Rho-dependent termination enables cellular pH homeostasis.

The termination factor Rho, an ATP-dependent RNA translocase, preempts pervasive transcription processes, thereby rendering genome integrity in bacteria. Here, we show that the loss of Rho function raised the intracellular pH to >8.0 in Escherichia coli. The loss of Rho function upregulates tryptophanase-A (TnaA), an enzyme that catabolizes tryptophan to produce indole, pyruvate, and ammonia. We demonstrate that the enhanced TnaA function had produced the conjugate base ammonia, raising the cellular pH in the Rho-dependent termination defective strains. On the other hand, the constitutively overexpressed Rho lowered the cellular pH to about 6.2, independent of cellular ammonia levels. Since Rho overexpression may increase termination activities, the decrease in cellular pH could result from an excess H+ ion production during ATP hydrolysis by overproduced Rho. Furthermore, we performed in vivo termination assays to show that the efficiency of Rho-dependent termination was increased at both acidic and basic pH ranges. Given that the Rho level remained unchanged, the alkaline pH increases the termination efficiency by stimulating Rho's catalytic activity. We conducted the Rho-mediated RNA release assay from a stalled elongation complex to show an efficient RNA release at alkaline pH, compared to the neutral or acidic pH, that supports our in vivo observation. Whereas acidic pH appeared to increase the termination function by elevating the cellular level of Rho. This study is the first to link Rho function to the cellular pH homeostasis in bacteria. IMPORTANCE The current study shows that the loss or gain of Rho-dependent termination alkalizes or acidifies the cytoplasm, respectively. In the case of loss of Rho function, the tryptophanase-A enzyme is upregulated, and degrades tryptophan, producing ammonia to alkalize cytoplasm. We hypothesize that Rho overproduction by deleting its autoregulatory DNA portion increases termination function, causing excessive ATP hydrolysis to produce H+ ions and cytoplasmic acidification. Therefore, this study is the first to unravel a relationship between Rho function and intrinsic cellular pH homeostasis. Furthermore, the Rho level increases in the absence of autoregulation, causing cytoplasmic acidification. As intracellular pH plays a critical role in enzyme function, such a connection between Rho function and alkalization will have far-reaching implications for bacterial physiology.

Tryptophan-Based Hyperproduction of Bioindigo by Combinatorial Overexpression of Two Different Tryptophan Transporters.

Indigo is a valuable, natural blue dye that has been used for centuries in the textile industry. The large-scale commercial production of indigo relies on its extraction from plants and chemical synthesis. Studies are being conducted to develop methods for environment-friendly and sustainable production of indigo using genetically engineered microbes. Here, to enhance the yield of bioindigo from an E. coli whole-cell system containing tryptophanase (TnaA) and flavin-containing monooxygenase (FMO), we evaluated tryptophan transporters to improve the transport of aromatic compounds, such as indole and tryptophan, which are not easily soluble and passable through cell walls. Among the three transporters, Mtr, AroP, and TnaB, AroP enhanced indigo production the most. The combination of each transporter with AroP was also evaluated, and the combination of AroP and TnaB showed the best performance compared to the single transporters and two transporters. Bioindigo production was then optimized by examining the culture medium, temperature, isopropyl ß-D-1-thiogalactopyranoside concentration, shaking speed (rpm), and pH. The novel strain containing aroP and tnaB plasmid with tnaA and FMO produced 8.77 mM (2.3 g/l) of bioindigo after 66 h of culture. The produced bioindigo was further recovered using a simple method and used as a watercolor dye, showing good mixing with other colors and color retention for a relatively long time. This study presents an effective strategy for enhancing indigo production using a combination of transporters.

Discovery of New Fusobacterium nucleatum Inhibitors to Attenuate Migratory Capability of Colon Cancer Cells by the Drug Repositioning Strategy.

Recent studies revealed that intestinal microbiota played important roles in colorectal cancer (CRC) carcinogenesis. Particularly, Fusobacterium nucleatum was confirmed to promote the proliferation and metastasis of CRC. Therefore, targeting F. nucleatum may be a potential preventive and therapeutic approach for CRC. Herein, 2,272 off-patent drugs were screened inhibitory activity against F. nucleatum. Among the hits, nitisinone was identified as a promising anti-F. nucleatum lead compound. Further optimization of nitisinone led to the discovery of more potent derivatives. Particularly, compounds 19q and 22c showed potent anti-F. nucleatum activity (MIC50 = 1 and 2 µg/mL, respectively) with low cytotoxicity. Among them, compound 19q effectively attenuated the migratory ability of MC-38 cells induced by F. nucleatum. Preliminary mechanism studies suggested that nitisinone and its derivatives might act by downregulating nitroreductase and tryptophanase. Thus, the development of small molecule F. nucleatum inhibitors represents an effective strategy to treat CRC.

Unusual Evolution of Cephalopod Tryptophan Indole-Lyases.

Tryptophan indole-lyase (TIL), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the hydrolysis of L-tryptophan (L-Trp) to indole and ammonium pyruvate. TIL is widely distributed among bacteria and bacterial TILs consist of a D2-symmetric homotetramer. On the other hand, TIL genes are also present in several metazoans. Cephalopods have two TILs, TILα and TILß, which are believed to be derived from a gene duplication that occurred before octopus and squid diverged. However, both TILα and TILß individually contain disruptive amino acid substitutions for TIL activity, and neither was active when expressed alone. When TILα and TILß were coexpressed, however, they formed a heterotetramer that exhibited low TIL activity. The loss of TIL activity of the heterotetramer following site-directed mutagenesis strongly suggests that the active heterotetramer contains the TILα/TILß heterodimer. Metazoan TILs generally have lower kcat values for L-Trp than those of bacterial TILs, but such low TIL activity may be rather suitable for metazoan physiology, where L-Trp is in high demand. Therefore, reduced activity may have been a less likely target for purifying selection in the evolution of cephalopod TILs. Meanwhile, the unusual evolution of cephalopod TILs may indicate the difficulty of post-gene duplication evolution of enzymes with catalytic sites contributed by multiple subunits, such as TIL.

Mechanism-based inhibition of gut microbial tryptophanases reduces serum indoxyl sulfate.

Indoxyl sulfate is a microbially derived uremic toxin that accumulates in late-stage chronic kidney disease and contributes to both renal and cardiovascular toxicity. Indoxyl sulfate is generated by the metabolism of indole, a compound created solely by gut microbial tryptophanases. Here, we characterize the landscape of tryptophanase enzymes in the human gut microbiome and find remarkable structural and functional similarities across diverse taxa. We leverage this homology through a medicinal chemistry campaign to create a potent pan-inhibitor, (3S) ALG-05, and validate its action as a transition-state analog. (3S) ALG-05 successfully reduces indole production in microbial culture and displays minimal toxicity against microbial and mammalian cells. Mice treated with (3S) ALG-05 show reduced cecal indole and serum indoxyl sulfate levels with minimal changes in other tryptophan-metabolizing pathways. These studies present a non-bactericidal pan-inhibitor of gut microbial tryptophanases with potential promise for reducing indoxyl sulfate in chronic kidney disease.

De novo tryptophanase-based indole production by metabolically engineered Corynebacterium glutamicum.

Indole has an increasing interest in the flavor and fragrance industry. It is used in dairy products, tea drinks, and fine fragrances due to its distinct floral odor typical of jasmine blossoms. The current production of indole based on isolation from coal tar is non-sustainable and its isolation from plants is often unprofitable due to low yields. To offer an alternative to the conventional production, biosynthesis of indole has been studied recently. A glucose-based indole production was achieved by employing the Corynebacterium glutamicum tryptophan synthase α-subunit (TrpA) or indole-3-glycerol phosphate lyase (IGL) from wheat Triticum aestivum in a genetically-engineered C. glutamicum strain. In addition, a highly efficient bioconversion process using C. glutamicum heterologously expressing tryptophanase gene (tnaA) from Providencia rettgeri as a biocatalyst was developed. In this work, de novo indole production from glucose was enabled by expressing the P. rettgeri tnaA in a tryptophan-producing C. glutamicum strain. By metabolic engineering of a C. glutamicum shikimate accumulating base strain, tryptophan production of 2.14 ± 0.02 g L-1 was achieved. Introduction of the tryptophanase form P. rettgeri enabled indole production, but to low titers, which could be improved by sequestering indole into the water-immiscible solvent tributyrin during fermentation and a titer of 1.38 ± 0.04 g L-1 was achieved. The process was accelerated by decoupling growth from production increasing the volumetric productivity about 4-fold to 0.08 g L-1 h-1. KEY POINTS: • Efficient de novo indole production via tryptophanases from glucose • Increased indole titers by product sequestration and improved precursor supply • Decoupling growth from production accelerated indole production.

Molecular Confirmation of an Indole-Negative Klebsiella oxytoca Isolated from a Patient with Cystitis.

Klebsiella oxytoca is an opportunistic pathogen that causes nosocomial infections. Here, we describe an unusual clinical strain of indole-negative K. oxytoca, GU175, isolated from the urine of a patient with cystitis. The GU175 strain was identified as K. pneumoniae with a probability of 99.40%, negative for indole production, and resistant to third-generation cephalosporins by using the MicroScan Walkaway 40 SI system with the Negative combo EN1 J panel. Biochemical characterization of this strain using lysine-indole motility medium was negative for indole production. However, identification tests using the MALDI Biotyper system and 16S rRNA gene sequence analysis revealed that GU175 is K. oxytoca. DNA sequence analysis of the tryptophanase operon comparing the GU175 strain with the revertant GU176 strain, which tested positive for indole, revealed a point mutation in the Shine-Dalgarno sequence upstream of tnaC in the GU175 strain. This is the first report of indole-negative K. oxytoca, which was attributed to a mutation in the DNA sequence of the tryptophanase operon isolated from a patient with a urinary tract infection. As indole-negative K. oxytoca can be misidentified as K. pneumoniae by biochemical characterization, clinical microbiologists should be aware of such misidentifications.

Genetically engineered bacterium-modified magnetic particles assisted chiral recognition and colorimetric determination of D/L-tryptophan in millets.

Chiral recognition of enantiomers has always been a thorny issue since they exhibit the same properties under an achiral environment. Herein, polydopamine-functionalized magnetic particles (MP@PDA) were synthesized to immobilize the genetically engineered bacterium Escherichia coli DH5α (MP@PDA-E. coli). L-tryptophan (Trp) instead of D-Trp can be stereo-specifically degraded by tryptophanase in E. coli. The degradation product indole reacts with 4-dimethylaminobenzaldehyde to generate a rose-red adduct. Thus, MP@PDA-E. coli was employed to fabricate a chiral colorimetric method for chiral recognition and determination of L-Trp. The method averts the purification of tryptophanase. More importantly, tryptophanase demonstrates excellent enantioselective ability for L-Trp. The method can not only quantitatively detect L-Trp but also realize the measurement of the enantiomer percentage in the enantiomeric mixture. The feasibility was verified by detecting L-Trp in millet samples from different origins. Furthermore, a portable device was fabricated to make the method more convenient.

Production of indole and hydrogen sulfide by the oxygen-tolerant mutant strain Clostridium sp. Aeroto-AUH-JLC108 contributes to form a hypoxic microenvironment.

In this study, the oxygen-tolerant mutant strain Clostridium sp. Aeroto-AUH-JLC108 was found to produce indole when grown aerobically. The tnaA gene coding for tryptophanase responsible for the production of indole was cloned. The tnaA gene from Aeroto-AUH-JLC108 is 1677 bp and has one point mutation (C36G) compared to the original anaerobic strain AUH-JLC108. Phylogenetic analyses based on the amino acid sequence showed significant homology to that of TnaA from Flavonifractor. Furthermore, we found that the tnaA gene also exhibited cysteine desulfhydrase activity. The production of hydrogen sulfide (H2S) was accompanied by decrease in the amount of the dissolved oxygen in the culture medium. Similarly, the amount of indole produced by strain Aeroto-AUH-JLC108 obviously decreased the oxidation-reduction potential (ORP) in BHI liquid medium. The results demonstrated that production of indole and H2S helped to form a hypoxic microenvironment for strain Aeroto-AUH-JLC108 when grown aerobically.

Identification of tryptophanase from Escherichia coli for the synthesis of S-allyl-l-cysteine and related S-substituted cysteine derivatives.

A wide variety of S-substituted cysteine derivatives occur in plant metabolites. For example, S-allyl-l-cysteine (SAC), mainly contained in garlic, gathers huge interest because of its favorable bioactivities for human health. However, conventional methods for preparing SAC suffer from several drawbacks with regard to efficiency and toxicity, which highlights the need for improved processes for SAC synthesis. This study aims to develop a novel bioprocess to produce SAC by microbial enzymes from easily available substrates. We found that Escherichia coli had the ability to synthesize SAC from allyl mercaptan, pyruvic acid, and ammonium sulfate. An enzyme purification through 3-step column chromatography, followed by determination of the N-terminal amino acid sequence revealed that tryptophanase (TnaA) was the enzyme responsible for SAC formation. Although the enzyme catalyzed the reversible reaction for synthesizing and degrading SAC, the degradation proceeded significantly faster than the synthesis. Interestingly, TnaA catalyzed the synthesis of a wide range of S-substituted cysteines with alkyl chains or aromatic rings, some of which are present in Allium and Petiveria plants. Our results showed a novel substrate specificity of TnaA toward various S-substituted cysteine. TnaA is a promising biocatalyst for developing a new process to supply various valuable S-substituted cysteine derivatives for medicinal and health-promoting applications.

Fermentative Production of Halogenated Tryptophan Derivatives with Corynebacterium glutamicum Overexpressing Tryptophanase or Decarboxylase Genes.

The aromatic amino acid l-tryptophan serves as a precursor for many valuable compounds such as neuromodulators, indoleamines and indole alkaloids. In this work, tryptophan biosynthesis was extended by halogenation followed by decarboxylation to the respective tryptamines or cleavage to the respective indoles. Either the tryptophanase genes tnaAs from E. coli and Proteus vulgaris or the aromatic amino acid decarboxylase genes AADCs from Bacillus atrophaeus, Clostridium sporogenes, and Ruminococcus gnavus were expressed in Corynebacterium glutamicum strains producing (halogenated) tryptophan. Regarding indoles, final titers of 16 mg L-1 7-Cl-indole and 23 mg L-1 7-Br-indole were attained. Tryptamine production led to a much higher titer of 2.26 g L-1 upon expression of AADC from B. atrophaeus. AADC enzymes were shown to be active with halogenated tryptophan in vitro and in vivo and supported production of 0.36 g L-1 7-Br-tryptamine with a volumetric productivity of 8.3 mg L-1 h-1 in a fed-batch fermentation.

From Tryptophan to Toxin: Nature's Convergent Biosynthetic Strategy to Aetokthonotoxin.

Aetokthonotoxin (AETX) is a cyanobacterial neurotoxin that causes vacuolar myelinopathy, a neurological disease that is particularly deadly to bald eagles in the United States. The recently characterized AETX is structurally unique among cyanotoxins and is composed of a pentabrominated biindole nitrile. Herein we report the discovery of an efficient, five-enzyme biosynthetic pathway that the freshwater cyanobacterium Aetokthonos hydrillicola uses to convert two molecules of tryptophan to AETX. We demonstrate that the biosynthetic pathway follows a convergent route in which two functionalized indole monomers are assembled and then reunited by biaryl coupling catalyzed by the cytochrome P450 AetB. Our results revealed enzymes with novel biochemical functions, including the single-component flavin-dependent tryptophan halogenase AetF and the iron-dependent nitrile synthase AetD.

(+)-Sesamin, a sesame lignan, is a potent inhibitor of gut bacterial tryptophan indole-lyase that is a key enzyme in chronic kidney disease pathogenesis.

The progression of chronic kidney disease (CKD) increases the risks of cardiovascular morbidity and end-stage kidney disease. Indoxyl sulfate (IS), which is derived from dietary l-tryptophan by the action of bacterial l-tryptophan indole-lyase (TIL) in the gut, serves as a uremic toxin that exacerbates CKD-related kidney disorder. A mouse model previously showed that inhibition of TIL by 2-aza-l-tyrosine effectively reduced the plasma IS level, causing the recovery of renal damage. In this study, we found that (+)-sesamin and related lignans, which occur abundantly in sesame seeds, inhibit intestinal bacteria TILs. Kinetic studies revealed that (+)-sesamin and sesamol competitively inhibited Escherichia coli TIL (EcTIL) with Ki values of 7 µM and 14 µM, respectively. These Ki values were smaller than that of 2-aza-l-tyrosine (143 µM). Molecular docking simulation of (+)-sesamin- (or sesamol-)binding to EcTIL predicted that these inhibitors potentially bind near the active site of EcTIL, where the cofactor pyridoxal 5'-phosphate is bound, consistent with the kinetic results. (+)-Sesamin is a phytochemical with a long history of consumption and is generally regarded as safe. Hence, dietary supplementation of (+)-sesamin encapsulated in enteric capsules could be a promising mechanism-based strategy to prevent CKD progression. Moreover, the present findings would provide a new structural basis for designing more potent TIL inhibitors for the development of mechanism-based therapeutic drugs to treat CKD.

Characterisation of novel functionality within the Blastocystis tryptophanase gene.

In recent years, the human gut microbiome has been recognised to play a pivotal role in the health of the host. Intestinal homeostasis relies on this intricate and complex relationship between the gut microbiota and the human host. While much effort and attention has been placed on the characterization of the organisms that inhabit the gut microbiome, the complex molecular cross-talk between the microbiota could also exert an effect on gastrointestinal conditions. Blastocystis is a single-cell eukaryotic parasite of emerging interest, as its beneficial or pathogenic role in the microbiota has been a subject of contention even to-date. In this study, we assessed the function of the Blastocystis tryptophanase gene (BhTnaA), which was acquired by horizontal gene transfer and likely to be of bacterial origin within Blastocystis. Bioinformatic analysis and phylogenetic reconstruction revealed distinct divergence of BhTnaA versus known bacterial homologs. Despite sharing high homology with the E. coli tryptophanase gene, we show that Blastocystis does not readily convert tryptophan into indole. Instead, BhTnaA preferentially catalyzes the conversion of indole to tryptophan. We also show a direct link between E. coli and Blastocystis tryptophan metabolism: In the presence of E. coli, Blastocystis ST7 is less able to metabolise indole to tryptophan. This study examines the potential for functional variation in horizontally-acquired genes relative to their canonical counterparts, and identifies Blastocystis as a possible producer of tryptophan within the gut.

Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment.

Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the ΔtnaA strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment. IMPORTANCE Persister and VBNC cells can phenotypically survive environmental stressors, such as antibiotic treatment, limitation of nutrients, and acid stress, and have been linked to chronic infections and antimicrobial resistance. It has recently been suggested that pH regulation might play a role in an organism's phenotypic survival to antibiotics; however, this hypothesis remains to be tested. Here, we demonstrate that even before antibiotic treatment, cells that will become persisters have a more acidic intracellular pH than clonal cells that will be either susceptible or VBNC upon antibiotic treatment. Moreover, after antibiotic treatment, persisters become more alkaline than VBNC and susceptible E. coli cells. This newly found phenotypic feature is remarkable because it distinguishes persister and VBNC cells that have often been thought to display the same dormant phenotype. We then show that this differential pH regulation is abolished in the absence of the enzyme tryptophanase via a major remodeling of bacterial metabolism and pH homeostasis. These new whole-genome transcriptome data should be taken into account when modeling bacterial metabolism at the crucial transition from exponential to stationary phase. Overall, our findings indicate that the manipulation of the intracellular pH represents a bacterial strategy for surviving antibiotic treatment. In turn, this suggests a strategy for developing persister-targeting antibiotics by interfering with cellular components, such as tryptophanase, that play a major role in pH homeostasis.

Tryptophanase gene deficiency improves the application of dioxygenase to 3-(2-hydroxyethyl)catechol production.

3-(2-Hydroxyethyl)catechol (HEC) is a polyphenol reported to exhibit skin-lightning and antioxidative effects, and hence is expected to be used as cosmetic and food additives and chemical products such as electronic materials. In this study, we established biocatalytic HEC production from 2-phenylethanol using the dioxygenase whose expression was induced by toluene, CumA, and its flanking dehydrogenase, CumB, from an isolated strain, Pseudomonas sp. K17. Escherichia coli cells coexpressing CumA and CumB were stained blue during cultivation in Luria-Bertani medium, and HEC was not produced upon using the cell-free extracts as biocatalysts, likely resulting from the inhibitory effects of the blue dyes. The disruption of the tryptophanase gene of E. coli was found to repress the generation of the blue dyes, and enhanced HEC production. The blue dyes were extracted from the cell-free extracts, and their molecular formula was C16H10N2O3, suggesting they were monooxygenated indigo or its isomers. Although repression of blue dye formation and enhancement of HEC production were observed when cells were cultivated with glucose, the percent yield of HEC was 84% at 20 h, whereas that with tryptophanase disruption strain was 84% at 4 h. It was suggested that tryptophanase gene disruption could contribute to more efficient HEC production.

The Tryptophan-Induced tnaC Ribosome Stalling Sequence Exposes High Amino Acid Cross-Talk That Can Be Mitigated by Removal of NusB for Higher Orthogonality.

A growing number of engineered synthetic circuits have employed biological parts coupling transcription and translation in bacterial systems to control downstream gene expression. One such example, the leader sequence of the tryptophanase (tna) operon, is a transcription-translation system commonly employed as an l-tryptophan inducible circuit controlled by ribosome stalling. While induction of the tna operon has been well-characterized in response to l-tryptophan, cross-talk of this modular component with other metabolites in the cell, such as other naturally occurring amino acids, has been less explored. In this study, we investigated the impact of natural metabolites and E. coli host factors on induction of the tna leader sequence. To do so, we constructed and biochemically validated an experimental assay using the tna operon leader sequence to assess differential regulation of transcription elongation and translation in response to l-tryptophan. Operon induction was then assessed following addition of each of the 20 naturally occurring amino acids to discover that several additional amino acids (e.g., l-alanine, l-cysteine, l-glycine, l-methionine, and l-threonine) also induce expression of the tna leader sequence. Following characterization of dose-dependent induction by l-cysteine relative to l-tryptophan, the effect on induction by single gene knockouts of protein factors associated with transcription and/or translation were interrogated. Our results implicate the endogenous cellular protein, NusB, as an important factor associated with induction of the operon by the alternative amino acids. As such, removal of the nusB gene from strains intended for tryptophan-sensing utilizing the tna leader region reduces amino acid cross-talk, resulting in enhanced orthogonal control of this commonly used synthetic system.

miR-375 prevents high-fat diet-induced insulin resistance and obesity by targeting the aryl hydrocarbon receptor and bacterial tryptophanase (tnaA) gene.

Background: Diet manipulation is the basis for prevention of obesity and diabetes. The molecular mechanisms that mediate the diet-based prevention of insulin resistance are not well understood. Here, as proof-of-concept, ginger-derived nanoparticles (GDNP) were used for studying molecular mechanisms underlying GDNP mediated prevention of high-fat diet induced insulin resistance. Methods: Ginger-derived nanoparticles (GDNP) were isolated from ginger roots and administered orally to C57BL/6 high-fat diet mice. Fecal exosomes released from intestinal epithelial cells (IECs) of PBS or GDNP treated high-fat diet (HFD) fed mice were isolated by differential centrifugation. A micro-RNA (miRNA) polymerase chain reaction (PCR) array was used to profile the exosomal miRs and miRs of interest were further analyzed by quantitative real time (RT) PCR. miR-375 or antisense-miR375 was packed into nanoparticles made from the lipids extracted from GDNP. Nanoparticles was fluorescent labeled for monitoring their in vivo trafficking route after oral administration. The effect of these nanoparticles on glucose and insulin response of mice was determined by glucose and insulin tolerance tests. Results: We report that HFD feeding increased the expression of AhR and inhibited the expression of miR-375 and VAMP7. Treatment with orally administered ginger-derived nanoparticles (GDNP) resulted in reversing HFD mediated inhibition of the expression of miR-375 and VAMP7. miR-375 knockout mice exhibited impaired glucose homeostasis and insulin resistance. Induction of intracellular miR-375 led to inhibition of the expression of AhR and VAMP7 mediated exporting of miR-375 into intestinal epithelial exosomes where they were taken up by gut bacteria and inhibited the production of the AhR ligand indole. Intestinal exosomes can also traffic to the liver and be taken up by hepatocytes, leading to miR-375 mediated inhibition of hepatic AhR over-expression and inducing the expression of genes associated with the hepatic insulin response. Altogether, GDNP prevents high-fat diet-induced insulin resistance by miR-375 mediated inhibition of the aryl hydrocarbon receptor mediated pathways over activated by HFD feeding. Conclusion: Collectively our findings reveal that oral administration of GDNP to HFD mice improves host glucose tolerance and insulin response via regulating AhR expression by GDNP induced miR-375 and VAMP7.

Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli.

Tyrian purple, mainly composed of 6,6'-dibromoindigo (6BrIG), is an ancient dye extracted from sea snails and was recently demonstrated as a biocompatible semiconductor material. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and the difficulty of regiospecific bromination. Here, we introduce an effective 6BrIG production strategy in Escherichia coli using tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO. Since tryptophan halogenases are expressed in highly insoluble forms in E. coli, a flavin reductase (Fre) that regenerates FADH2 for the halogenase reaction was used as an N-terminal soluble tag of SttH. A consecutive two-cell reaction system was designed to overproduce regiospecifically brominated precursors of 6BrIG by spatiotemporal separation of bromination and bromotryptophan degradation. These approaches led to 315.0 mg l-1 6BrIG production from tryptophan and successful synthesis of regiospecifically dihalogenated indigos. Furthermore, it was demonstrated that 6BrIG overproducing cells can be directly used as a bacterial dye.

Diet posttranslationally modifies the mouse gut microbial proteome to modulate renal function.

Associations between chronic kidney disease (CKD) and the gut microbiota have been postulated, yet questions remain about the underlying mechanisms. In humans, dietary protein increases gut bacterial production of hydrogen sulfide (H2S), indole, and indoxyl sulfate. The latter are uremic toxins, and H2S has diverse physiological functions, some of which are mediated by posttranslational modification. In a mouse model of CKD, we found that a high sulfur amino acid-containing diet resulted in posttranslationally modified microbial tryptophanase activity. This reduced uremic toxin-producing activity and ameliorated progression to CKD in the mice. Thus, diet can tune microbiota function to support healthy host physiology through posttranslational modification without altering microbial community composition.