Controlled production of a polyhydroxyalkanoate (PHA) tetramer containing different mole fraction of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3 HV), 4 HV and 5 HV units by engineered Cupriavidus necator.

Polyhydroxyalkanoates (PHAs) are promising alternatives to existing petrochemical-based plastics because of their bio-degradable properties. However, the limited structural diversity of PHAs has hindered their application. In this study, high mole-fractions of Poly (39 mol% 3HB-co-17 mol% 3 HV-co-44 mol% 4 HV) and Poly (25 mol% 3HB-co-75 mol% 5 HV) were produced from 4- hydroxyvaleric acid and 5-hydroxyvaleric acid, using Cupriavidus necator PHB-4 harboring the gene phaCBP-M-CPF4 with modified sequences. In addition, the complex toxicity of precursor mixtures was tested, and it was confirmed that the engineered C. necator was capable of synthesizing Poly (32 mol% 3HB-co-11 mol% 3 HV-co-25 mol% 4 HV-co-32 mol% 5 HV) at low mixture concentrations. Correlation analyses of the precursor ratio and the monomeric mole fractions indicated that each mole fractions could be precisely controlled using the precursor proportion. Physical property analysis confirmed that Poly (3HB-co-3 HV-co-4 HV) is a rubber-like amorphous polymer and Poly (3HB-co-5 HV) has a high tensile strength and elongation at break. Poly (3HB-co-3 HV-co-4 HV-co-5 HV) had a much lower glass transition temperature than the co-, terpolymers containing 3 HV, 4 HV and 5 HV. This study expands the range of possible physical properties of PHAs and contributes to the realization of custom PHA production by suggesting a method for producing PHAs with various physical properties through mole-fraction control of 3 HV, 4 HV and 5 HV.

The multiple effects of fecal microbiota transplantation on diarrhea-predominant irritable bowel syndrome (IBS-D) patients with anxiety and depression behaviors.

BACKGROUND: Anxiety and depression are complications in Irritable bowel syndrome (IBS) patients. In this study, we recruited 18 IBS patients with mild-modest anxiety and depression behaviors, and after the screening, we defined the FMT treatment group (n = 9) and the control group (n = 9). The IBS symptom severity scale (IBS-SSS), Hamilton Anxiety Rating Scale (HAM-A), Hamilton Depression Rating Scale (HAM-D), Irritable Bowel Syndrome Quality of Life (IBS-QOL) and Bristol stool scale (BSS) were evaluated one week before FMT (baseline), one-week-, one-month-, two-month-, and three-month-following FMT. Meanwhile, we determined the SCFAs in the patient's feces and serum and continued the metagenomic analysis of the microorganisms in the patient's feces. RESULTS: The results showed that the patient's anxiety and depression behavior gradually improved with FMT treatment. Moreover, the illness and quality of life had also been relieved significantly. The content of isovaleric acid and valeric acid was significantly reduced in the FMT group compared to the Col group. Metagenomic analysis showed that FMT treatment decreased the abundance of Faecalibacterium, Eubacterium and Escherichia. From KEGG functional analysis, we confirmed that the top five abundant pathways were "bacterial chemotaxis, "flagellar assembly", "glycine, serine and threonine metabolism", "apoptosis", and "bacterial invasion of epithelial cells". CONCLUSIONS: FMT treatment can effectively alleviate the anxiety and depression behaviors of IBS-D patients and reduce the IBS-SSS score, indicating that FMT can improve patients' symptoms. The high throughput sequencing results show that Bifidobacterium and Escherichia play the most critical role in the formation and recovery of IBS-D patients. The GC/MS data indicated that faeces isovaleric acid and valeric acid might be more suitable as a metabolic indicator of IBS-D remission. Trial registration ChiCTR, ChiCTR1900024924, Registered 3 August 2019, https://www.chictr.org.cn/showproj.aspx?proj=41676 .

Enantiomers of 4-aminopentanoic acid act as false GABAergic neurotransmitters and impact mouse behavior.

Imbalance in the metabolic pathway linking excitatory and inhibitory neurotransmission has been implicated in multiple psychiatric and neurologic disorders. Recently, we described enantiomer-specific effects of 2-methylglutamate, which is not decarboxylated to the corresponding methyl analogue of gamma-aminobutyric acid (GABA): 4-aminopentanoic acid (4APA). Here, we tested the hypothesis that 4APA also has enantiomer-specific actions in brain. Mouse cerebral synaptosome uptake (nmol/mg protein over 30 min) of (R)-4APA or (S)-4APA was time and temperature dependent; however, the R enantiomer had greater uptake, reduction of endogenous GABA concentration, and release following membrane depolarization than did the S enantiomer. (S)-4APA exhibited some weak agonist (GABAA α4ß3δ, GABAA α5ß2γ2, and GABAB B1/B2) and antagonist (GABAA α6ß2γ2) activity while (R)-4APA showed weak agonist activity only with GABAA α5ß2γ2. Both 4APA enantiomers (100 mg/kg IP) were detected in mouse brain 10 min after injection, and by 1 hr had reached concentrations that were stable over 6 hr; both enantiomers were cleared rapidly from mouse serum over 6 hr. Two-month-old mice had no mortality following 100-900 mg/kg IP of each 4APA enantiomer but did have similar dose-dependent reduction in distance moved in a novel cage. Neither enantiomer at 30 or 100 mg/kg impacted outcomes in 23 measures of well-being, activity chamber, or withdrawal from hot plate. Our results suggest that enantiomers of 4APA are active in mouse brain, and that (R)-4APA may act as a novel false neurotransmitter of GABA. Future work will focus on disease models and on possible applications as neuroimaging agents.

Screening of the strictly xylose-utilizing Bacillus sp. SM01 for polyhydroxybutyrate and its co-culture with Cupriavidus necator NCIMB 11599 for enhanced production of PHB.

Polyhydroxybutyrate (PHB) is a biodegradable plastic that can be used as an alternative to petrochemical-based plastics. PHB is produced by various microorganisms such as Ralstonia, Halomonas, and Bacillus species. However, there are very few strains that produce PHB using xylose, an abundant and inexpensive carbon source. In this study, ten xylose-utilizing PHB producers isolated from South Korean marine environments were screened and characterized. Among these isolates, Bacillus sp. SM01, a newly identified strain, produced the highest amount of PHB using xylose. Under optimal conditions, the maximum dry cell weight (DCW) was 3.41 ± 0.09 g/L, with 62% PHB content, and Bacillus sp. SM01 showed Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production with propionate; however, the growth of Bacillus sp. SM01 was greatly inhibited by the presence of glucose. Co-culturing Bacillus sp. SM01 with Cupriavidus necator NCIMB 11599 resulted in increased DCW, PHB production, and utilization of glucose and xylose, the main sugar of lignocellulosic biomass, compared with the monoculture. Our results indicated that this co-culture system can be used to increase PHB production and overcome the limitation of sugar consumption associated with Bacillus sp. SM01 and C. necator.

Nicotinamide Ameliorates Dextran Sulfate Sodium-Induced Chronic Colitis in Mice through Its Anti-Inflammatory Properties and Modulates the Gut Microbiota.

Vitamin B (nicotinamide (NAM)), one of the most important nutritional components for humans, exerts anti-inflammatory activity. This study was aimed at investigating the effect of NAM on the gut microbiota and short-chain fatty acids (SCFAs) in mice with chronic colitis. Colitis was induced in C57BL/6 male mice by administration of 1.5% dextran sulfate sodium (DSS), and the mice were intraperitoneally injected with normal saline (NS) or NAM. NAM treatment ameliorated weight loss and changes in colon length, disease activity index (DAI) score, and histologic scores. Moreover, enzyme-linked immunosorbent assay (ELISA) analysis of LPL cells revealed that the level of interleukin- (IL-) 6, IL-12p70, IL-1ß, tumor necrosis factor- (TNF-) α, interferon- (IFN-) γ, IL-21, and IL-17A was increased, while IL-10 was reduced, in the chronic colitis group compared to the control group, but the levels of all these factors were restored after NAM treatment. Then, 16S rRNA sequencing of the large intestinal content was performed, and analysis of alpha diversity and beta diversity showed that the richness of the gut microbiota was decreased in the DSS group compared to the control group and restored after NAM treatment. In addition, NAM modulated specific bacteria, including Odoribacter, Flexispira, and Bifidobacterium, in the NAM+chronic colitis group. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis indicated that NAM treatment restored disruptions in the functions of the gut microbiota (replication and repair, cell motility) in mice with DSS-induced colitis. Furthermore, NAM also restored the reduction in valeric acid in mice with DSS-induced chronic colitis. Our results suggest that NAM treatment could alleviate DSS-induced chronic colitis in mice by inhibiting inflammation and regulating the composition and function of gut microbiota.

Optimization of immobilization conditions of Bacillus atrophaeus FSHM2 lipase on maleic copolymer coated amine-modified graphene oxide nanosheets and its application for valeric acid esterification.

The thermoalkalophilic lipase from Bacillus atrophaeus (BaL) was immobilized onto amine-functionalized graphene oxide nanosheets coated with the poly (maleic anhydride-alt-1-octadecene) copolymer (GO-NH2-PMAO) and activated with glutaraldehyde as spacer arm through interfacial activation and subsequent multipoint covalent attachment. Experimental design method was applied for optimization of immobilization conditions including GO-NH2-PMAO concentration, buffer concentration, pH, sonication time, enzyme concentration, glutaraldehyde concentration, time, and temperature. The optimum specific activity of the immobilized BaL (105.95 ± 2.37 U/mg) reached at 5 mg/mL for GO-NH2-PMAO, 25 mM of buffer, pH 6.0, 60 min sonication time, 100 mM glutaraldehyde, 60 U/mL of enzyme, and 4 h of immobilization time at 25 °C, which was very close to the predicted amount (106.08 ± 1.42 U/mg). Maximum immobilization yield (81.35%) and efficiency (277.63%) were determined in optimal immobilization conditions. The obtained results clearly indicated that the immobilized BaL exhibited better stability at extreme temperature and pH than the free BaL. At temperature of 90 °C and pH 11, more than 90% of the initial activity of the immobilized BaL was retained. Furthermore, the immobilized BaL retained about 90% of its initial activity after 10 days of storage and 6 cycles of application. The esterification studies showed that maximum bioconversion of valeric acid to pentyl valerate using the free BaL (34.5%) and the immobilized BaL (96.3%) occurred in the xylene medium after 48 h of incubation at 60 °C. Therefore, the BaL immobilized on GO-NH2-PMAO was introduced as an effective biocatalyst to synthesize green apple flavour ester.

Effects of feeding mother liquor, by-product of monosodium glutamate, on digestibility, energy and nitrogen balances, and rumen condition in Thai native bulls.

Four Thai native bulls were used to evaluate the availability of mother liquor (ML), by-product of monosodium glutamate, as a replacement of soybean meal (SBM) consisting of 10% in concentrate. The SBM was replaced by the ML at 0% (C), 20% (T1), 40% (T2), and 60% (T3), and the experiment was a randomized block design experiment. The animals were fed concentrate and roughage (60:40, on a dry matter [DM] basis). There were no significant differences in the digestibility of DM, crude protein, ether extract, acid detergent fiber expressed exclusive of residual ash and non-fibrous carbohydrate, and energy and nitrogen balances among the treatments. However, the digestibility of the neutral detergent fiber in T2 was lower than the other treatments (p < .05). The valeric acid of T2 was lower than those of C and T1 and the iso-valeric acid of T3 was the lowest (p < .05), followed by those of T2, T1, and C at 4 hr post-feeding. No significant differences were observed in the ruminal total VFA concentrations, pH, and NH3 -N among the treatments. These results suggested that SBM could be replaced by the ML up to 60% without adverse effects on nitrogen and energy balance, rumen conditions, and blood metabolites in Thai native bulls.

Production of polyhydroxyalkanoate from acetate by metabolically engineered Aeromonas hydrophilia.

Aeromonas hydrophila 4AK4 normally produces the copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) using lauric acid as the carbon source. In this study we reported the metabolic engineering of A. hydrophila 4AK4 for the production of polyhydroxyalkanoate (PHA) using acetate as a main carbon source. Recombinant A. hydrophila overexpressing ß-ketothiolase and acetoacetyl-CoA reductase could accumulate poly-3-hydroxybutyrate (PHB) from acetate with a polymer content of 1.39 wt%. Further overexpression of acetate kinase/phosphotransacetylase and acetyl-CoA synthetase improved PHB content to 8.75 wt% and 19.82 wt%, respectively. When acetate and propionate were simultaneously supplied as carbon sources, the engineered A. hydrophila overexpressing ß-ketothiolase, acetoacetyl-CoA reductase, and acetyl-CoA synthetase was found able to produce the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV). The recombinant grew to 3.79 g/L cell dry weight (CDW) containing 15.02 wt% PHBV. Our proposed metabolic engineering strategies illustrate the feasibility for producing PHA from acetate by A. hydrophila.

Integrated strain engineering and bioprocessing strategies for high-level bio-based production of 3-hydroxyvalerate in Escherichia coli.

As petro-based production generates numerous environmental impacts and their associated technological concerns, bio-based production has been well recognized these days as a modern alternative to manufacture chemical products in a more renewable, environmentally friendly, and sustainable manner. Herein, we report the development of a microbial bioprocess for high-level and potentially economical production of 3-hydroxyvalerate (3-HV), a valuable special chemical with multiple applications in chemical, biopolymer, and pharmaceutical industries, from glycerol, which can be cheaply and renewably refined as a byproduct from biodiesel production. We used our recently derived 3-HV-producing Escherichia coli strains for bioreactor characterization under various culture conditions. In the parental strain, 3-HV biosynthesis was limited by the intracellular availability of propionyl-CoA, whose formation was favored by anaerobic conditions, which often compromised cell growth. With appropriate strain engineering, we demonstrated that 3-HV can be effectively produced under both microaerobic (close to anaerobic) and aerobic conditions, which determine the direction of dissimilated carbon flux toward the succinate node in the tricarboxylic acid (TCA) cycle. We first used the ∆sdhA single mutant strain, in which the dissimilated carbon flux was primarily directed to the Sleeping beauty mutase (Sbm) pathway (via the reductive TCA branch, with enhanced cell growth under microaerobic conditions, achieving 3.08 g L-1 3-HV in a fed-batch culture. In addition, we used the ∆sdhA-∆iclR double mutant strain, in which the dissimilated carbon flux was directed from the TCA cycle to the Sbm pathway via the deregulated glyoxylate shunt, for cultivation under rather aerobic conditions. In addition to demonstrating effective cell growth, this strain has shown impressive 3-HV biosynthesis (up to 10.6 g L-1), equivalent to an overall yield of 18.8% based on consumed glycerol, in aerobic fed-batch culture. This study not only represents one of the most effective bio-based production of 3-HV from structurally unrelated carbons to date, but also highlights the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.Key points• TCA cycle engineering was applied to enhance 3-HV biosynthesis in E. coli. • Effects of oxygenic conditions on 3-HV in E. coli biosynthesis were investigated. • Bioreactor characterization of 3-HV biosynthesis in E. coli was performed.

Volatile disease markers of American foulbrood-infected larvae in Apis mellifera.

The honey bee, which lives in the crowded environment of a social hive, is vulnerable to disease infection and spread. Despite efforts to develop various diagnostic methods, American foulbrood (AFB) caused by Paenibacillus larvae infection has caused enormous damage to the apicultural industry. Here, we investigated the volatile organic compounds derived from AFB. After inoculation of the AFB pathogen in honey bee larvae under lab conditions, we identified propionic acid, valeric acid, and 2-nonanone as volatile disease markers (VDMs) of AFB infection using GC/MS. Electrophysiological recordings demonstrated that middle-aged bees, the hygienic-aged bees, are more sensitive to these VDMs than the foragers. Thus, these VDMs have the potential to be efficient and significant cues for worker detection of AFB infected larvae in bee hives. This study supports the idea that the specific olfactory sensitivity of different worker bees depends on their tasks. Taken together, our finding is crucial and sufficient to develop novel disease volatile markers associated with honey bee diseases to diagnose and study the molecular and neural correlates of given hygienic behavior detecting these volatile chemicals by honey bees.

Short-chain fatty acids accompanying changes in the gut microbiome contribute to the development of hypertension in patients with preeclampsia.

Preeclampsia (PE) is regarded as a pregnancy-associated hypertension disorder that is related to excessive inflammatory responses. Although the gut microbiota (GM) and short-chain fatty acids (SCFAs) have been related to hypertension, their effects on PE remain unknown. We determined the GM abundance and faecal SCFA levels by 16S ribosomal RNA (rRNA) sequencing and gas chromatography, respectively, using faecal samples from 27 patients with severe PE and 36 healthy, pregnant control subjects. We found that patients with PE had significantly decreased GM diversity and altered GM abundance. At the phylum level, patients with PE exhibited decreased abundance of Firmicutes albeit increased abundance of Proteobacteria; at the genus level, patients with PE had lower abundance of Blautia, Eubacterium_rectale, Eubacterium_hallii, Streptococcus, Bifidobacterium, Collinsella, Alistipes, and Subdoligranulum, albeit higher abundance of Enterobacter and Escherichia_Shigella. The faecal levels of butyric and valeric acids were significantly decreased in patients with PE and significantly correlated with the above-mentioned differential GM abundance. We predicted significantly increased abundance of the lipopolysaccharide (LPS)-synthesis pathway and significantly decreased abundance of the G protein-coupled receptor (GPCR) pathway in patients with PE, based on phylogenetic reconstruction of unobserved states (PICRUSt). Finally, we evaluated the effects of oral butyrate on LPS-induced hypertension in pregnant rats. We found that butyrate significantly reduced the blood pressure (BP) in these rats. In summary, we provide the first evidence linking GM dysbiosis and reduced faecal SCFA to PE and demonstrate that butyrate can directly regulate BP in vivo, suggesting its potential as a therapeutic agent for PE.

Gut commensal derived-valeric acid protects against radiation injuries.

BACKGROUND: Hematopoietic and intestinal systems side effects are frequently found in patients who suffered from accidental or medical radiation exposure. In this case, we investigated the effects of gut microbiota produced-valeric acid (VA) on radiation-induced injuries. METHODS: Mice were exposed to total body irradiation (TBI) or total abdominal irradiation (TAI) to mimic accidental or clinical scenarios. High-performance liquid chromatography (HPLC) was performed to assess short-chain fatty acids (SCFAs) in fecal pellets. Oral gavage with VA was used to mitigate radiation-induced toxicity. Gross examination was performed to assess tissue injuries of thymus, spleen and small intestine. High-throughput sequencing was used to characterize the gut microbiota profile. Isobaric tags for relative and absolute quantitation (iTRAQ) were performed to analyze the difference of protein profile. Hydrodynamic-based gene delivery assay was performed to silence KRT1 in vivo. RESULTS: VA exerted the most significant radioprotection among the SCFAs. In detail, VA replenishment elevated the survival rate of irradiated mice, protected hematogenic organs, improved gastrointestinal (GI) tract function and intestinal epithelial integrity in irradiated mice. High-throughput sequencing and iTRAQ showed that oral gavage of VA restored the enteric bacteria taxonomic proportions, reprogrammed the small intestinal protein profile of mice following TAI exposure. Importantly, keratin 1 (KRT1) played a pivotal role in the radioprotection of VA. CONCLUSIONS: Our findings provide new insights into gut microbiota-produced VA and underpin that VA might be employed as a therapeutic option to mitigate radiation injury in pre-clinical settings.

Perfusion process combining low temperature and valeric acid for enhanced recombinant factor VIII production.

Perfusion operation mode remains the preferred platform for production of labile biopharmaceuticals (e.g., blood factors) and is also being increasingly adopted for production of stable products (e.g., monoclonal antibodies). Regardless of the product, process development typically aims at maximizing production capacity. In this work, we investigated the impact of perfusion cultivation conditions on process productivity for production of human factor VIII (FVIII). Recombinant CHO cells were cultivated in bioreactors coupled to inclined settlers and the effects of reducing the temperature to 31°C with or without valeric acid (VA) supplementation were evaluated. Increases in cell specific productivity (qp ) up to 2.4-fold (FVIII concentration) and up to 3.0-fold (FVIII biological activity) were obtained at 31°C with VA compared to the control at 37°C. Biological activity is the most important quality attribute for FVIII and was positively affected by mild hypothermia in combination with the chemical inducer. The low temperature conditions resulted in enhanced product transcript levels, suggesting that the higher qp is related to the increased mRNA levels. Furthermore, a high-producer subclone was evaluated under the perfusion conditions optimized for the parental clone (31°C with VA), yielding increases in qp of 6-fold and 15-fold compared to the parental clone cultivated under the same condition and at 37°C, respectively. The proposed perfusion strategy enables increased product formation without increasing production costs, being potentially applicable to perfusion production of other CHO-derived biopharmaceuticals. To the best of our knowledge, this is the first report showing the benefits of perfusion combining mild hypothermia with VA supplementation.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

Production of characteristic volatile markers and their relation to Staphylococcus aureus growth status in pork.

The 3-methyl-butanal and 3-methyl-butanoic acid are known as fingerprint compounds from Staphylococcus aureus. In this study, we investigated production of these two volatile biomarkers and their correlation to S. aureus growth in pork. Both 3-methyl-butanal and 3-methyl-butanoic acid presented high specificity for S. aureus in either media or pork. In sterile minced pork and pork broth, production of volatile biomarkers and the growth of S. aureus were significantly correlated for most single cultures. However, for mixed cultures, only 3-methyl-butanoic acid indicated correlations with growth of S. aureus. Similar trending was also discovered in raw pork, where production of 3-methyl-butanoic acid was significantly correlated with the growth of S. aureus, but not for 3-methyl-butanal. In summary, 3-methyl-butanoic acid was a more stable metabolic marker than 3-methyl-butanal which could be used as an indicator for the presence of S. aureus in pork. This rapid, convenient and cost-effective detection approach could be applied in meat industry to achieve specific detection of S. aureus.

Heterologous production of 3-hydroxyvalerate in engineered Escherichia coli.

3-Hydroxyacids are a group of valuable fine chemicals with numerous applications, and 3-hydroxybutyrate (3-HB) represents the most common species with acetyl-CoA as a precursor. Due to the lack of propionyl-CoA in most, if not all, microorganisms, bio-based production of 3-hydroxyvalerate (3-HV), a longer-chain 3-hydroxyacid member with both acetyl-CoA and propionyl-CoA as two precursors, is often hindered by high costs associated with the supplementation of related carbon sources, such as propionate or valerate. Here, we report the derivation of engineered Escherichia coli strains for the production of 3-HV from unrelated cheap carbon sources, in particular glucose and glycerol. Activation of the sleeping beauty mutase (Sbm) pathway in E. coli enabled the intracellular formation of non-native propionyl-CoA. A selection of enzymes involved in 3-HV biosynthetic pathway from various microorganisms were explored for investigating their effects on 3-HV biosynthesis in E. coli. Glycerol outperformed glucose as the carbon source, and glycerol dissimilation for 3-HV biosynthesis was primarily mediated through the aerobic GlpK-GlpD route. To further enhance 3-HV production, we developed metabolic engineering strategies to redirect more dissimilated carbon flux from the tricarboxylic acid (TCA) cycle to the Sbm pathway, resulting in an enlarged intracellular pool of propionyl-CoA. Both the presence of succinate/succinyl-CoA and their interconversion step in the TCA cycle were identified to critically limit the carbon flux redirection into the Sbm pathway and, therefore, 3-HV biosynthesis. A selection of E. coli host TCA genes encoding enzymes near the succinate node were targeted for manipulation to evaluate the contribution of the three TCA routes (i.e. oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt) to the redirected carbon flux into the Sbm pathway. Finally, the carbon flux redirection into the Sbm pathway was enhanced by simultaneously deregulating glyoxylate shunt and blocking the oxidative TCA cycle, significantly improving 3-HV biosynthesis. With the implementation of these biotechnological and bioprocessing strategies, our engineered E. coli strains can effectively produce 3-HV up to 3.71 g l-1 with a yield of 24.1% based on the consumed glycerol in shake-flask cultures.

A novel lipase from Aspergillus oryzae catalyzed resolution of (R,S)-ethyl 2-bromoisovalerate.

In this study, a novel lipase M5 derived from Aspergillus oryzae WZ007 was prone to exhibit high hydrolytic activity and stereoselectivity towards racemic substrate (R,S)-ethyl 2-bromoisovalerate. (R)-ethyl 2-bromoisovalerate was obtained by enzymatic resolution, which is the key chiral intermediate for highly efficient enantiomerically fluvalinate. The results showed that the enzymatic reaction was carried out in 120mM racemic substrate for 3 hours, the enantiomeric excess reached 98.6%, the conversion was 51.7%, and E value above 120. Therefore, the novel lipase M5 has the ability to efficiently produce (R)-ethyl 2-bromoisovalerate, which greatly reduces the industrial production cost of the highly efficient counterpart of fluvalinate.

Impact of Steviol Glycosides and Erythritol on the Human and Cebus apella Gut Microbiome.

Leaf extracts of Stevia rebaudiana, composed of more than 10 steviol glycosides (SGs), are used as non-nutritive, table sugar (sucrose) alternatives due to their high level of sweetness and low caloric impact. They are often combined with the sugar alcohol erythritol to increase volume and reduce aftertaste. Little is known of the impact of sugar alternatives on the human gut microbiota in terms of the diversity, composition, and metabolic products. Testing of SGs and erythritol using six representatives of the gut microbiota in vitro found no impact on bacterial growth, yet treatment with erythritol resulted in an enhancement of butyric and pentanoic acid production when tested using a human gut microbial community. Furthermore, administration of SGs and erythritol to a Cebus apella model resulted in changes to the gut microbial structure and diversity. Overall, the study did not find a negative impact of SGs and erythritol on the gut microbial community.

5-(Hydroxyphenyl)-γ-Valerolactone-Sulfate, a Key Microbial Metabolite of Flavan-3-ols, Is Able to Reach the Brain: Evidence from Different in Silico, In Vitro and In Vivo Experimental Models.

Phenolic compounds have been recognized as promising compounds for the prevention of chronic diseases, including neurodegenerative ones. However, phenolics like flavan-3-ols (F3O) are poorly absorbed along the gastrointestinal tract and structurally rearranged by gut microbiota, yielding smaller and more polar metabolites like phenyl-γ-valerolactones, phenylvaleric acids and their conjugates. The present work investigated the ability of F3O-derived metabolites to cross the blood-brain barrier (BBB), by linking five experimental models with increasing realism. First, an in silico study examined the physical-chemical characteristics of F3O metabolites to predict those most likely to cross the BBB. Some of these metabolites were then tested at physiological concentrations to cross the luminal and abluminal membranes of brain microvascular endothelial cells, cultured in vitro. Finally, three different in vivo studies in rats injected with pure 5-(3',4'-dihydroxyphenyl)-γ-valerolactone, and rats and pigs fed grapes or a F3O-rich cocoa extract, respectively, confirmed the presence of 5-(hydroxyphenyl)-γ-valerolactone-sulfate (3',4' isomer) in the brain. This work highlighted, with different experimental models, the BBB permeability of one of the main F3O-derived metabolites. It may support the neuroprotective effects of phenolic-rich foods in the frame of the "gut-brain axis".

The impact of dietary fermentable carbohydrates on a postinflammatory model of irritable bowel syndrome.

BACKGROUND: A low fermentable carbohydrate (FODMAP) diet is used in quiescent inflammatory bowel disease when irritable bowel syndrome-like symptoms occur. There is concern that the diet could exacerbate inflammation by modifying microbiota and short-chain fatty acid (SCFA) production. We examined the effect of altering dietary FODMAP content on inflammation in preclinical inflammatory models. METHODS: C57BL/6 mice were given 3% dextran sodium sulfate (DSS) in drinking water for 5 days and recovered for 3 weeks (postinflammatory, n = 12), or 5 days (positive-control, n = 12). Following recovery, DSS-treated or control mice (negative-control, n = 12) were randomized to 2-week low- (0.51 g/100 g total FODMAP) or high-FODMAP (4.10 g) diets. Diets mimicked human consumption containing fructose, sorbitol, galacto-oligosaccharide, and fructan. Colons were assessed for myeloperoxidase (MPO) activity and histological damage. Supernatants were generated for perforated patch-clamp recordings and cytokine measurement. Cecum contents were analyzed for microbiota, SCFA, and branched-chain fatty acids (BCFA). Data were analyzed by two-way ANOVA with Bonferroni. KEY RESULTS: Inflammatory markers were higher in the positive-control compared with negative-control and postinflammatory groups, but no differences occurred between the two diets within each treatment (MPO P > .99, histological scores P > .99, cytokines P > .05), or the perforated patch-clamp recordings (P > .05). Microbiota clustered mainly based on DSS exposure. No difference in SCFA content occurred. Higher total BCFA occurred with the low-FODMAP diet in positive-control (P < .01) and postinflammatory groups (P < .01). CONCLUSIONS AND INFERENCES: In this preclinical study, reducing dietary FODMAPs did not exacerbate nor mitigate inflammation. Microbiota profile changes were largely driven by inflammation rather than diet. Low FODMAP intake caused a shift toward proteolytic fermentation following inflammation.