Metabolic Adaptations in Cereibacter sphaeroides: Bypassing Pyruvate Carboxylase for Carbon Assimilation
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Date
2025-05
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The Ohio State University
Abstract
Central carbon metabolism is a network of biochemical pathways encompassing intermediates and reactions that every carbon substrate must pass through to become cellular material. The entry point into this network varies by substrate, thereby determining the pathways used to replenish essential metabolite pools. Cereibacter sphaeroides serves as a model microorganism for studying energy metabolism and carbon assimilation due to its versatile growth modes and metabolic pathways.
Pyruvate carboxylase, encoded by the pycA gene (rsp_2090) in C. sphaeroides 2.4.1, is of particular interest given its role in carboxylating pyruvate to form oxaloacetate, a precursor metabolite and branching point for biosynthetic pathways. Substrates such as lactate require pyruvate carboxylase to synthesize oxaloacetate, which is essential for producing certain amino acids and pyrimidines. However, succinate as a substrate can bypass pyruvate carboxylase as oxaloacetate is synthesized through fumarate and malate.
Using succinate as a carbon substrate, it was possible to isolate the in-frame pycA deletion mutant CSΔpycAHB through assembly PCR and homologous recombination. As expected, CSΔpycAHB was unable to grow with media containing L-lactate as the carbon source. However, after prolonged incubation with L-lactate, suppression was observed, as the culture reached a final optical density comparable to the wild type. Upon re-inoculation into L-lactate medium, growth resumed even after intermediate transfer to succinate, suggesting genetic suppression. Two independent suppressors were isolated using succinate medium plates and surprisingly these two suppressor strains exhibited different growth patterns. Although they were derived from the same parent strain, the doubling time of suppressor HBΔsupp12/24-2 with succinate during phototrophic growth was 1.7-fold longer than HBΔsupp12/24-1 and wild type, allowing to consider the existence of more than one bypass.
These findings give insight into the metabolic flexibility of C. sphaeroides and its ability to adapt to the loss of pyruvate carboxylase through different bypasses or mechanisms. Understanding these metabolic adaptations is crucial for comprehending microbial resilience and has broader implications for metabolic engineering, synthetic biology, and biofuel production.
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Keywords
Cereibacter sphaeroides, pyruvate carboxylase, precursor metabolites, carbon assimilation, metabolism