Tryptophan Auxotrophic Strain Research Articles

Synthetic mutualism in engineered E. coli mutant strains as functional basis for microbial production consortia.

In nature, microorganisms often reside in symbiotic co-existence providing nutrition, stability, and protection for each partner by applying "division of labor." This principle may also be used for the overproduction of targeted compounds in bioprocesses. It requires the engineering of a synthetic co-culture with distributed tasks for each partner. Thereby, the competition on precursors, redox cofactors, and energy-which occurs in a single host-is prevented. Current applications often focus on unidirectional interactions, that is, the product of partner A is used for the completion of biosynthesis by partner B. Here, we present a synthetically engineered Escherichia coli co-culture of two engineered mutant strains marked by the essential interaction of the partners which is achieved by implemented auxotrophies. The tryptophan auxotrophic strain E. coli ANT-3, only requiring small amounts of the aromatic amino acid, provides the auxotrophic anthranilate for the tryptophan producer E. coli TRP-3. The latter produces a surplus of tryptophan which is used to showcase the suitability of the co-culture to access related products in future applications. Co-culture characterization revealed that the microbial consortium is remarkably functionally stable for a broad range of inoculation ratios. The range of robust and functional interaction may even be extended by proper glucose feeding which was shown in a two-compartment bioreactor setting with filtrate exchange. This system even enables the use of the co-culture in a parallel two-level temperature setting which opens the door to access temperature sensitive products via heterologous production in E. coli in a continuous manner.

Allosteric networks regulate function in multi‐enzyme complexes

Networks of noncovalent amino acid interactions propagate allosteric signals throughout proteins. Tryptophan synthase (TS) is an allosterically controlled bienzyme in which the indole product of the alpha subunit (αTS) is transferred through a 25 Å hydrophobic tunnel to the active site of the beta subunit (βTS). Previous nuclear magnetic resonance and molecular dynamics simulations identified allosteric networks in αTS important for its function. Here, we show that these allosteric networks change across the catalytic cycle of αTS, such that chemical catalysis is associated with specific conformational dynamics and networks not observed in the substrate‐ and products‐bound states. Moreover, substitution of a distant, surface‐exposed network residue in αTS enhances tryptophan production, not by activating αTS function, but through dynamically controlling the opening of the indole channel and stimulating βTS activity. While stimulation is modest, the substitution also enhances cell growth in a tryptophan‐auxotrophic strain of Escherichia coli compared to complementation with wild‐type αTS, emphasizing the biological importance of the network. Network residues thus represent ideal targets to optimize mutagenesis strategies in protein engineering studies directed towards changing enzyme function and regulation, especially in multi‐enzyme complexes.

Substitution of a Surface-Exposed Residue Involved in an Allosteric Network Enhances Tryptophan Synthase Function in Cells.

Networks of noncovalent amino acid interactions propagate allosteric signals throughout proteins. Tryptophan synthase (TS) is an allosterically controlled bienzyme in which the indole product of the alpha subunit (αTS) is transferred through a 25 Å hydrophobic tunnel to the active site of the beta subunit (βTS). Previous nuclear magnetic resonance and molecular dynamics simulations identified allosteric networks in αTS important for its function. We show here that substitution of a distant, surface-exposed network residue in αTS enhances tryptophan production, not by activating αTS function, but through dynamically controlling the opening of the indole channel and stimulating βTS activity. While stimulation is modest, the substitution also enhances cell growth in a tryptophan-auxotrophic strain of Escherichia coli compared to complementation with wild-type αTS, emphasizing the biological importance of the network. Surface-exposed networks provide new opportunities in allosteric drug design and protein engineering, and hint at potential information conduits through which the functions of a metabolon or even larger proteome might be coordinated and regulated.

Evaluation of the in vitro and in vivo genotoxicity of a Dioscorea Rhizome water extract.

Dioscorea Rhizome is commonly used in traditional herbal medicines for the treatment of diabetes, hyperthyroidism, liver damage, neuropathy, and asthma. Here, we investigated the genotoxicity potential of D. Rhizome water extract (DRWE) using three standard battery systems in accordance with the test guidelines of the Organisation for Economic Cooperation and Development and Ministry of Food and Drug Safety as well as the principles of Good Laboratory Practice. A bacterial reverse mutation test (Ames test) was performed using the direct plate incorporation method in the presence or absence of a metabolic activation system (S9 mixture). The tester strains used included four histidine auxotrophic strains of Salmonella typhimurium, TA100, TA1535, TA98, and TA1537, along with a tryptophan auxotrophic strain of Escherichia coli, WP2 uvrA. An in vitro chromosome aberration test was performed using CHL/IU cells originally derived from the lung of a female Chinese hamster in the presence or absence of the S9 mixture. An in vivo mouse bone marrow micronucleus test was performed using male ICR mice. The micronucleus was confirmed after observation of the micro-nucleated polychromatic. The Ames test showed that DRWE did not induce gene mutations at any dose level in any of the tested strains. Additionally, DRWE did not result in any chromosomal aberrations specified in the in vitro chromosomal aberration and in vivo micronucleus tests. These results showed that DRWE exhibited neither mutagenic nor clastogenic potential in either the in vitro or in vivo test systems.

Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein

BackgroundTrichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum.ResultsThe gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method.We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing.ConclusionsWe developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

Identification of a regulatory element for yeast tryptophan permease Tat2 ubiquitination using high hydrostatic pressure

ABSTRACTTryptophan uptake in the yeast Saccharomyces cerevisiae is extremely sensitive to high pressure; therefore, the growth of tryptophan auxotrophic strains is impaired. Degradation of tryptophan permease Tat2 is enhanced at 25 MPa, depending on Rsp5 ubiquitin ligase. Any defect in Tat2 ubiquitination confers high pressure growth capacity, which is a luminous phenotype of the yeast used to explore the mechanism by which Rsp5 mediates Tat2 ubiquitination. Here we show that the N-terminal four (K17, K20, K29, and K31) among five lysines are required for efficient Tat2 degradation under high pressure. We found that a domain spanning D70 to S76 is also critical for Tat2 degradation at 25 MPa probably because of the recognition by Bul1, an adaptor protein of Rsp5. Defects in Tat2 ubiquitination do not produce any remarkable mutant phenotype at 0.1 MPa. Therefore, we suggest that high pressure is a unique and advanced tool to explore ubiquitination-dependent Tat2 regulation.

Acetaminophen reduces the protein levels of high affinity amino acid permeases and causes tryptophan depletion

In yeast, toxicity of acetaminophen (APAP), a frequently used analgesic and antipyretic drug, depends on ubiquitin-controlled processes. Previously, we showed a remarkable overlap in toxicity profiles between APAP and tyrosine, and a similarity with drugs like rapamycin and quinine, which induce degradation of the amino acid permease Tat2. Therefore, we investigated in yeast whether APAP reduced the expression levels of amino acid permeases. The protein levels of Tat2, Tat1, Mup1 and Hip1 were reduced, while the expression of the general permease Gap1 was increased, consistent with a nutrient starvation response. Overexpression of Tat1 and Tat2, but not Mup1, Hip1 and Gap1 conferred resistance to APAP. A tryptophan auxotrophic strain trp1Δ was more sensitive to APAP than wild-type and addition of tryptophan completely restored the growth restriction of trp1∆ upon APAP exposure, while tyrosine had an additive effect on APAP toxicity. Furthermore, intracellular aromatic amino acid concentrations were reduced upon APAP exposure. This effect was less prominent in ubiquitin-deficient yeast strains that were APAP resistant and showed a reduced degradation of high affinity amino acid permeases. APAP-induced changes in intracellular amino acid concentrations were also detected in hepatoma HepG2 cells indicating significance for humans.

The YPR153W gene is essential for the pressure tolerance of tryptophan permease Tat2 in the yeast Saccharomyces cerevisiae

ABSTRACTIn the yeast Saccharomyces cerevisiae, hydrostatic pressure at 25 MPa is known to be nonlethal but significantly impairs the uptake of tryptophan by the permease Tat2, thereby inhibiting the growth of strains that require tryptophan from the medium. Here, we found that the lack of the YPR153W gene, so far poorly characterized for its role in yeast, caused a serious adverse effect on the growth at 10–25 MPa in the strain that required tryptophan. Deletion for YPR153W resulted in an increased rate of pressure-induced degradation of Tat2, suggesting that Tat2 is destabilized in the YPR153W deletion mutant at 25 MPa. Overexpression of the TAT2 gene enabled the deletion mutant to grow at 25 MPa. These results suggest that Ypr153w is essential for the stability and proper transport function of Tat2 under pressure at 10–25 MPa.ABBREVIATIONS: Trp+: the phenotype of tryptophan prototrophic strains that synthesize tryptophan de novo; Trp−: the phenotype of tryptophan auxotrophic strains that require tryptophan from the medium; YPR153W: an yeast poorly characterized gene; Ypr153w: a protein encoded by the YPR153W gene; ypr153wΔ: a deletion mutant for the YPR153W gene; Tat2: the high-affinity tryptophan permease; MCT10: a human aromatic amino acid transporter

Evaluation of the genotoxicity of ginseng leaf extract UG0712.

Although ginseng (genus Panax) leaf extract contains high concentrations of bioactive constituents, its effects have been reported in few preclinical studies, and information regarding its toxicity is not sufficient to allow for its clinical use. We evaluated the genotoxicity of UG0712, which is a powdered extract of ginseng leaves. UG0712 did not increase the number of revertant colonies in 4 histidine auxotrophic strains of Salmonella typhimurium (TA100, TA1535, TA98, and TA1537) or in a tryptophan auxotrophic strain of Escherichia coli (WP2uvrA(pKM101)) at any concentration evaluated, either in the absence or presence of the metabolic activation system. There was no significant increase in the number of metaphase cells with structural or numerical aberrations in the UG0712-treated groups compared to the concurrent vehicle control at any dose, regardless of the presence of the metabolic activation system. Oral administration of the extract at doses up to 2,000 mg/kg in male mice did not increase the frequency of micronucleated polychromatic erythrocytes in the bone marrow, and did not result in any significant clinical signs, body weight loss, gross findings, or mortality. These results suggest that UG0712 does not act as a mutagenic or genotoxic material at the concentrations evaluated.

Regulation of Yeast Nutrient Permease Endocytosis by ATP-binding Cassette Transporters and a Seven-transmembrane Protein, RSB1

Ceramide is produced by the condensation of a long chain base with a very long chain fatty acid. In Saccharomyces cerevisiae, one of the two major long chain bases is called phytosphingosine (PHS). PHS has been shown to cause toxicity in tryptophan auxotrophic strains of yeast because this bioactive ceramide precursor causes diversion of the high affinity tryptophan permease Tat2 to the vacuole rather than the plasma membrane. Loss of the integral membrane protein Rsb1 increased PHS sensitivity, which was suggested to be due to this protein acting as an ATP-dependent long chain base efflux protein. More recent experiments demonstrated that loss of the genes encoding the ATP-binding cassette transporter proteins Pdr5 and Yor1 elevated PHS tolerance. This increased resistance was suggested to be due to increased expression of RSB1. Here, we provide an alternative view of PHS resistance influenced by Rsb1 and Pdr5/Yor1. Rsb1 has a seven-transmembrane domain topology more consistent with that of a regulatory protein like a G-protein-coupled receptor rather than a transporter. Importantly, an rsb1Δ cell does not exhibit higher internal levels of PHS compared with isogenic wild-type cells. However, tryptophan transport is increased in pdr5Δ yor1 strains and reduced in rsb1Δ cells. Localization and vacuolar degradation of Tat2 are affected in these genetic backgrounds. Finally, internalization of FM4-64 dye suggests that loss of Pdr5 and Yor1 slows normal endocytic rates. Together, these data argue that Rsb1, Pdr5, and Yor1 regulate the endocytosis of Tat2 and likely other membrane transporter proteins.

Candida glabrata Persistence in Mice Does Not Depend on Host Immunosuppression and Is Unaffected by Fungal Amino Acid Auxotrophy

Candida glabrata has emerged as an important fungal pathogen of humans, causing life-threatening infections in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, even with high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leads to fungal burdens that are transiently increased over those in immunocompetent mice. However, even immunocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immune responses to microabscesses were mild as monitored by clinical parameters, including blood enzyme levels, histology, myeloperoxidase, and cytokine levels. Furthermore, we investigated the suitability of amino acid auxotrophic C. glabrata strains for in vitro and in vivo studies of fitness and/or virulence. Histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitro growth in rich medium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parental wild-type strain during the first week of infection and was only mildly reduced 4 weeks after infection, suggesting that C. glabrata is capable of utilizing a broad range of host-derived nutrients during infection. These data suggest that C. glabrata histidine, leucine, or tryptophan auxotrophic strains are suitable for the generation of knockout mutants for in vivo studies. Notably, our work indicates that C. glabrata has successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts.

Involvement of alkylhydroxybenzenes, microbial autoregulators, in controlling the expression of stress regulons

Alkylhydroxybenzenes (AHB) were found to control the activation of protective functions of microorganisms by inducing stress gene expression and increasing the frequency of the intrapopulation phase transitions which are responsible for the phenotypic variability of bacteria. We established the dependence of the regulatory effects of AHB on their structure (alkyl radical length) and concentration. A reversion assay using the tryptophan auxotrophic strain Bacillus subtilis trpA5 B 1733 indicated a relationship between the reversion frequency that was 40–120 times higher than the background value and phase transition’s intensity (with R → S transition rates up to 87% in contrast to 2% in the control experiment) induced by specific doses (5–100 mg/ml) of long-chain AHB such as C12-AHB acting for a short time (1 h) on vegetative (dividing or stationary-phase) B. subtilis cells. Using four test strains constructed from Escherichia coli C600 thi, thr, leuΔ(pro-lac) with transcriptional or translational vectors containing the hybrid umuD-lacZ or osmE-lacZ operons, we demonstrated that AHB perform the regulatory functions involved in controlling the SOS response gene expression and the general rpoS -dependent stationary-phase regulon, respectively. The dose-dependent effect of long-chain AHB (within the 50–100 µg/ml range) resulting in a two- to threefold increase in the stress gene expression, similar to the effect of natural stress factors such as UV irradiation and starvation, provides evidence that AHB function as alarmones (danger signals). From the fact that the osm E gene is upregulated by 35–70 µg/ml C12-AHB (its regulation level is increased up to twofold), it follows that C12-AHB controls rpoS-dependent regulation and the transition to the stationary phase. The effect of the short-chain homologue C7-AHB substantially differs from that of C12-AHB. C7-AHB in concentrations of 10–100µg/ml causes a significant decrease in osmE and umuD expression. A 30-min preincubation of cells with 10–100µg/ml C7-AHB protected them from UV irradiation, as was observed as a 3.6-fold decrease in umuD expression. Comparative analysis of the marker gene’s expression values in the strains with the transcriptional and translational vectors demonstrates that AHB nonspecifically activate stress regulons at the transcription level.

Regulation and dynamics of yeast tryptophan permeases studied using hydrostatic pressure

Abstract. The yeast Saccharomyces cerevisiae possesses 24 amino acid permeases and their homologs, which exhibit different properties with respect to substrate specificity, capacity, and regulation. These permeases are regulated in response to physiological and nutritional changes at the transcriptional and posttranslational levels. Two permeases, Tat1 and Tat2, mediate the import of tryptophan across the plasma membrane. There is evidence showing that tryptophan uptake is the Achilles' heel of yeast physiology since a variety of adverse conditions impair its uptake, and hence limit the growth of tryptophan-auxotrophic strains. Recent studies in yeast cell biology have yielded information on the posttranslational regulatory systems of the two tryptophan permeases by ubiquitination. When cells are subjected to nutrient starvation, toxic chemicals, low temperature, or high hydrostatic pressure, Tat2 is rapidly degraded in a manner dependent on Rsp5 ubiquitin ligase. Hydrostatic pressure is a thermodynamic parameter that has recently received renewed attention in biophysics, biochemistry, and microbiology. Researchers have attempted to analyze system volume changes associated with protein unfolding, enzymatic reactions, or membrane protein functions. Here I review the literature on yeast tryptophan permeases and the properties of these permease proteins using hydrostatic pressure to investigate the dynamic structural changes of tryptophan permeases.

Alkanindiges hongkongensis sp. nov. A novel Alkanindiges species isolated from a patient with parotid abscess

A bacterium was isolated from the abscess pus of a 72-year-old patient with Warthin's tumor and parotid abscess. The cells were aerobic, non-motile, Gram-negative but difficult to be destained, non-sporulating, coccobacillus. The bacterium grew poorly on sheep blood agar and MacConkey agar as non-hemolytic colonies of 0.5 mm in diameter after 24 h of incubation at 37 °C in ambient air. Growth was enhanced by Tween 80. It produces catalase but not cytochrome oxidase. Sequencing of the cloned 16S rRNA PCR products of the bacterium revealed three different 16S rRNA gene sequences, with 12–31 bp differences among them. Phylogenetic analysis showed that the bacterium is closely related to Alkanindiges illinoisensis, with 5.0–5.9% differences between the 16S rRNA gene sequence of the bacterium and that of A. illinoisensis. Tryptophan auxotrophic strain of Acinetobacter trpE27 transformed with DNA extracted from the bacterium was unable to grow on tryptophan deficient medium, indicating that the bacterium was not a strain of Acinetobacter. The G+C content of the bacterium (mean±SD) was 46.9±4.3%. A new species, Alkanindiges hongkongensis sp. nov., is proposed, for which HKU9 T is the type strain. Isolates with “small colonies” that are apparently Acinetobacter-like species should be carefully identified. Growth enhancement with aliphatic hydrocarbons should be looked for and 16S rRNA gene sequencing performed in order to find more potential cases of Alkanindiges infections, as well as to define the epidemiology, clinical spectrum, and outcome of infections associated with this genus.

Multiple ubiquitin-specific protease genes are involved in degradation of yeast tryptophan permease Tat2 at high pressure

When Saccharomyces cerevisiae cells are exposed to high hydrostatic pressure, tryptophan permease Tat2 is degraded in a manner dependent on Rsp5 ubiquitin ligase. Consequently, cell growth is arrested in tryptophan auxotrophic strains. Here we show that of 17 ubiquitin-specific protease genes ( UBP), deletion of DOA4, UBP6 or UBP14 causes stabilization of Tat2 and hence the cells can grow at 25 MPa. These disruptant cells displayed marked sensitivity to the arginine analogue canavanine. Internal free ubiquitin decreased 2- to 5-fold upon UBP deletion, although overproduction of ubiquitin did not affect their high-pressure growth and canavanine sensitivity. These results suggest that multiple ubiquitin-specific proteases are involved in pressure-induced degradation of Tat2, rather than free ubiquitin depletion.

Domain-specific spectroscopy of 5-hydroxytryptophan-containing variants of Escherichia coli DnaJ

Tryptophan-containing variants of Escherichia coli DnaJ protein were constructed in order to examine the hypothetical domain structure by fluorescence quenching and denaturant-induced unfolding. Two residues in the J-domain and one in the Gly/Phe-rich region were targeted for replacement and the proteins were expressed in a tryptophan auxotrophic strain in the presence of 5-hydroxytryptophan (5-HW). Fluorescence quenching with iodide of 5-HW in the variant proteins suggests that the Gly/Phe-rich region is more accessible to solvent than the J-domain. This is consistent with the proposal that the Gly/Phe-rich region is unstructured. Unfolding of the 5-HW-containing variants was monitored by fluorescence, and the results showed that the unfolding of the J-domain is cooperative and the unfolding of the Gly/Phe-rich region is not cooperative.

Sexual development of Aspergillus nidulans in tryptophan auxotrophic strains.

The interplay between sexual development and amino acid biosynthesis in the ascomycete Aspergillus nidulans was studied. The growth and differentiation of four tryptophan auxotrophic strains that are unable to regulate their tryptophan biosynthesis, were examined. These strains are sterile on medium containing low tryptophan concentrations. Fruitbody formation was restored by supplementation with high concentrations of tryptophan and was promoted by supplementation with indole or auxin. Tryptophan supplementation resulted in auxin production of A. nidulans. Fertility of ascospores of the tryptophan auxotrophic strains could only be partially re-established. trpC transcript levels and enzyme activities remained stable in cleistothecia and conidiophore extracts as compared to those of mycelium, while levels of gene transcripts involved in glycolysis were lower in fruitbodies and conidiospores. Auxotrophic strains unable to form fruitbodies at intermediate amino acid supplementation levels turned on the cross-pathway regulatory system that is induced by amino acid starvation. We conclude that there is a connection between the genetic network of cross-pathway control and sexual development in A. nidulans.