RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Tryptophan is an essential amino acid required for tumor cell growth and is also the precursor to kynurenine, an immunosuppressive molecule that plays a role in limiting anticancer immunity. Tryptophanase (TNase) is an enzyme expressed by different bacterial species that converts tryptophan into indole, pyruvate and ammonia, but is absent in the Salmonella strain VNP20009 that has been used as a therapeutic delivery vector. We cloned the Escherichia coli TNase operon tnaCAB into the VNP20009 (VNP20009-tnaCAB), and were able to detect linear production of indole over time, using Kovács reagent. In order to conduct further experiments using the whole bacteria, we added the antibiotic gentamicin to stop bacterial replication. Using a fixed number of bacteria, we found that there was no significant effect of gentamicin on stationary phase VNP20009-tnaCAB upon their ability to convert tryptophan to indole over time. We developed a procedure to extract indole from media while retaining tryptophan, and were able to measure tryptophan spectrophotometrically after exposure to gentamicin-inactivated whole bacterial cells. Using the tryptophan concentration equivalent to that present in DMEM cell culture media, a fixed number of bacteria were able to deplete 93.9% of the tryptophan in the culture media in 4 h. In VNP20009-tnaCAB depleted tissue culture media, MDA-MB-468 triple negative breast cancer cells were unable to divide, while those treated with media exposed only to VNP20009 continued cell division. Re-addition of tryptophan to conditioned culture media restored tumor cell growth. Treatment of tumor cells with molar equivalents of the TNase products indole, pyruvate and ammonia only caused a slight increase in tumor cell growth. Using an ELISA assay, we confirmed that TNase depletion of tryptophan also limits the production of immunosuppressive kynurenine in IFNγ-stimulated MDA-MB-468 cancer cells. Our results demonstrate that Salmonella VNP20009 expressing TNase has improved potential to stop tumor cell growth and reverse immunosuppression.

RESUMEN

Indigo dye is an organic compound with a distinctive blue color. Most of the indigo currently used in industry is produced via chemical synthesis, which generates a large amount of wastewater. Therefore, several studies have recently been conducted to find ways to produce indigo eco-friendly using microorganisms. Here, we produced indigo using recombinant Escherichia coli with both an indigo-producing plasmid and a cyclopropane fatty acid (CFA)-regulating plasmid. The CFA-regulating plasmid contains the cfa gene, and its expression increases the CFA composition of the phospholipid fatty acids of the cell membrane. Overexpression of cfa showed cytotoxicity resistance of indole, an intermediate product formed during the indigo production process. This had a positive effect on indigo production and cfa originated from Pseudomonas sp. B 14-6 was used. Optimal conditions for indigo production were determined by adjusting the expression strain, culture temperature, shaking speed, and isopropyl ß-D-1-thiogalactopyranoside concentration. Treatment with Tween 80 at a particular concentration to increase the permeability of the cell membrane had a positive effect on indigo production. The strain with the CFA plasmid produced 4.1 mM of indigo after 24 h of culture and produced 1.5-fold higher indigo than the control strain without the CFA plasmid that produced 2.7 mM.

Asunto(s)

RESUMEN

High hydrostatic pressure (HHP) treatment is one of the most widely accepted non-thermal food processing methods, but HHP-resistance development in pathogenic or spoilage bacteria might compromise the safety and stability of HHP-treated foods. Charting the possible routes and mechanisms of HHP resistance development in foodborne bacteria is therefore essential to anticipate or prevent the appearance of resistant variants. While upregulation of the RpoS-governed general stress response is a well-established route for increased HHP resistance in Escherichia coli, previous work revealed that mutations causing attenuated cAMP/CRP activity or aggregation-prone TnaA variants can evolve to overcome the HHP-hypersensitivity of an E. coli ΔrpoS mutant. In this study, further directed evolution and genetic analysis approaches allowed us to demonstrate that both kinds of mutants tend to co-emerge and compete with each other in E. coli ΔrpoS populations evolving towards HHP resistance, because of the higher HHP resistance of cAMP/CRP mutants and the faster growth rate of the TnaA mutants. Moreover, closer scrutiny of evolving populations revealed RpoS, cAMP/CRP and TnaA independent routes of HHP resistance development, based on downregulation of YegW or RppH activity.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Indigo is an important pigment widely used in industries of food, cosmetics, and textile. In this work, the styrene monooxygenase StyAB from Pseudomonas putida was co-expressed with the tryptophanase TnaA and the chaperone groES-groEL in Escherichia coli for indigo production. Over-expression of the gene styAB endowed the recombinant E. coli AB with the capacity of indigo biosynthesis from indole and tryptophan. Tryptophan fermentation in E. coli AB generated about five times more indigo than that from indole, and the maximum 530 mg/L of indigo was obtained from 1.2 mg/mL of tryptophan. The gene TnaA was then co-expressed with styAB, and the tryptophanase activity significantly increased in the recombinant E. coli ABT. However, TnaA expression led to a decrease in the activity of StyAB and indigo yield in E. coli ABT. Furthermore, the plasmid pGro7 harboring groES-groEL was introduced into E. coli AB, which obviously promoted the activity of StyAB and accelerated indigo biosynthesis in the recombinant E. coli ABP. In addition, the maximum yield of indigo was further increased to 550 mg/L from 1.2 mg/mL of tryptophan in E. coli ABP. The genetic manipulation strategy proposed in this work could provide new insights into construction of indigo biosynthesis cell factory for industrial production.

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

BACKGROUND: The nitrogen containing aromatic compound indole is known for its floral odor typical of jasmine blossoms. Due to its characteristic scent, it is frequently used in dairy products, tea drinks and fine fragrances. The demand for natural indole by the flavor and fragrance industry is high, yet, its abundance in essential oils isolated from plants such as jasmine and narcissus is low. Thus, there is a strong demand for a sustainable method to produce food-grade indole. RESULTS: Here, we established the biotechnological production of indole upon L-tryptophan supplementation in the bacterial host Corynebacterium glutamicum. Heterologous expression of the tryptophanase gene from E. coli enabled the conversion of supplemented L-tryptophan to indole. Engineering of the substrate import by co-expression of the native aromatic amino acid permease gene aroP increased whole-cell biotransformation of L-tryptophan to indole by two-fold. Indole production to 0.2 g L-1 was achieved upon feeding of 1 g L-1 L-tryptophan in a bioreactor cultivation, while neither accumulation of side-products nor loss of indole were observed. To establish an efficient and robust production process, new tryptophanases were recruited by mining of bacterial sequence databases. This search retrieved more than 400 candidates and, upon screening of tryptophanase activity, nine new enzymes were identified as most promising. The highest production of indole in vivo in C. glutamicum was achieved based on the tryptophanase from Providencia rettgeri. Evaluation of several biological aspects identified the product toxicity as major bottleneck of this conversion. In situ product recovery was applied to sequester indole in a food-grade organic phase during the fermentation to avoid inhibition due to product accumulation. This process enabled complete conversion of L-tryptophan and an indole product titer of 5.7 g L-1 was reached. Indole partitioned to the organic phase which contained 28 g L-1 indole while no other products were observed indicating high indole purity. CONCLUSIONS: The bioconversion production process established in this study provides an attractive route for sustainable indole production from tryptophan in C. glutamicum. Industrially relevant indole titers were achieved within 24 h and indole was concentrated in the organic layer as a pure product after the fermentation.

Asunto(s)

RESUMEN

Tryptophanase encoded by the gene tnaA is a pyridoxal phosphate-dependent enzyme that catalyses the conversion of tryptophan to indole, which is commonly used as an intra- and interspecies signalling molecule, particularly by microbes. However, the production of indole is rare in eukaryotic organisms. A nucleotide and protein database search revealed tnaA is commonly reported in various Gram-negative bacteria, but that only a few Gram-positive bacteria and archaea possess the gene. The presence of tnaA in eukaryotes, particularly protozoans and marine organisms, demonstrates the importance of this gene in the animal kingdom. Here, we document the distribution of tnaA and its acquisition and expansion among different taxonomic groups, many of which are usually categorized as non-indole producers. This study provides an opportunity to understand the intriguing role played by tnaA, and its distribution among various types of organisms.

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

Asunto(s)

RESUMEN

The current chemical process for industrial indigo production puts a heavy burden on the environment. An attractive option would be to develop an alternative biotechnological process which does not rely on a petrochemical. This study describes a new biotransformation approach in which l-tryptophan is used as starting material. Its conversion to indigo can be achieved through recombinant overexpression of a bifunctional fusion enzyme, flavin-containing monooxygenase (FMO) fused to tryptophanase (TRP). First, TRP converts l-tryptophan into pyruvate, ammonia and indole. The formed indole serves as substrate for FMO, resulting in indigo formation, while pyruvate fuels the cells for regenerating the required NADPH. To optimize this bioconversion, different fusion constructs were tested. Fusing TRP to FMO at either the N-terminus (TRP-FMO) or the C-terminus (FMO-TRP) resulted in similar high expression levels of bifunctional fusion enzymes. Using whole cells and l-tryptophan as a precursor, high production levels of indigo could be obtained, significantly higher when compared with cells containing only overexpressed FMO. The TRP-FMO containing cells gave the highest yield of indigo resulting in full conversion of 2.0 g l-tryptophan into 1.7 g indigo per liter of culture. The process developed in this study provides an alternative biotransformation approach for the production of indigo starting from biobased starting material.

Asunto(s)

RESUMEN

We have reported that bicarbonate (NaHCO3 ) potentiates the activity of aminoglycosides in Escherichia coli, but the action mechanism was not identified. To eventually understand how NaHCO3 can potentiate antibiotics, we thought that a rational first step was to examine the effect of NaHCO3 separately and to inspect initial gene expression changes triggered by it. In this work, we started by confirming that NaHCO3 can reduce the number of viable E. coli bacteria. We then investigated, via RNAseq, gene expression changes induced by NaHCO3 . There were upregulated and downregulated genes, among the top upregulated genes c. 10-fold increase in expression) was tnaA, the gene encoding tryptophanase, the enzyme that degrades tryptophan to indole. Considering that higher expression of tnaA likely led to increases in indole, we tested the effect of indole and found both growth inhibition and synergy with NaHCO3 . We suggest that indole may participate in growth inhibition of E. coli. The RNAseq analysis also revealed upregulation (≥4-fold) of genes encoding proteins for the acquisition of iron and downregulation (≥16-fold) of genes encoding iron-sulphur-holding proteins; hence NaHCO3 apparently triggered also an iron-deficit response. We suggest that iron deficiency may also be involved in growth inhibition by NaHCO3 . SIGNIFICANCE AND IMPACT OF THE STUDY: Bicarbonate (NaHCO3 ) can enhance the activity of various antibiotics. This work investigated its action mechanism. We carried out a transcriptional analysis in Escherichia coli with the aim of defining initial bacterial changes potentially linked to the enhancing activity of NaHCO3 . Our approach differed from the longer term exposure to NaHCO3 recently used by other researchers, who noticed changes in the bacterial proton motive force. Based on our analysis, we propose two routes possibly linked to the effect of NaHCO3 . Conceivably, those routes are potential targets that could be manipulated by alternative means to augment the effect of antibiotics.

Asunto(s)

RESUMEN

Gut microbiota produces a wide and diverse array of metabolites that are an integral part of the host metabolome. The emergence of the gut microbiome-brain axis concept has prompted investigations on the role of gut microbiota dysbioses in the pathophysiology of brain diseases. Specifically, the search for microbe-related metabolomic signatures in human patients and animal models of psychiatric disorders has pointed out the importance of the microbial metabolism of aromatic amino acids. Here, we investigated the effect of indole on brain and behavior in rats. Indole is produced by gut microbiota from tryptophan, through the tryptophanase enzyme encoded by the tnaA gene. First, we mimicked an acute and high overproduction of indole by injecting this compound in the cecum of conventional rats. This treatment led to a dramatic decrease of motor activity. The neurodepressant oxidized derivatives of indole, oxindole and isatin, accumulated in the brain. In addition, increase in eye blinking frequency and in c-Fos protein expression in the dorsal vagal complex denoted a vagus nerve activation. Second, we mimicked a chronic and moderate overproduction of indole by colonizing germ-free rats with the indole-producing bacterial species Escherichia coli. We compared emotional behaviors of these rats with those of germ-free rats colonized with a genetically-engineered counterpart strain unable to produce indole. Rats overproducing indole displayed higher helplessness in the tail suspension test, and enhanced anxiety-like behavior in the novelty, elevated plus maze and open-field tests. Vagus nerve activation was suggested by an increase in eye blinking frequency. However, unlike the conventional rats dosed with a high amount of indole, the motor activity was not altered and neither oxindole nor isatin could be detected in the brain. Further studies are required for a comprehensive understanding of the mechanisms supporting indole effects on emotional behaviors. As our findings suggest that people whose gut microbiota is highly prone to produce indole could be more likely to develop anxiety and mood disorders, we addressed the issue of the inter-individual variability of indole producing potential in humans. An in silico investigation of metagenomic data focused on the tnaA gene products definitively proved this inter-individual variability.

RESUMEN

We report the enzymatic synthesis of the derivatives of L-tryptophan methylated in indole moiety and labeled with deuterium and tritium in the 2-position of side chain. For kinetic studies twelve isotopomers, i.e., 1'-methyl-[2-2H]-, 1'-methyl-[2-3H]-, 1'-methyl-[2-2H/3H]-, 2'-methyl-[2-2H]-, 2'-methyl-[2-3H]-, 2'-methyl-[2-2H/3H]-, 5'-methyl-[2-2H]-, 5'-methyl-[2-3H]-, 5'-methyl-[2-2H/3H]-, 7'-methyl-[2-2H]-, 7'-methyl-[2-3H]-, and 7'-methyl-[2-2H/3H]-L-tryptophan are obtained by the enzymatic coupling of the appropriate methylated indole moiety with S-methyl-L-cysteine catalyzed by the enzyme tryptophanase.