gas-fermenting cell factories.

synthetic biology↗

bioRxiv2026

Measure Catabolism: Real-time shifts in microbial metabolism through online pressure measurements

Microbial activity is often inferred from cell density measurements; however, biomass formation is merely an indirect, cumulative result of metabolism, known as anabolism. Microbial activity is more accurately indicated by energy conservation or catabolism. This is especially true under low or no-growth conditions, where anabolism remains constant, and shifts in catabolic fluxes go unnoticed with biomass measurements alone. In anaerobic and gas-based metabolic processes, net gas exchange is linked to energy conservation, and catabolism can then be quantified through headspace measurements. We introduce a sealed-vial, non-invasive workflow that uses high-resolution headspace pressure measurements to estimate gas exchange rates and catabolic reactions, enabling real-time visualisation of metabolic shifts throughout an entire batch cultivation cycle. The method was applied to carbon monoxide (CO) fermentations of three Clostridium autoethanogenum strains (JA1-1, LAbrini, and LAbrini_mut) cultivated in serum bottles. Two of them were indistinguishable by OD-derived max. Pressure-derived gas uptake rates resolved multiple exponential phases of gas consumption and identified specific shifts in the metabolism, consistent with transitions from mixotrophic to autotrophic growth. Small but significant differences in terminal headspace pressure were detected, providing an experimentally accessible end-state parameter for phenotypic characterisation that would be obscured by routine intrusive headspace sampling. Finally, pressure-derived catabolic rates further enabled estimates of relative product formation during the main autotrophic phase. The strains were successfully characterised and distinguished by identifying several exponential phases of gas consumption and their rates, as well as differences in the final absolute pressure threshold. This provided phenotypic characterisation and insights not obtainable from OD measurements alone. The work establishes a practical framework for catabolism-resolved microbial characterisation in sealed batch vials through high-resolution online pressure (gas exchange). The assumption that pressure measurements correlate with CO2 and catabolic rates is sensitive to solubility/buffering and temperature/vapour effects, but these limitations are addressable through controls and complementary analytics.

microbiology↗

bioRxiv2023

Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions - hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950) - directly adjacent and expressed at similar levels to the C1-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both {Delta}hp and {Delta}nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

synthetic biology↗