Bile salt hydrolase acyltransferase activity expands bile acid diversity.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Therapeutic Effects of the In Vitro Cultured Human Gut Microbiota as Transplants on Altering Gut Microbiota and Improving Symptoms Associated with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a brain-based neurodevelopmental disorder characterized by behavioral abnormalities. Accumulating studies show that the gut microbiota plays a vital role in the pathogenesis of ASD, and gut microbiota transplantation (GMT) is a promising technique for the treatment of ASD. In clinical applications of GMT, it is challenging to obtain effective transplants because of the high costs of donor selection and heterogeneity of donors' gut microbiota, which can cause different clinical responses. In vitro batch culture is a fast, easy-to-operate, and repeatable method to culture gut microbiota. Thus, the present study investigates the feasibility of treating ASD with in vitro cultured gut microbiota as transplants. We cultured gut microbiota via the in vitro batch culture method and performed GMT in the maternal immune activation (MIA)-induced ASD mouse model with original donor microbiota and in vitro cultured microbiota. Open field, three-chamber social, marble burying, and self-grooming tests were used for behavioral improvement assessment. Serum levels of chemokines were detected. Microbial total DNA was extracted from mouse fecal samples, and 16S rDNA was sequenced using Illumina. Our results showed that GMT treatment with original and cultured donor gut microbiota significantly ameliorated anxiety-like and repetitive behaviors and improved serum levels of chemokines including GRO-α (CXCL1), MIP-1α (CCL3), MCP-3 (CCL7), RANTES (CCL5), and Eotaxin (CCL11) in ASD mice. Meanwhile, the gut microbial communities of the two groups that received GMT treatment were changed compared with the ASD mice groups. In the group treated with in vitro cultured donor gut microbiota, there was a significant decrease in the relative abundance of key differential taxa, including S24-7, Clostridiaceae, Prevotella_other, and Candidatus Arthromitus. The relative abundance of these taxa reached close to the level of healthy mice. Prevotella_other also decreased in the group treated with original donor gut microbiota, with a significant increase in Ruminococcaceae and Oscillospira. The present study demonstrated that GMT with in vitro cultured microbiota also improved behavioral abnormalities and chemokine disorders in an ASD mouse model compared with GMT with original donor gut microbiota. In addition, it significantly modified several key differential taxa in gut microbial composition.

Metabolic profiles analysis of 1,3-propanediol production process by Clostridium butyricum through repeated batch fermentation coupled with activated carbon adsorption.

1,3-propanediol production by Clostridium butyricum is a low productivity process due to the long time seed cultivation and thus hinders its industrial scale production. In the present study, repeated batch fermentation coupled with activated carbon adsorption strategy was first established which conduced not only to saving the time of seed cultivation and enhancing the productivity, but also to reducing the costs for the seed cultivation to achieve the purpose of 1,3-propanediol continuous production. The concentration of 1,3-propanediol from first to fourth cycle was 42.89, 45.78, 44.48, 42.39 (g/L), and the corresponding volumetric productivity was 2.14, 1.91, 1.85, 2.12 (g/L · h-1 ) respectively. More importantly, a relatively complete schematic diagram of the proposed metabolic pathways was firstly mapped out based on the intracellular metabolites analysis through GC-MS. At the same time, metabolic pathway and principal components analyses were carried out to give us deep insight into metabolic state. Many metabolites occurred to response to the stress in Cycle II. Even resting body formed and lipid accumulated owing to the worsening environment in the group without activated carbon in Cycle III. Thus, it demonstrated that activated carbon provided a favorable microenvironment for Clostridium butyricum in the repeated batch fermentation process to achieve the purpose of 1,3-propanediol continuous production.

The bioenergetics response of the coral Pocillopora damicornis to temperature changes during its reproduction stage.

Sexual reproduction of reef-building corals is vital for coral reef ecosystem recovery. Corals allocate limited energy to growth and reproduction, when being under environmental disturbance, which ultimately shapes the community population dynamics. In the present study, energetic and physiological parameters of both parental colonies and larvae of the coral Pocillopora damicornis were measured during their reproduction stage under four temperatures; 28 °C (low-temperature acclimation, LA), 29 °C (control temperature, CT), 31 °C (high-temperature acclimation, HA), and 32 °C (heat stress, HS). The results showed temperature changes altered the larvae release timing and fecundity in P. damicornis. Parental colonies exposed to the LA treatment exhibited reduced investment in reproduction and released fewer larvae, while retaining more energy for their development. However, each larva acquired higher energy and symbiont densities enabling survival through longer planktonic periods before settlement. In contrast, parental colonies exposed to the HA treatment had increased investment for reproduction and larvae output, while per larva gained less energy to mitigate the threat of higher temperature. Furthermore, the energy allocation processes restructured fatty acids concentration and composition in both parental colonies and larvae as indicated by shifts in membrane fluidity under adaptable temperature changes. Notably, parental colonies from the HS treatment expended more energy in response to heat stress, resulting in adverse effects, especially after larval release. Our study expands the current knowledge on the energy allocation strategies of P. damicornis and how it is impacted by temperature. Parental colonies employed different energy allocation strategies under distinct temperature regimes to optimize their development and offspring success, but under heat stress, both were compromised. Lipid metabolism is essential for the success of coral reproduction and further understanding their response to heat stress can improve intervention strategies for coral reef conservation in warmer future oceans.

Responses of human gut microbiota abundance and amino acid metabolism in vitro to berberine.

The intestine is a potential location for berberine (BBR) to exert its therapeutic effects, but the understanding of the influences of BBR on the gut microbiota is limited. Through in vitro fermentation of human intestinal microbiota, we investigated the effects of BBR on microbiota composition and metabolism. The result indicated that BBR reduced the production of acetic acid and propionic acid and had no effect on the content of butyric acid. Analysis of the 16S rRNA gene-based community revealed that BBR increased the abundance of Faecalibacterium and decreased the abundance of Bifidobacterium, Streptococcus and Enterococcus. Through metabolomics analysis, BBR treatment regulated various amino acid metabolism pathways of intestinal microbiota, especially tyrosine, serine and L-glutamate. Our study presented direct impacts of BBR on the intestinal microbiota, which provided the probable targets of the therapies by BBR and supported further exploration of the underlying mechanisms.

Identification and antioxidant activity of bovine bone collagen-derived novel peptides prepared by recombinant collagenase from Bacillus cereus.

Millions of tons of collagen-rich bovine bone are produced as byproducts of the consumption of beef. Hydrolyzing bovine bone collagen (BBC) is an effective measure for both increasing its added value and protecting the environment. In this study, a kind of recombinant bacterial collagenase mining from Bacillus cereus was successfully performed and applied to hydrolyze BBC to collagen-soluble peptides (CPP). Response surface methodology (RSM) was applied to optimize the processing conditions of antioxidant CPP, attaining a distinguished ABTS free radical scavenging activity of 99.21 ± 0.35% while keeping DPPH free radical scavenging activity and reducing power at high levels under the optimal condition. Furthermore, we identified five new antioxidant peptides by LC-MS/MS with typical collagen repeated Gly-Xaa-Yaa sequence units within the CPP. These results suggest that our recombinant collagenase is a powerful tool for degrading collagen and the CPP are promising candidates for antioxidant and related functional food applications.

Preservation of the fecal samples at ambient temperature for microbiota analysis with a cost-effective and reliable stabilizer EffcGut.

With the increasing researches on the role of gut microbiota in human health and disease, appropriate storage method of fecal samples at ambient temperature would conveniently guarantee the precise and reliable microbiota results. Nevertheless, less choice of stabilizer that is cost-efficient and feasible to be used in longer preservation period obstructed the large-scale metagenomics studies. Here, we evaluated the efficacy of a guanidine isothiocyanate-based reagent method EffcGut and compared it with the other already used storage method by means of 16S rRNA gene sequencing technology. We found that guanidine isothiocyanate-based reagent method at ambient temperature was not inferior to OMNIgene·GUT OM-200 and it could retain the similar bacterial community as that of -80 °C within 24 weeks. Furthermore, bacterial diversity and community structure difference were compared among different sample fraction (supernatant, suspension and precipitate) preserved in EffcGut and -80 °C. We found that supernatant under the preservation of EffcGut retained the similar community structure and composition as that of the low temperature preservation method.