Butyrate Synthesis Research Articles

A tailored series of engineered yeasts for the cell-dependent treatment of inflammatory bowel disease by rational butyrate supplementation

ABSTRACT Intestinal microbiota dysbiosis and metabolic disruption are considered essential characteristics in inflammatory bowel disorders (IBD). Reasonable butyrate supplementation can help patients regulate intestinal flora structure and promote mucosal repair. Here, to restore microbiota homeostasis and butyrate levels in the patient’s intestines, we modified the genome of Saccharomyces cerevisiae to produce butyrate. We precisely regulated the relevant metabolic pathways to enable the yeast to produce sufficient butyrate in the intestine with uneven oxygen distribution. A series of engineered strains with different butyrate synthesis abilities was constructed to meet the needs of different patients, and the strongest can reach 1.8 g/L title of butyrate. Next, this series of strains was used to co-cultivate with gut microbiota collected from patients with mild-to-moderate ulcerative colitis. After receiving treatment with engineered strains, the gut microbiota and the butyrate content have been regulated to varying degrees depending on the synthetic ability of the strain. The abundance of probiotics such as Bifidobacterium and Lactobacillus increased, while the abundance of harmful bacteria like Candidatus Bacilloplasma decreased. Meanwhile, the series of butyrate-producing yeast significantly improved trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice by restoring butyrate content. Among the series of engineered yeasts, the strain with the second-highest butyrate synthesis ability showed the most significant regulatory and the best therapeutic effect on the gut microbiota from IBD patients and the colitis mouse model. This study confirmed the existence of a therapeutic window for IBD treatment by supplementing butyrate, and it is necessary to restore butyrate levels according to the actual situation of patients to restore intestinal flora.

The gut microbiome regulates the clinical efficacy of sulfasalazine therapy for IBD-associated spondyloarthritis

Sulfasalazine is a prodrug known to be effective for the treatment of inflammatory bowel disease (IBD)-associated peripheral spondyloarthritis (pSpA), but the mechanistic role for the gut microbiome in regulating its clinical efficacy is not well understood. Here, treatment of 22 IBD-pSpA subjects with sulfasalazine identifies clinical responders with a gut microbiome enriched in Faecalibacterium prausnitzii and the capacity for butyrate production. Sulfapyridine promotes butyrate production and transcription of the butyrate synthesis gene but in F. prausnitzii invitro, which is suppressed by excess folate. Sulfasalazine therapy enhances fecal butyrateproduction and limits colitis in wild-type and gnotobiotic mice colonized with responder, but not non-responder, microbiomes. F. prausnitzii is sufficient to restore sulfasalazine protection from colitis in gnotobiotic mice colonized with non-responder microbiomes. These findings reveal a mechanistic link between the efficacy of sulfasalazine therapy and the gut microbiome with the potential to guide diagnostic and therapeutic approaches for IBD-pSpA.

Microbial butyrate capacity is reduced in inflamed mucosa in patients with ulcerative colitis

Reduced butyrate-production capacity has been reported in fecal microbial communities in patients with active ulcerative colitis. However, the butyrate-production capacity of the mucosal microbiome from active vs quiescent mucosa in ulcerative colitis has been unexplored. We sought to determine the diversity and relative abundance of mucosal bacterial and fungal communities from endoscopically active vs quiescent mucosa in patients with UC, and aimed to predict contributions of mucosal microbial communities to butyrate synthesis. Systematic, segmental right- and left-sided biopsies were obtained from endoscopically active (n = 13) or quiescent (n = 17) colonic mucosa, among 15 patients with pan-colonic ulcerative colitis. Dietary fiber intake of patients was performed using the validated five-item FiberScreen questionnaire. Amplicon sequencing of mucosal bacteria and fungi was performed. The diversity and relative abundance of mucosal bacterial and fungal taxa were quantified, and predicted contributions to butyrate synthesis were ascertained. Bacterial alpha and beta diversity were similar between active vs quiescent mucosa. Butyrogenic taxa were significantly increased in quiescence, including Butyricimonas, Subdoligranulum, and Alistipes. Predicted butyrate kinase activity was significantly and concomitantly increased in quiescent mucosa. Fiber intake was positively correlated with butyrogenic microbes. Compared to mucosal bacterial prevalence, mucosal fungi were detected in low prevalence. Butyrogenic microbes are relatively increased in quiescent mucosa in ulcerative colitis, and may be related to increased fiber intake during quiescence. Manipulation of the mucosal microbiome towards butyrate-producing bacteria may be associated with endoscopic quiescence.

Differences in the gut microbiome of young adults with schizophrenia spectrum disorder: using machine learning to distinguish cases from controls

While an association between the gut microbiome and schizophrenia spectrum disorders (SSD) has been suggested, the existing evidence is still inconclusive. To this end, we analyzed bacteria and bacterial genes in feces from 52 young adult SSD patients and 52 controls using fecal shotgun metagenomic sequencing. Compared to controls, young SSD patients were found to have significantly lower α-diversity and different β-diversity both regarding bacterial species (i.e., taxonomic diversity) and bacterial genes (i.e., functional diversity). Furthermore, the α-diversity measures ‘Pielou’s evenness’ and ‘Shannon’ were significantly higher for both bacterial species, bacterial genes encoding enzymes and gut brain modules in young SSD patients on antipsychotic treatment (young SSD not on antipsychotics=9 patients, young SSD on antipsychotics=43 patients). We also applied machine learning classifiers to distinguish between young SSD patients and healthy controls based on their gut microbiome. Results showed that taxonomic and functional data classified young SSD individuals with an accuracy of ≥ 70% and with an area under the receiver operating characteristic curve (AUROC) of ≥ 0.75. Differential abundance analysis on the most important features in the classifier models revealed that most of the species with higher abundance in young SSD patients had their natural habitat in the oral cavity. In addition, many of the modules with higher abundance in young SSD patients were amino acid biosynthesis modules. Moreover, the abundances of gut-brain modules of butyrate synthesis and acetate degradation were lower in the SSD patients compared to controls. Collectively, our findings continue to support the presence of gut microbiome alterations in SSD and provide support for the use of machine learning algorithms to distinguish patients from controls based on gut microbiome profiles.

DOP39 Constructing butyrate-producing yeast as a cell-dependent treatment option for Inflammatory Bowel Disease

Abstract Background Intestinal microbiota dysbiosis and metabolic disruption are essential characteristics of inflammatory bowel disorders (IBD). Butyrate has been proven to regulate immune response and gut microbiota. It has been confirmed that the butyrate content in the intestinal cavity of IBD patients decreases (Fig. 1A). Methods In this study, gene editing was used to reconstruct the butyrate synthesis pathway and regulate related metabolic pathways in Saccharomyces cerevisiae to produce butyrate and enrich its synthesis in anaerobic environments to cope with complex intestinal conditions. A series of engineered strains were co-cultured with gut microbiota samples from six patients with mild-to-moderate ulcerative colitis and 16S ribosomal RNA sequencing was used to analyze the regulatory effect of engineered strains on intestinal microbiota in vitro. The TNBS-induced IBD mice model was used to explore the protective effect of butyrate-engineered yeasts in vivo. Results A series of engineered strains with different butyrate synthesis abilities was constructed to meet the needs of different patients. S. cerevisiae was engineered by introducing an exogenous butyrate synthesis pathway and regulating yeast metabolism to increase the availability of the precursors. Engineered yeast can produce up to 1.8 g/L butyrate (Fig. 1B). The modification of related metabolic pathways also focused on enhancing the synthesis of butyrate under anaerobic conditions to enable engineered yeasts to adapt to the intestinal environment. This series of engineered yeasts with different butyrate synthesis abilities regulated the intestinal microbiota of IBD patients to varying degrees, enhancing the abundance of related probiotics such as Lactobacillus and Bifidobacterium and reducing the abundance of harmful bacteria like Candidatus Bacilloplasma (Fig. 1C). Meanwhile, strains improved TNBS-induced colitis in mice by restoring butyrate content. Remarkably, the engineered strain with the second-highest butyrate yield showed the most significant therapeutic effect (Fig. 1D). Conclusion This study realized butyrate synthesis in S. cerevisiae and regulated related metabolic modules to adjust the butyrate production to achieve the best therapeutic effect on colitis in vivo and in vitro. In addition, the strain with the second-highest butyrate production has the best therapeutic effect. This highlights the dual role of butyrate in epithelial cell repair and suggests a therapeutic window for IBD treatment through butyrate supplementation. Selecting the most suitable strain for each patient's condition is essential for restoring butyrate levels, regulating microbiota, and promoting mucosal repair.

Tributyrin oral administration significantly attenuates the loss of intestinal butyrate‐producing bacteria and the ensuing cognitive dysfunction associated with Alzheimer’s Disease pathogenesis in 3xTg‐AD mice

AbstractBackgroundAlzheimer’s Disease (AD) development is linked to the loss of overall gut microbial diversity. However, the specific alterations are only beginning to be examined. In this regard, decreases in intestinal butyrate‐producing bacteria have been indicated by pre‐clinical and clinical studies in AD. The current work, employing the 3xTg‐AD mouse model examined the causal association of loss of butyrate‐producing bacterial communities with cognitive dysfunction associated with AD pathogenesis.MethodCecal samples from tributyrin (TB) treated and untreated female 3xTg‐AD mice were used to perform metataxonomic analysis by sequencing the 16S rRNA gene (V4 region) on a large‐scale Illumina MiSeq platform. Inferred metagenomics analysis was performed using the PICRUSt2 pipeline. TB treatment (oral gavage, 2mg/kg, twice a week) was initiated at 6‐months of age until 17‐months. Spatial and short‐term memory functions were tested using the Y‐maze and Novel Object Recognition Test (NORT).ResultMetataxonomic analysis showed an age‐dependent, significant decline in alpha diversity represented by measures of OTUs, Chao‐1 and Shannon Index from 2‐ to 18‐months. Additionally, age‐dependent changes in gut microbial composition were demonstrated by a significant decrease in the Firmicutes‐to‐Bacteroidota (F/B) ratio. Further analysis of specific features of the gut microbiome demonstrated an age‐dependent decrease in total butyrate‐producing families within the Firmicutes phylum; particularly, a significant loss of critical butyrate producing family Ruminococcacea was identified. Predictive functional analysis using PICRUSt2 also showed a significant reduction in the abundance of specific bacterial genes involved in butyrate synthesis. Importantly, these age‐dependent markers of gut microbial dysbiosis coincided with diminished measures of spatial and short‐term memory. Notably, administration of TB, a butyrate pro‐drug, significantly attenuated the age‐dependent (i) decline in gut microbial diversity & butyrate‐producing bacteria and (ii) loss of spatial and short‐term memory.ConclusionThe findings from this study identify that decline in total butyrate‐producing families is a key pathogenic feature of AD‐associated microbial dysbiosis and cognitive dysfunction. Importantly, these findings have the potential to inform the development of treatment strategies targeting gut microbial dysbiosis using TB, to prevent onset and development of cognitive decline in AD.

Engraftment of essential functions through multiple fecal microbiota transplants in chronic antibiotic-resistant pouchitis—a case study using metatranscriptomics

BackgroundIleal pouch-anal anastomosis (IPAA) is the standard of care after total proctocolectomy for ulcerative colitis (UC). Around 50% of patients will experience pouchitis, an idiopathic inflammatory condition. Antibiotics are the backbone of treatment of pouchitis; however, antibiotic-resistant pouchitis develops in 5–10% of those patients. It has been shown that fecal microbiota transplantation (FMT) is an effective treatment for UC, but results for FMT antibiotic-resistant pouchitis are inconsistent.MethodsTo uncover which metabolic activities were transferred to the recipients during FMT and helped the remission, we performed a longitudinal case study of the gut metatranscriptomes from three patients and their donors. The patients were treated by two to three FMTs, and stool samples were analyzed for up to 140 days.ResultsReduced expression in pouchitis patients compared to healthy donors was observed for genes involved in biosynthesis of amino acids, cofactors, and B vitamins. An independent metatranscriptome dataset of UC patients showed a similar result. Other functions including biosynthesis of butyrate, metabolism of bile acids, and tryptophan were also much lower expressed in pouchitis. After FMT, these activities transiently increased, and the overall metatranscriptome profiles closely mirrored those of the respective donors with notable fluctuations during the subsequent weeks. The levels of the clinical marker fecal calprotectin were concordant with the metatranscriptome data. Faecalibacterium prausnitzii represented the most active species contributing to butyrate synthesis via the acetyl-CoA pathway. Remission occurred after the last FMT in all patients and was characterized by a microbiota activity profile distinct from donors in two of the patients.ConclusionsOur study demonstrates the clear but short-lived activity engraftment of donor microbiota, particularly the butyrate biosynthesis after each FMT. The data suggest that FMT triggers shifts in the activity of patient microbiota towards health which need to be repeated to reach critical thresholds. As a case study, these insights warrant cautious interpretation, and validation in larger cohorts is necessary for generalized applications. In the long run, probiotics with high taxonomic diversity consisting of well characterized strains could replace FMT to avoid the costly screening of donors and the risk of transferring unwanted genetic material.1wvY14mjpJT86R5RsB8tdiVideo

Investigation of the Relationship Between Serum Low-Density Lipoprotein Cholesterol Levels with Genetic Polymorphisms, Gut Microbiota, and Nutrition.

Background: To prevent cardiovascular disease (CVD), it is important to determine the factors that are associated with its development. High serum low-density lipoprotein (LDL) cholesterol (LDL-C) levels are a modifiable prevention and treatment target known to contribute to the development of CVD, but the factors affecting blood cholesterol levels, including LDL-C, remain controversial. Objective: In this study, the factors (genetic, nutritional, and gut microbiota) thought to be effective on serum LDL-C levels were discussed from a holistic perspective, and the effects of the relationship between these factors on LDL-C levels were examined. Methods: The study was carried out with 609 adults (48% male) who applied to a private health institution between 2016 and 2022. Results: It was observed that serum LDL-C levels were positively correlated with body mass index (BMI) (P = 0.000) and different ApoE alleles had significant effects on LDL-C levels. It was observed that the highest LDL-C levels were in the ɛ4+ group, followed by ɛ3+ and ɛ2+ groups, respectively (P = 0.000). Results showed that dietary cholesterol and fiber consumption did not significantly affect serum LDL-C levels (P = 0.705 and P = 0.722, respectively). It was also observed that enterotypes and the butyrate synthesis potential of intestinal microbiota did not cause significant changes in serum LDL-C levels (P = 0.369 and P = 975, respectively). Conclusion: Serum LDL-C levels are affected by modifiable factors such as BMI and nonmodifiable factors such as APOE genotype. By identifying these factors and conducting further studies on them, new ways to improve serum LDL-C levels, which is an important factor in the development of CVD, can be identified. In addition, no significant effect of gene-nutrient or microbiota-nutrient interactions on serum LDL-C levels was detected. Further research is needed, especially on the relationship between intestinal microbiota and serum LDL levels.

Mining and application of lipase from Clostridium acetobutylicum with higher catalytic activity for butyl butyrate production

Butyl butyrate is extensively applied in beverage, fragrances, food and daily cosmetics industries. However, the most commonly used commercial enzymes for butyl butyrate biosynthesis have low specificity as they can catalyze the synthesis of a variety of short-chain fatty acid esters (SCFAE). Therefore, it is necessary to find lipases with higher specificity for the esterification synthesis of butyl butyrate. In this study, the endogenous lipase of Clostridium acetobutylicum was first excavated and analyzed, and then successfully displayed on the surface of Escherichia coli. When it was applied in butyl butyrate synthesis, its catalytic efficiency reached 85 % of that of commercial enzyme. Importantly, its specificity for butyl butyrate synthesis was higher, and lower titer of by-products was generated than commercial lipase. Additionally, the catalytic activity lasted for more than 35 days when it was immobilized by sodium alginate hydrogel. The successful cell surface-displayed of the lipase can effectively eliminate the addition of commercial lipase in the process of butyl butyrate biosynthesis.

Effects of cashew nutshell extract and monensin on microbial fermentation in a dual-flow continuous culture

The objective of this study was to compare cashew nutshell extract (CNSE) to monensin and evaluate changes in in vitro mixed ruminal microorganism fermentation, nutrient digestibility, and microbial nitrogen outflow. Treatments were randomly assigned to 8 fermenters in a replicated 4 × 4 Latin square design with 4 experimental periods of 10 d (7 d for diet adaptation and 3 d for sample collection). Basal diets contained 43.5:56.5 forage: concentrate ratio and each fermenter was fed 106 g of DM/d divided equally between 2 feeding times. Treatments were control (CON, basal diet without additives), 2.5 μM monensin (MON), 0.1 mg CNSE granule/g DM (CNSE100), and 0.2 mg CNSE granule/g DM (CNSE200). On d 8 to10, samples were collected for pH, lactate, NH3-N, volatile fatty acids (VFA), mixed protozoa counts, organic matter (OM), and neutral detergent fiber (NDF) digestibility. Data were analyzed with the GLIMMIX procedure of SAS. Orthogonal contrasts were used to test the effects of (1) ADD (CON vs. MON, CNSE100, and CNSE200); (2) MCN (MON vs. CNSE100 and CNSE200); and (3) DOSE (CNSE100 vs. CNSE200). We observed that butyrate concentration in all treatments was lower compared with CON and the concentration for MON was lower compared with CNSE treatments. Protozoal population in all treatments was lower compared with CON. No effects were observed for pH, lactate, NH3-N, total VFA, OM, or N utilization. Within the 24-h pool, protozoal generation time, tended to be lower, while NDF digestibility tended to be greater in response to all additives. Furthermore, the microbial N flow, and the efficiency of N use tended to be lower for the monensin treatment compared with CNSE treatments. Overall, our results showed that both monensin and CNSE decreased butyrate synthesis and protozoal populations, while not affecting OM digestibility and tended to increase NDF digestibility; however, such effects are greater with monensin than CNSE nutshell.

Enzymatic synthesis of citronellyl butyrate by lipase B from Candida antarctica immobilized on magnetic cashew apple bagasse lignin

The enzymatic biotransformation of terpenic alcohols allows the selective production of new compounds with improved bioactivity. However, few studies are performed using enzymes mainly immobilized on natural material. In this context, Candida antarctica lipase B (CAL-B) was immobilized on lignin extracted from cashew apple bagasse conjugated with magnetic nanoparticles, and used in the synthesis of citronellyl butyrate. The immobilization process took place predominantly by adsorption and interfacial activation, followed by covalent bonds. The enzymatic load of 5 mgPROTEIN/gSUPPORT was chosen because it generated a catalyst (Lig-MNPs_CALB) with the highest catalytic activity (12.6 U/g) and yield of 71.5%. Lig-MNPs_CALB maintained 65% of its activity after 96 h of exposure at 50 °C and 73% after 60 days stored at 4 °C. In the synthesis of citronellyl butyrate by CAL-B in both forms, the optimal esterification conditions were 50 °C, 1:1 molar ratio (alcohol:acid), 10 mgPROTEIN/mL and 200 RPM. The esterification yield was higher in the process catalyzed by Lig-MNPs_CALB (96.4%) compared to catalyzed by free CAL-B (55%) at 24 h, and the Lig-MNPs_CALB maintained its activity for 10 cycles. Also, the antibacterial activity of citronellol in its esterified form has been improved, and citronellyl butyrate showed antifungal activity.

Quantitative Proteomic Analysis of Outer Membrane Vesicles from Fusobacterium nucleatum Cultivated in the Mimic Cancer Environment.

Fusobacterium nucleatum is a Gram-negative bacterium that has been identified as an important pathogenic gut bacterium associated with colorectal cancer. Compared with the normal intestine, the pH value of the tumor microenvironment is weakly acidic. The metabolic changes of F. nucleatum in the tumor microenvironment, especially the protein composition of its outer membrane vesicles, remain unclear. Here, we systematically analyzed the effect of environmental pH on the proteome of outer membrane vesicles (OMVs) from F. nucleatum by tandem mass tag (TMT) labeling-high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 991 proteins were identified in acidic OMVs (aOMVs) and neutral OMVs (nOMVs), including known virulence proteins and putative virulence proteins. Finally, 306 upregulated proteins and 360 downregulated proteins were detected in aOMVs, and approximately 70% of the expression of OMV proteins was altered under acidic conditions. A total of 29 autotransporters were identified in F. nucleatum OMVs, and 13 autotransporters were upregulated in aOMVs. Interestingly, three upregulated autotransporters (D5REI9, D5RD69, and D5RBW2) show homology to the known virulence factor Fap2, suggesting that they may be involved in various pathogenic pathways such as the pathway for binding with colorectal cancer cells. Moreover, we found that more than 70% of MORN2 domain-containing proteins may have toxic effects on host cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that a number of proteins were significantly enriched in multiple pathways involving fatty acid synthesis and butyrate synthesis. Seven metabolic enzymes involved in fatty acid metabolism pathways were identified in the proteomic data, of which 5 were upregulated and 2 were downregulated in aOMVs, while 14 metabolic enzymes involved in the butyric acid metabolic pathway were downregulated in aOMVs. In conclusion, we found a key difference in virulence proteins and pathways in the outer membrane vesicles of F. nucleatum between the tumor microenvironment pH and normal intestinal pH, which provides new clues for the prevention and treatment of colorectal cancer. IMPORTANCE F. nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. OMVs have been demonstrated to play key roles in pathogenesis by delivering toxins and other virulence factors to host cells. By employing quantitative proteomic analysis, we found that the pH conditions could affect the protein expression of the outer membrane vesicles of F. nucleatum. Under acidic conditions, approximately 70% of the expression of proteins in OMVs was altered. Several virulence factors, such as type 5a secreted autotransporter (T5aSSs) and membrane occupation and recognition nexus (MORN) domain-containing proteins, were upregulated under acidic conditions. A large number of proteins showed significant enrichments in multiple pathways involving fatty acid synthesis and butyrate synthesis. Proteomics analysis of the outer membrane vesicles secreted by pathogenic bacteria in the acidic tumor microenvironment is of great significance for elucidating the pathogenicity mechanism and its application in vaccine and drug delivery vehicles.

Modulation of the gut microbiome with nisin

Nisin is a broad spectrum bacteriocin used extensively as a food preservative that was identified in Lactococcus lactis nearly a century ago. We show that orally-ingested nisin survives transit through the porcine gastrointestinal tract intact (as evidenced by activity and molecular weight determination) where it impacts both the composition and functioning of the microbiota. Specifically, nisin treatment caused a reversible decrease in Gram positive bacteria, resulting in a reshaping of the Firmicutes and a corresponding relative increase in Gram negative Proteobacteria. These changes were mirrored by the modification in relative abundance of pathways involved in acetate, butyrate (decreased) and propionate (increased) synthesis which correlated with overall reductions in short chain fatty acid levels in stool. These reversible changes that occur as a result of nisin ingestion demonstrate the potential of bacteriocins like nisin to shape mammalian microbiomes and impact on the functionality of the community.

Gut Microbial Metabolite Butyrate and Its Therapeutic Role in Inflammatory Bowel Disease: A Literature Review.

Background and objective: Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a chronic inflammatory disorder characterized by aberrant immune responses and compromised barrier function in the gastrointestinal tract. IBD is associated with altered gut microbiota and their metabolites in the colon. Butyrate, a gut microbial metabolite, plays a crucial role in regulating immune function, epithelial barrier function, and intestinal homeostasis. In this review, we aim to present an overview of butyrate synthesis and metabolism and the mechanism of action of butyrate in maintaining intestinal homeostasis and to discuss the therapeutic implications of butyrate in IBD. Methods: We searched the literature up to March 2023 through PubMed, Web of Science, and other sources using search terms such as butyrate, inflammation, IBD, Crohn's disease, and ulcerative colitis. Clinical studies in patients and preclinical studies in rodent models of IBD were included in the summary of the therapeutic implications of butyrate. Results: Research in the last two decades has shown the beneficial effects of butyrate on gut immune function and epithelial barrier function. Most of the preclinical and clinical studies have shown the positive effect of butyrate oral supplements in reducing inflammation and maintaining remission in colitis animal models and IBD patients. However, butyrate enema showed mixed effects. Butyrogenic diets, including germinated barley foodstuff and oat bran, are found to increase fecal butyrate concentrations and reduce the disease activity index in both animal models and IBD patients. Conclusions: The current literature suggests that butyrate is a potential add-on therapy to reduce inflammation and maintain IBD remission. Further clinical studies are needed to determine if butyrate administration alone is an effective therapeutic treatment for IBD.

De novo biosynthesis of butyl butyrate in engineered Clostridium tyrobutyricum

Butyl butyrate has broad applications in foods, cosmetics, solvents, and biofuels. Microbial synthesis of bio-based butyl butyrate has been regarded as a promising approach recently. Herein, we engineered Clostridium tyrobutyricum ATCC 25755 to achieve de novo biosynthesis of butyl butyrate from fermentable sugars. Through introducing the butanol synthetic pathway (enzyme AdhE2), screening alcohol acyltransferases (AATs), adjusting transcription of VAAT and adhE2 (i.e., optimizing promoter), and efficient supplying butyryl-CoA, an excellent engineered strain, named MUV3, was obtained with ability to produce 4.58 g/L butyl butyrate at 25 °C with glucose in serum bottles. More NADH is needed for butyl butyrate synthesis, thus mannitol (the more reduced substrate) was employed to produce butyl butyrate. Ultimately, 62.59 g/L butyl butyrate with a selectivity of 95.97%, and a yield of 0.21 mol/mol was obtained under mannitol with fed-batch fermentation in a 5 L bioreactor, which is the highest butyl butyrate titer reported so far. Altogether, this study presents an anaerobic fermentative platform for de novo biosynthesis of butyl butyrate in one step, which lays the foundation for butyl butyrate biosynthesis from renewable biomass feedstocks.

Construction of a Microbial Consortium for the De Novo Synthesis of Butyl Butyrate from Renewable Resources.

Butyl butyrate has shown wide applications in food, cosmetic, and biofuel sectors. Currently, biosynthesis of butyl butyrate still requires exogenous addition of precursors and lipase, which increases the production cost and limits further large-scale development. In this study, a microbial consortium was first designed to realize direct butyl butyrate production from lignocellulose. The highest butyl butyrate concentration of 34.42 g/L was detected in the solvent phase from 60 g/L glucose using a microbial coculture system composed of Clostridium acetobutylicum NJ4 and Clostridium tyrobutyricum LD with the elimination of butyric acid supplementation. Meanwhile, 13.52 g/L butyl butyrate was synthesized from 60 g/L glucose using a microbial consortium composed of three strains including strain NJ4, strain LD, and Escherichia coli BL21- pET-29a(+)-LE without the addition of any exogenous precursors and lipase. In addition, 2.94 g/L butyl butyrate could be directly produced from 60 g/L microcrystalline cellulose when Trichoderma asperellum was added to the above-mentioned three-strain microbial consortium. This four-strain microbial consortium represents the first study regarding the direct butyl butyrate production from lignocellulose without the supplementation of exogenous precursors and lipase, which may be extended to the biosynthesis of other short-chain esters, such as ethyl acetate and butyl lactate.

Deficient butyrate-producing capacity in the gut microbiome is associated with bacterial network disturbances and fatigue symptoms in ME/CFS

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by unexplained debilitating fatigue, cognitive dysfunction, gastrointestinal disturbances, and orthostatic intolerance. Here, we report a multi-omic analysis of a geographically diverse cohort of 106 cases and 91 healthy controls that revealed differences in gut microbiome diversity, abundances, functional pathways, and interactions. Faecalibacterium prausnitzii and Eubacterium rectale, which are both recognized as abundant, health-promoting butyrate producers in the human gut, were reduced in ME/CFS. Functional metagenomics, qPCR, and metabolomics of fecal short-chain fatty acids confirmed a deficient microbial capacity for butyrate synthesis. Microbiome-based machine learning classifier models were robust to geographic variation and generalizable in a validation cohort. The abundance of Faecalibacterium prausnitzii was inversely associated with fatigue severity. These findings demonstrate the functional nature of gut dysbiosis and the underlying microbial network disturbance in ME/CFS, providing possible targets for disease classification and therapeutic trials.

Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum

Acetogenic bacteria are a unique biocatalyst that highly promises to develop the sustainable bioconversion of carbon oxides (e.g., CO and CO2) into multicarbon biochemicals. Genotype-phenotype relationships are important for engineering their metabolic capability to enhance their biocatalytic performance; however, systemic investigation on the fitness contribution of individual gene has been limited. Here, we report genome-scale CRISPR interferencescreening using 41,939 guide RNAs designed from the E.limosum genome, one of the model acetogenic species, where all genes were targeted for transcriptional suppression. We investigated the fitness contributions of 96% of the total genes identified, revealing the gene fitness and essentiality for heterotrophic and autotrophic metabolisms. Our data show that the Wood-Ljungdahl pathway, membrane regeneration, membrane protein biosynthesis, and butyrate synthesis are essential for autotrophic acetogenesis in E.limosum. Furthermore, we discovered genes that are repression targets that unbiasedly increased autotrophic growth rates fourfold and acetoin production 1.5-fold compared to the wild-type strain under CO2-H2 conditions. These results provide insight for understanding acetogenic metabolism and genome engineering in acetogenic bacteria.

THE GUT MICROBIOME REGULATES EFFICACY OF SULFASALAZINE THERAPY FOR SPONDYLOARTHRITIS IN INFLAMMATORY BOWEL DISEASE

Abstract Joint inflammation, or spondyloarthritis (SpA), is the most common extra-intestinal manifestation of inflammatory bowel disease (IBD), but the specific role for therapies targeting SpA is not well defined. One of the earliest medications used for the treatment of IBD is sulfasalazine (SAS). SAS is a prodrug composed of two chemical moieties, the anti-inflammatory 5-aminosalicilate and the antibiotic sulfapyridine. The efficacy of SAS in peripheral arthritis is thought to depend on its “antibacterial” properties, however the impact of SAS on the IBD-SpA microbiome and how it may improve extra-intestinal symptoms is unknown. Therefore, our study aims to diagnostically evaluate the role for the fecal microbiome in clinical response to SAS therapy and identify microbial and immunologic therapeutic targets associated with clinical response. We have designed an observational study to longitudinally follow IBD patients with SpA who have a medical indication for SAS therapy. Clinical data and fecal samples from 22 patients were collected before initiation of SAS and at week 12 after initiation of SAS. Eleven IBD-SpA patients were concomitantly enrolled as controls and followed by 12 weeks. Metagenomic sequencing was used to define the effect of SAS on the IBD-SpA fecal microbiome and to evaluate its relationship with improvement in joint symptoms. Mouse models and in vitro assays were used to test the sufficiency of the SAS effect observed in patients. The fecal microbiome of SAS-responders was distinct from that observed in non-responders and 6 pre-treatment microbial markers (including the short chain fatty acid (SCFA) producer Faecalibacterium prausnitzii) predicted SAS-response (AUC = 0.9). Fecal metabolome of SAS responders had lower thymine and higher deoxyuridine compared to non-responders consistent with evidence of a folate trap in response to SAS treatment. SAS therapy in SPF mouse-model of chemically-induced colitis alleviated colitis in GPR 109a-dependent fashion consistent with a synergistic role for SCFA. In vitro assays revealed SAS direct regulation of F. prausnitzii metabolic function and butyrate synthesis. CONCLUSIONS: Collectively, these findings highlight the potential role for microbial diagnostics to improve SAS efficacy, and drug modulation of microbial markers to potentiate therapy for IBD patients with SpA.

The butyrate-producing and spore-forming bacterial genus Coprococcus as a potential biomarker for neurological disorders.

The host-intestinal microbiome interaction has gained much scientific attention in the past two decades, boosted by advances in DNA sequencing and cultivation techniques. An accumulating amount of evidence shows that gut microbes play crucial roles in gut homeostasis, immune system education, and are associated with quality-of-life indicators. Beneficial health factors are associated with the digestion of dietary fibres in the colon and the subsequent production of short-chain fatty acids, including acetate, propionate, and butyrate. Coprococcus is a butyrate-producing genus in the phylum Firmicutes, and its abundance is inversely correlated with several neuropsychological and neurodegenerative disorders. Case-control studies provide strong evidence of decreased abundance of Coprococcus spp. in depressed individuals. The species Coprococcus eutactus has the unique capacity to use two separate pathways for butyrate synthesis and has been found to be depleted in children with delayed language development and adults with Parkinson's disease. The combined literature on Coprococcus and the gut microbiota-brain axis points towards enhanced butyrate production and reduced colonisation of pathogenic clades as factors explaining its association with health effects. The genus Coprococcus is a promising candidate for a mental health biomarker and an interesting lead for novel dietary-based preventive therapies for specific neurological disorders.