The reverse β-oxidation pathway in Megasphaera indica links valerate and hexanoate biosynthesis to the suppression of gut-colonizing fungi
Abstract
The gastrointestinal expansion of opportunistic fungal pathogens has been shown as an important reservoir for severe invasive infections in immunocompromised population. In healthy individuals, such expansion is constrained by short chain fatty acids (SCFAs) which are linked with the presence of gut commensal bacteria. Although the antifungal activities and the biosynthesis of acetate, propionate, and butyrate have been well-characterized, the gut bacterial producers and biosynthetic pathways of valerate and hexanoate remain largely unknown. Here, we systematically profiled SCFA production across a phylogenetically diverse collection of healthy human gut bacterial isolates and identified Megasphaera indica as a prominent producer of valerate and hexanoate. We found that the reverse β-oxidation pathway genes were highly expressed in Megasphaera indica and knockout of the key pathway thiolase genes thlA1-thlA2 abolished biosynthesis of both valerate and hexanoate. In mice experiments, wild-type and reverse β-oxidation-deficient Megasphaera indica colonized the intestine at comparable levels, but the mutant strain failed to produce valerate or hexanoate in vivo. Finally, we showed that disruption of the reverse β-oxidation pathway markedly attenuated the inhibitory activity of Megasphaera indica against diverse fungi, including Candida albicans, Candida parapsilosis and Nakaseomyces glabratus. Together, our findings elucidate the gut bacterial metabolic pathway that links valerate and hexanoate biosynthesis to the suppression of opportunistic gut fungi, providing critical insights for microbiota-based antifungal strategies.
References
1 Fan, D. et al. Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization. Nature medicine 21, 808-814 (2015).
2 Zhai, B. et al. High-resolution mycobiota analysis reveals dynamic intestinal translocation preceding invasive candidiasis. Nature Medicine 26, 59-64, doi:10.1038/s41591-019-0709-7 (2020).
3 Witchley, J. N. et al. Candida albicans morphogenesis programs control the balance between gut commensalism and invasive infection. Cell host & microbe 25, 432-443. e436 (2019).
4 Mishra, A. A. et al. Microbiota-derived short-chain fatty acids mediate Candida albicans gastrointestinal colonization resistance. Cell Host & Microbe (2026).
5 Organization, W. H. WHO fungal priority pathogens list to guide research, development and public health action. (World Health Organization, 2022).
6 Ioannou, P. & Papakitsou, I. Kodamaea ohmeri infections in humans: A systematic review. Mycoses 63, 636-643 (2020).
7 Mendoza-Reyes, D. F., Gómez-Gaviria, M. & Mora-Montes, H. M. Candida lusitaniae: biology, pathogenicity, virulence factors, diagnosis, and treatment. Infection and Drug Resistance, 5121-5135 (2022).
8 Ioannou, P., Baliou, S. & Kofteridis, D. P. Fungemia by Wickerhamomyces anomalus—A narrative review. Pathogens 13, 269 (2024).
9 Chen, J. et al. Clinical characteristics and outcomes of candidemia caused by Meyerozyma guilliermondii complex in cancer patients undergoing surgery. Mycopathologia 185, 975-982 (2020).
10 Yasuma-Mitobe, K. et al. Intracellular acidification by microbiota-derived valeric acid facilitates trans-kingdom ecology limiting Candida parapsilosis colonization. Cell Host & Microbe (2026).
11 Lin, Y. et al. Altered mycobiota signatures and enriched pathogenic Aspergillus rambellii are associated with colorectal cancer based on multicohort fecal metagenomic analyses. Gastroenterology 163, 908-921, doi:10.1053/j.gastro.2022.06.038 (2022).
12 Jacobsen, I. D. et al. Fungi of the murine gut: episodic variation and proliferation during antibiotic treatment. PLos One 8, doi:10.1371/journal.pone.0071806 (2013).
13 Matsuo, K. et al. Fecal microbiota transplantation prevents Candida albicans from colonizing the gastrointestinal tract. Microbiology and Immunology 63, 155-163, doi:10.1111/1348-0421.12680 (2019).
14 McCrory, C., Lenardon, M. & Traven, A. Bacteria-derived short-chain fatty acids as potential regulators of fungal commensalism and pathogenesis. Trends in Microbiology 32, 1106-1118, doi:10.1016/j.tim.2024.04.004 (2024).
15 Cottier, F., Tan, A. S. M., Xu, X., Wang, Y. & Pavelka, N. MIG1 regulates resistance of Candida albicans against the fungistatic effect of weak organic acids. Eukaryotic cell 14, 1054-1061 (2015).
16 Louis, P. & Flint, H. J. Formation of propionate and butyrate by the human colonic microbiota. Environmental microbiology 19, 29-41 (2017).
17 Mishra, A. A. et al. Microbiota-derived short-chain fatty acids mediate Candida albicans gastrointestinal 2 colonization resistance. doi:10.1101/2025.09.08.674977 (2025).
18 Mukhopadhya, I. & Louis, P. Gut microbiota-derived short-chain fatty acids and their role in human health and disease. Nature Reviews Microbiology, 1-17 (2025).
19 Lee, J. S., Wang, R. X., Alexeev, E. E. & Colgan, S. P. Intestinal inflammation as a dysbiosis of energy procurement: new insights into an old topic. Gut Microbes 13, 1880241 (2021).
20 Reichardt, N. et al. Phylogenetic distribution of three pathways for propionate production within the human gut microbiota. The ISME journal 8, 1323-1335 (2014).
21 Louis, P. & Flint, H. J. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS microbiology letters 294, 1-8 (2009).
22 Louis, P. et al. Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the human colon. Journal of bacteriology 186, 2099-2106 (2004).
23 Guo, C.-J. et al. Depletion of microbiome-derived molecules in the host using Clostridium genetics. Science 366, eaav1282 (2019).
24 Qiao, S. et al. Gut Parabacteroides merdae protects against cardiovascular damage by enhancing branched-chain amino acid catabolism. Nature Metabolism 4, 1271-1286 (2022).
25 Barker, H. A., D'Ari, L. & Kahn, J. Enzymatic reactions in the degradation of 5-aminovalerate by Clostridium aminovalericum. Journal of Biological Chemistry 262, 8994-9003, doi:https://doi.org/10.1016/S0021-9258(18)48036-2 (1987).
26 Barker, H. A. Pathway of 5-Aminovalerate Degradation by Clostridium aminovalericum. Curr Top Cell Regul., 349-354, doi:10.1016/b978-0-12-152826-3.50033-4. (1985).
27 Vögeli, B. et al. Cell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria. Nature communications 13, 3058 (2022).
28 Høverstad, T., Fausa, O., Bjørneklett, A. & Bøhmer, T. Short-chain fatty acids in the normal human feces. Scandinavian journal of gastroenterology 19, 375-381 (1984).
29 Francisco, E. C. et al. Emergence of cryptic species and clades of Meyerozyma guilliermondii species complex exhibiting limited in vitro susceptibility to antifungals in patients with candidemia. Microbiology Spectrum 11, e05115-05122 (2023).
30 Cabral, L. d. S. & Weimer, P. J. Megasphaera elsdenii: its role in ruminant nutrition and its potential industrial application for organic acid biosynthesis. Microorganisms 12, 219 (2024).
31 Yuille, S., Reichardt, N., Panda, S., Dunbar, H. & Mulder, I. E. Human gut bacteria as potent class I histone deacetylase inhibitors in vitro through production of butyric acid and valeric acid. PloS one 13, e0201073 (2018).
32 Kim, H., Jeon, B. S. & Sang, B.-I. An efficient new process for the selective production of odd-chain carboxylic acids by simple carbon elongation using Megasphaera hexanoica. Scientific reports 9, 11999 (2019).
33 McDonald, J. A. et al. Inhibiting growth of Clostridioides difficile by restoring valerate, produced by the intestinal microbiota. Gastroenterology 155, 1495-1507. e1415 (2018).
34 Bornstein, B. & Barker, H. The energy metabolism of Clostridium kluyveri and the synthesis of fatty acids. (1948).
35 Liu, C. et al. GMrepo v3: a curated human gut microbiome database with expanded disease coverage and enhanced cross-dataset biomarker analysis. Nucleic Acids Research, gkaf1190 (2025).
36 Zhou, S. et al. A symbiotic filamentous gut fungus ameliorates MASH via a secondary metabolite–CerS6–ceramide axis. Science 388, eadp5540 (2025).
37 Jeon, B. S. et al. Molecular Chain Elongation Mechanism for n‐Caproate Biosynthesis by Megasphaera Hexanoica. Advanced Science, e06069 (2025).
38 Li, Z.-M. et al. Gut-derived fungemia due to Kodamaea ohmeri combined with invasive pulmonary aspergillosis: a case report. BMC Infectious Diseases 22, 903 (2022).
39 Sun, S. et al. Therapeutic manipulation of gut microbiota by polysaccharides of Wolfiporia cocos reveals the contribution of the gut fungi-induced PGE2 to alcoholic hepatic steatosis. Gut microbes 12, 1830693 (2020).
40 Huertas-Díaz, L. et al. Megasphaera contributes to lactate-driven valerate production in the human gut. Microbiome 13, 210 (2025).
41 Wu, J. et al. The intestinal fungus Aspergillus tubingensis promotes polycystic ovary syndrome through a secondary metabolite. Cell Host & Microbe 33, 119-136. e111 (2025).
42 Gong, X. et al. Metabolic engineering of commensal bacteria for gut butyrate delivery and dissection of host-microbe interaction. Metabolic Engineering 80, 94-106 (2023).
43 Wu, L. et al. Data-driven prediction of colonization outcomes for complex microbial communities. Nature Communications 15, 2406 (2024).
44 Zhang, K. et al. Quantification of personalized glycemic sensitivity to food and its potential for precision nutrition in a series of n-of-1 trials. The American Journal of Clinical Nutrition (2025).
45 Chaumeil, P.-A., Mussig, A. J., Hugenholtz, P. & Parks, D. H. (Oxford University Press, 2020).
46 Hillmann, B. et al. Evaluating the information content of shallow shotgun metagenomics. Msystems 3, 10.1128/msystems. 00069-00018 (2018).
47 White TJ, B. T., Lee S, Taylor JW. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications. 315-322 (Academic Press, Inc., 1990).
48 Rolling, T., Zhai, B., Frame, J., Hohl, T. M. & Taur, Y. Customization of a DADA2-based pipeline for fungal internal transcribed spacer 1 (ITS1) amplicon data sets. JCI Insight 7, doi:10.1172/jci.insight.151663 (2022).
49 Tatusova, T. et al. Update on RefSeq microbial genomes resources. Nucleic Acids Research 43, D599-D605, doi:10.1093/nar/gku1062 (2015).
50 Paul J McMurdie, S. H. Phyloseq: a bioconductor package for handling and analysis of high-throughput phylogenetic sequence data. Pac Symp Biocomput, 235-246 (2012).
51 Maestrone, G. & Semar, R. Establishment and treatment of cutaneous Candida albicans infection in the rabbit. (1968).
52 Qu, Z. et al. Selective utilization of medicinal polysaccharides by human gut Bacteroides and Parabacteroides species. Nature communications 16, 638 (2025).
53 Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884-i890 (2018).
54 Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nature methods 9, 357-359 (2012).
55 Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC bioinformatics 12, 323 (2011).
56 Liu Xue, D. L., Luo Mingyue, He Yuzhang , Zhu Ning, Tan Yan. An exogenous gene expression vector of Megasphaera indica and its construction method and primers [一种马氏巨球菌外源基因表达载体及其构建方法和引物]. China patent (2024).
57 Raymond, F. et al. The initial state of the human gut microbiome determines its reshaping by antibiotics. Isme j 10, 707-720, doi:10.1038/ismej.2015.148 (2016).
58 Nielsen, H. B. et al. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes. Nat Biotechnol 32, 822-828, doi:10.1038/nbt.2939 (2014).
59 Zeller, G. et al. Potential of fecal microbiota for early-stage detection of colorectal cancer. Mol Syst Biol 10, 766, doi:10.15252/msb.20145645 (2014).
60 Fukuyama, J. et al. Multidomain analyses of a longitudinal human microbiome intestinal cleanout perturbation experiment. PLoS Comput Biol 13, e1005706, doi:10.1371/journal.pcbi.1005706 (2017).
61 Liu, H. et al. Ecological dynamics of the gut microbiome in response to dietary fiber. Isme j 16, 2040-2055, doi:10.1038/s41396-022-01253-4 (2022).
62 Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120 (2014).
63 Lu, J. et al. Metagenome analysis using the Kraken software suite. Nature protocols 17, 2815-2839 (2022).
64 Parks, D. H. et al. GTDB release 10: a complete and systematic taxonomy for 715 230 bacterial and 17 245 archaeal genomes. Nucleic Acids Research, gkaf1040 (2025).
65 Lu, J., Breitwieser, F. P., Thielen, P. & Salzberg, S. L. Bracken: estimating species abundance in metagenomics data. PeerJ Computer Science 3, e104 (2017).
Metrics
DOI:
Submission ID:
Downloads
Posted
How to Cite
Download Citation
Declaration of Competing Interests
Details of all competing interests to be disclosed are as follows:
Copyright
The copyright holder for this preprint is the author/funder.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.