Microbiota-directed fibre activates both targeted and secondary metabolic shifts in the distal gut

Beneficial modulation of the gut microbiome has high-impact implications not only in humans, but also in livestock that sustain our current societal needs. In this context, we have tailored an acetylated galactoglucomannan (AcGGM) fibre to match unique enzymatic capabilities of Roseburia and Faecalibacterium species, both renowned butyrate-producing gut commensals. Here, we test the accuracy of AcGGM within the complex endogenous gut microbiome of pigs, wherein we resolve 355 metagenome-assembled genomes together with quantitative metaproteomes. In AcGGM-fed pigs, both target populations differentially express AcGGM-specific polysaccharide utilization loci, including novel, mannan-specific esterases that are critical to its deconstruction. However, AcGGM-inclusion also manifests a “butterfly effect”, whereby numerous metabolic changes and interdependent cross-feeding pathways occur in neighboring non-mannanolytic populations that produce short-chain fatty acids. Our findings show how intricate structural features and acetylation patterns of dietary fibre can be customized to specific bacterial populations, with potential to create greater modulatory effects at large.

The authors appear to have carefully and respectfully considered each comment from the four reviewers. Their responses are detailed and appropriate, and in many cases include additional work, figures, and/or clarifications. This revised version reads much better and addresses most of the requested changes.
This work is scientifically sound and presents an interesting study of broad interest. The authors have addresses all of my concerns in appropriate detail. There is still some minor issues with the overall novelty and whether the conclusions are a bit over-reaching, but I am satisfied overall with the revisions and do not have further corrections.
Reviewer #4 (Remarks to the Author): I am satisfied with the changes that the authors have made in response to my comments and suggested edits. I agree that these changes have significantly improved and strengthened the manuscript. I only have a few additional edits suggested below: Line 164: Legend for Figure 2 needs updating to reflect that this figure now shows both absolute and relative abundances of butyrate L345: While this figure displays the diversity of organisms and pathways contributing to butyrate production, I would appreciate a clearer delineation between how the substrates (e.g., B-mannans) relate to the GH enzymes and EC numbers. As currently presented, it's unclear which enzymes work on which substrates.
Line 644: given that both 16s rRNA gene sequencing and metagenomic analyses are performed in these papers, I would try to avoid confusion associated with '16s metagenomic analyses' -just refer to it as 16S rRNA gene community analyses or something similar.

COMMENTS FROM REVIEWER(S):
Reviewer #2 (Remarks to the Author): Overall I think the authors have done a very thorough job of responding to the reviewers comments and even carrying out further experiments and I commend them on this. I think the work provides a uniquely in-depth and state of the art analysis of the effects of fibre supplementation on the endogenous microbiota structure and function and is in this regard of significant interest to many researchers in the microbiota field. RESPONSE: We thank reviewer #2 for this very positive comment! a) One point I think does need further clarification is regarding the authors interpretation of the effect of supplementation on butyrate levels. One of the main reasons AcGGM was chosen is stated that it potentially has the ability to specifically promote growth of specific butyrate producers in the gut and this is good as higher butyrate is a good thing for gut health.

However the data show the absolute levels of butyrate do not increase significantly (although the trend is increasing butyrate), but the ratio of But:Ac does i.e. all that has happened is acetate levels have dropped. So statements like Line 158 '…suggesting that AcGGM inclusion promotes butyrogenic fermentation' are not actually true. AcGGM supplementation actually results in lower levels of total SCFAs (driven by lower acetate) vs no supplementation. In addition the authors have not explored or at least mentioned the effects supplementation has on propionate levels. Why is this? It should at least be
shown and ideally discussed. RESPONSE: We realize that this was a little imprecisely written and have made some changes to comply with the comments from reviewer #2.
Original text: "Measurements of major short-chain fatty acids (SCFAs) in the cecum and colon showed a trend of incremental increases of absolute butyrate levels as AcGGM levels were increased (Fig. 2). Butyrate is commonly measured in the colon at levels that are approximately one-third of acetate, the most abundant SCFA. Compared to control animals, the mean molar ratio of butyrate to acetate in samples from pigs fed 4% AcGGM increased in both the cecum (from 0.29:1 to 0.37:1 mM, p-value: 0.03) and colon (from 0.35:1 to 0.45:1 mM, p-value: 0.002) (Supplementary Dataset 1), suggesting that AcGGM inclusion promotes butyrogenic fermentation." Changed to (L152): "Measurements of major short chain fatty acids (SCFAs) in the cecum and colon showed a trend of incremental increases of absolute and relative butyrate levels as AcGGM levels were increased (Fig.  2). However, despite SCFA data suggesting that AcGGM inclusion promotes butyrogenic fermentation, it also showed that there was no statistically significant increase in total SCFA levels ( Supplementary  Dataset 1). Similarly, the levels of propionic acid were not affected by AcGGM inclusion (Supplementary Dataset 1). While changes in SCFA and microbiome composition (Fig. 2-3) resulted from AcGGM inclusion, we observed no adverse effects on the host's physiology, with the average weight, feed conversion ratio, blood cell composition, T cell population, and colon morphology not differing between the control and AcGGM treatments ( Supplementary Fig. 1, Supplementary Dataset  1)."