A rapid genome-proteome approach to identify rate-limiting steps in the butyrate production pathway in probiotic Clostridium butyricum, CBM588.

Wood, Liam; Omorotionmwan, Bunmi B.; Blanchard, Adam M.; Dowle, Adam; Tooth, David; Bailey, Tom S.; Griffin, Ruth

Abstract

Objectives:
Clostridium butyricum ferments non-digested dietary fibre in the colon to produce butyric acid. Butyrate is a four-carbon, short-chain fatty acid (SCFA) which has multiple health benefits. Many microbial products of pharmaceutical or industrial interest, such as butyrate, are produced in low quantities due to rate-limiting steps in their metabolic pathway, including low abundance or low activity of one or more enzymes in the pathway. By identifying the former, appropriate enzymes can be over-expressed to increase product yields, however, methods to determine these enzymes are laborious. To improve butyrate production in C. butyricum probiotic strain, CBM588, a novel rapid genome-proteome approach was deployed.

Methods:
First, whole genome sequencing was performed and the 8 genes involved in butyrate production identified on the chromosome. Second, the relative abundance of these enzymes was investigated by liquid chromatography-mass spectrometry (LC-MS) analysis of total cytosolic proteins from early stationary phase cultures.

Results:
Phosphotransbutyrylase (Ptb), butyrate kinase (Buk) and crotonase (Crt) were found to be the least abundant. Over-expression episomally of the corresponding genes individually or of the ptb-buk bicistron led to significant increases in butyrate titre per density of culture from 10 to 24 h, compared to the wild type.

Conclusions:
Our findings pave the way for over-expressing these genes chromosomally to generate a recombinant probiotic with improved butyrate production and potentially enhanced gut health properties.
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Citation

Wood, L., Omorotionmwan, B. B., Blanchard, A. M., Dowle, A., Tooth, D., Bailey, T. S., & Griffin, R. (2025). A rapid genome-proteome approach to identify rate-limiting steps in the butyrate production pathway in probiotic Clostridium butyricum, CBM588. Anaerobe, Article 102940. https://doi.org/10.1016/j.anaerobe.2025.102940

• Journal Article Type: Article
• Acceptance Date: Jan 22, 2025
• Online Publication Date: Feb 21, 2025
• Publication Date: Apr 1, 2025
• Deposit Date: Feb 28, 2025
• Publicly Available Date: Feb 28, 2025
• Journal: Anaerobe
• Print ISSN: 1075-9964
• Electronic ISSN: 1095-8274
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Article Number: 102940
• DOI: https://doi.org/10.1016/j.anaerobe.2025.102940
• Keywords: Genome, Proteome, Rate-limiting, Enzymes, Pathway, Clostridium butyricum, Butyrate
• Public URL: https://nottingham-repository.worktribe.com/output/45303404
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S1075996425000034?via%3Dihub
• Additional Information: This article is maintained by: Elsevier; Article Title: A rapid genome-proteome approach to identify rate-limiting steps in the butyrate production pathway in probiotic Clostridium butyricum, CBM588.; Journal Title: Anaerobe; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.anaerobe.2025.102940; Content Type: article; Copyright: © 2025 The Authors. Published by Elsevier Ltd.