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Essential amino acid, tryptophan which intake from food plays a critical role in numerous metabolic functions, exhibiting extensive biological functions and applications. Tryptophan is beneficial for the food sector by enhancing nutritional content and promoting the development of functional foods. A putative gene encoding tryptophan synthase was the first identified in Sphingobacterium soilsilvae Em02, a cellulosic bacterium making it inherently more environmentally friendly. The gene was cloned and expressed in exogenous host Escherichia coli, to elucidate its function. The recombinant tryptophan synthase with a molecular weight 42 KDa was expressed in soluble component. The enzymatic activity to tryptophan synthase in vivo was assessed using indole and L-serine and purified tryptophan synthase. The optimum enzymatic activity for tryptophan synthase was recorded at 50 ºC and pH 7.0, which was improved in the presence of metal ions Mg 2+ , Sr 2+ and Mn 2+ , whereas Cu 2+ , Zn 2+ and Co 2+ proved to be inhibitory. Using site-directed mutagenesis, the consensus pattern HK-S-[GGGSN]-E-S in the tryptophan synthase was demonstrated with K100Q, S202A, G246A, E361A and S385A as the active sites. Tryptophan synthase has been demonstrated to possess the defining characteristics of the β-subunits. The tryptophan synthase may eventually be useful for tryptophan production on a larger scale. Its diverse applications highlight the potential for improving both the quality and health benefits of food products, making it an essential component in advancing food science and technology., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Indigo, as a water-soluble non-azo colorant, is widely used in textile, food, pharmaceutical and other industrial fields. Currently, indigo is primarily synthesized by chemical methods, which causes environmental pollution, potential safety hazards, and other issues. Therefore, there is an urgent need to find a safer and greener synthetic method. In this study, a dual-enzyme cascade pathway was constructed with the tryptophan synthase (tryptophanase, Ec TnaA) from Escherichia coli and flavin-dependent monooxygenase (flavin-dependent monooxygenase, Ma FMO) from Methylophaga aminisulfidivorans to synthesize indigo with L-tryptophan as substrate. A recombinant strain EM -IND01 was obtained. The beneficial mutant Ma FMO D197E was obtained by protein engineering of the rate-limiting enzyme Ma FMO. Ma FMO D197E showed the specific activity and k cat / K m value 2.36 times and 1.34 times higher than that of the wild type, respectively. Furthermore, Ma FMO D197E was introduced into the strain EM -IND01 to construct the strain EM -IND02. After the fermentation conditions were optimized, the strain achieved the indigo titer of (1 288.59±7.50) mg/L, the yield of 0.86 mg/mg L-tryptophan, and the productivity of 26.85 mg/(L·h) in a 5 L fermenter. Protein engineering was used to obtain mutants with increased Ma FMO activity in this study, which laid a foundation for industrial production of indigo.

Protein engineering often targets binding pockets or active sites which are enriched in epistasis-nonadditive interactions between amino acid substitutions-and where the combined effects of multiple single substitutions are difficult to predict. Few existing sequence-fitness datasets capture epistasis at large scale, especially for enzyme catalysis, limiting the development and assessment of model-guided enzyme engineering approaches. We present here a combinatorially complete, 160,000-variant fitness landscape across four residues in the active site of an enzyme. Assaying the native reaction of a thermostable β-subunit of tryptophan synthase (TrpB) in a nonnative environment yielded a landscape characterized by significant epistasis and many local optima. These effects prevent simulated directed evolution approaches from efficiently reaching the global optimum. There is nonetheless wide variability in the effectiveness of different directed evolution approaches, which together provide experimental benchmarks for computational and machine learning workflows. The most-fit TrpB variants contain a substitution that is nearly absent in natural TrpB sequences-a result that conservation-based predictions would not capture. Thus, although fitness prediction using evolutionary data can enrich in more-active variants, these approaches struggle to identify and differentiate among the most-active variants, even for this near-native function. Overall, this work presents a large-scale testing ground for model-guided enzyme engineering and suggests that efficient navigation of epistatic fitness landscapes can be improved by advances in both machine learning and physical modeling., Competing Interests: Competing interests statement:The authors declare no competing interest.

L-tryptophan (l-Trp), a vital amino acid for the survival of various organisms, is synthesized by the enzyme tryptophan synthase (TS) in organisms such as eubacteria, archaebacteria, protista, fungi, and plantae. TS, a pyridoxal 5'-phosphate (PLP)-dependent enzyme, comprises α and β subunits that typically form an α 2 β 2 tetramer. The enzyme's activity is regulated by the conformational switching of its α and β subunits between the open (T state) and closed (R state) conformations. Many microorganisms rely on TS for growth and replication, making the enzyme and the l-Trp biosynthetic pathway potential drug targets. For instance, Mycobacterium tuberculosis , Chlamydiae bacteria, Streptococcus pneumoniae , Francisella tularensis , Salmonella bacteria, and Cryptosporidium parasitic protozoa depend on l-Trp synthesis. Antibiotic-resistant salmonella strains have emerged, underscoring the need for novel drugs targeting the l-Trp biosynthetic pathway, especially for salmonella-related infections. A single amino acid mutation can significantly impact enzyme function, affecting stability, conformational dynamics, and active or allosteric sites. These changes influence interactions, catalytic activity, and protein-ligand/protein-protein interactions. This study focuses on the impact of mutating the βGln114 residue on the catalytic and allosteric sites of TS. Extensive molecular dynamics simulations were conducted on E(PLP), E(AEX 1 ), E(A-A), and E(C 3 ) forms of TS using the WT, βQ114A, and βQ114N versions. The results show that both the βQ114A and βQ114N mutations increase protein backbone root mean square deviation fluctuations, destabilizing all TS forms. Conformational and hydrogen bond analyses suggest the significance of βGln114 drifting away from cofactor/intermediates and forming hydrogen bonds with water molecules necessary for l-Trp biosynthesis. The βQ114A mutation creates a gap between βAla114 and cofactor/intermediates, hindering hydrogen bond formation due to short side chains and disrupting β-sites. Conversely, the βQ114N mutation positions βAsn114 closer to cofactor/intermediates, forming hydrogen bonds with O3 of cofactors/intermediates and nearby water molecules, potentially disrupting the l-Trp biosynthetic mechanism.

Tryptophan synthase (TRPS) is a complex enzyme responsible for tryptophan biosynthesis. It occurs in bacteria, plants, and fungi as an αββα heterotetramer. Although encoded by independent genes in bacteria and plants, in fungi, TRPS is generated by a single gene that concurrently expresses the α and β entities, which are linked by an elongated peculiar segment. We conducted 1 µs all-atom molecular dynamics simulations on Hemileia vastatrix TRPS to address two questions: (i) the role of the linker segment and (ii) the comparative mode of action. Since there is not an experimental structure, we started our simulations with homology modeling. Based on the results, it seems that TRPS makes use of an already-existing tunnel that can spontaneously move the indole moiety from the α catalytic pocket to the β one. Such behavior was completely disrupted in the simulation without the linker. In light of these results and the αβ dimer's low stability, the full-working TRPS single genes might be the result of a particular evolution. Considering the significant losses that Hemileia vastatrix causes to coffee plantations, our next course of action will be to use the TRPS to look for substances that can block tryptophan production and therefore control the disease.

Indole in the gut is formed from dietary tryptophan by a bacterial tryptophan-indole lyase. Indole not only triggers biofilm formation and antibiotic resistance in gut microbes but also contributes to the progression of kidney dysfunction after absorption by the intestine and sulfation in the liver. As tryptophan is an essential amino acid for humans, these events seem inevitable. Despite this, we show in a proof-of-concept study that exogenous indole can be converted to an immunomodulatory tryptophan metabolite, indole-3-lactic acid (ILA), by a previously unknown microbial metabolic pathway that involves tryptophan synthase β subunit and aromatic lactate dehydrogenase. Selected bifidobacterial strains converted exogenous indole to ILA via tryptophan (Trp), which was demonstrated by incubating the bacterial cells in the presence of (2- 13 C)-labeled indole and l-serine. Disruption of the responsible genes variedly affected the efficiency of indole bioconversion to Trp and ILA, depending on the strains. Database searches against 11,943 bacterial genomes representing 960 human-associated species revealed that the co-occurrence of tryptophan synthase β subunit and aromatic lactate dehydrogenase is a specific feature of human gut-associated Bifidobacterium species, thus unveiling a new facet of bifidobacteria as probiotics. Indole, which has been assumed to be an end-product of tryptophan metabolism, may thus act as a precursor for the synthesis of a host-interacting metabolite with possible beneficial activities in the complex gut microbial ecosystem.

Tryptophan synthetase (TSase), which functions as a tetramer, is a typical enzyme with a substrate channel effect, and shows excellent performance in the production of non-standard amino acids, histamine, and other biological derivatives. Based on previous work, we fused a mutant CE protein (colistin of E. coli , a polypeptide with antibacterial activity) sequence with the sequence of TSase to explore whether its catalytic activity could be enhanced, and we also analyzed whether the addition of a DNA scaffold was a feasible strategy. Here, dCE (CE protein without DNase activity) protein tags were constructed and fused to the TrapA and TrapB subunits of TSase, and the whole cell was used for the catalytic reaction. The results showed that after the dCE protein tag was fused to the TrapB subunit, its whole cell catalytic activity increased by 50%. Next, the two subunits were expressed separately, and the proteins were bound in vitro to ensure equimolar combination between the two subunits. After the dCE label was fused to TrapB, the activity of TSase assembled with TrapA also improved. A series of experiments revealed that the enzyme fused with dCE9 showed higher activity than the wild-type protein. In general, the activity of assembly TSase was optimal when the temperature was 50 °C and the pH was about 9.0. After a long temperature treatment, the enzyme maintained good activity. With the addition of exogenous nucleic acid, the activity of the enzyme increased. The maximum yield was 0.58 g/L, which was almost three times that of the wild-type TSase (0.21 g/L). The recombinant TSase constructed in this study with dCE fusion had the advantages of higher heat resistance and higher activity, and confirmed the feasibility of adding a nucleic acid scaffold, providing a new idea for the improvement of structurally similar enzymes.

We report here a new application, CustomProteinSearch (CusProSe), whose purpose is to help users to search for proteins of interest based on their domain composition. The application is customizable. It consists of two independent tools, IterHMMBuild and ProSeCDA. IterHMMBuild allows the iterative construction of Hidden Markov Model (HMM) profiles for conserved domains of selected protein sequences, while ProSeCDA scans a proteome of interest against an HMM profile database, and annotates identified proteins using user-defined rules. CusProSe was successfully used to identify, in fungal genomes, genes encoding key enzyme families involved in secondary metabolism, such as polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), hybrid PKS-NRPS and dimethylallyl tryptophan synthases (DMATS), as well as to characterize distinct terpene synthases (TS) sub-families. The highly configurable characteristics of this application makes it a generic tool, which allows the user to refine the function of predicted proteins, to extend detection to new enzymes families, and may also be applied to biological systems other than fungi and to other proteins than those involved in secondary metabolism., (© 2023. The Author(s).)

The P1-like phage plasmid (PP) has been widely used as a molecular biology tool, but its role as an active accessory cargo element is not fully understood. In this study, we provide insights into the structural features and gene content similarities of 77 P1-like PPs in the RefSeq database. We also describe a P1-like PP carrying a bla CTX-M-55 gene, JL22, which was isolated from a clinical strain of Escherichia coli from a duck farm. P1-like PPs were very similar and conserved based on gene content similarities, with only eight highly variable regions. Importantly, two kinds of replicon types, namely, IncY and p0111, were identified and can be used to specifically identify the P1-like phage. JL22 is similar to P1, acquiring an important foreign DNA fragment with two obvious features, namely, the plasmid replication gene repA' (p0111) replacing the gene repA (IncY) and a 4,200-bp fragment mobilized by IS 1380 and IS 5 and containing a bla CTX-M-55 gene and a trpB gene encoding tryptophan synthase (indole salvaging). The JL22 phage could be induced but had no lytic capacities. However, a lysogenic recipient and intact structure of JL22 virions were observed, showing that the extended-spectrum β-lactamase bla CTX-M-55 gene was successfully transferred. Overall, conserved genes can be a good complement to improve the identification efficiency and accuracy in future screening for P1-like PPs. Moreover, the highly conserved structures may be important for their prevalence and dissemination. IMPORTANCE As a PP, P1 DNA exists as a low-copy-number plasmid and replicates autonomously with a lysogenization style. This unique mode of P1-like elements probably indicates a stable contribution to antibiotic resistance. After analyzing these elements, we show that P1-like PPs are very similar and conserved, with only eight highly variable regions. Moreover, we observed the occurrence of replicon IncY and p0111 only in the P1-like PP community, implying that these conserved regions, coupled with IncY and p0111, can be an important complement in future screening of P1-like PPs. Identification and characterization of JL22 confirmed our findings that major changes were located in variable regions, including the first detection of bla CTX-M-55 in such a mobile genetic element. This suggests that these variable regions may facilitate foreign DNA mobilization. This study features a comprehensive genetic analysis of P1-like PPs, providing new insights into the dissemination mechanisms of antibiotic resistance through P1 PPs.

The reverse genetic approach has uncovered indole synthase (INS) as the first enzyme in the tryptophan (trp)-independent pathway of IAA synthesis. The importance of INS was reevaluated suggesting it may interact with tryptophan synthase B (TSB) and therefore involved in the trp-dependent pathway. Thus, the main aim of this study was to clarify the route of INS through the analysis of Arabidopsis genome. Analysis of the top 2000 co-expression gene lists in general and specific conditions shows that TSA is strongly positively co-expressed with TSB in general, hormone, and abiotic conditions with mutual ranks of 89, 38, and 180 respectively. Moreover, TSA is positively correlated with TSB (0.291). However, INS was not found in any of these coexpressed gene lists and negatively correlated with TSB (- 0.046) suggesting unambiguously that these two routes are separately and independently operated. So far, the remaining steps in the INS pathway have remained elusive. Among all enzymes reported to have a role in IAA synthesis, amidase was found to strongly positively co-expressed with INS in general and light conditions with mutual ranks of 116 and 141 respectively. Additionally, amidase1 was found to positively correlate with INS (0.297) and negatively coexpressed with TSB concluding that amidase may exclusively involve in the trp-independent pathway., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Chlamydia trachomatis ( C. trachomatis ) is the most common etiological agent of bacterial sexually transmitted infections (STIs) and a worldwide public health issue. The natural course with C. trachomatis infection varies widely between individuals. Some infections clear spontaneously, others can last for several months or some individuals can become reinfected, leading to severe pathological damage. Importantly, the underlying mechanisms of C. trachomatis infection are not fully understood. C. trachomatis has the ability to adapt to immune response and persist within host epithelial cells. Indoleamine-2,3-dioxygenase (IDO) induced by interferon-gamma (IFN-γ) degrades the intracellular tryptophan pool, to which C. trachomatis can respond by converting to a non-replicating but viable state. C. trachomatis expresses and encodes for the tryptophan synthase (TS) genes ( trpA and trpB ) and tryptophan repressor gene ( trpR ). Multiple genes interact to regulate tryptophan synthesis from exogenous indole, and persistent C. trachomatis can recover its infectivity by converting indole into tryptophan. In this review, we discuss the characteristics of chlamydial infections, biosynthesis and regulation of tryptophan, the relationship between tryptophan and C. trachomatis , and finally, the links between the tryptophan/IFN-γ axis and C. trachomatis persistence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Hou, Yuan and Chen.)

To adapt to changing environments, plants have evolved elaborate regulatory mechanisms balancing their growth with stress responses. It is currently unclear whether and how the tryptophan (Trp), the growth-related hormone auxin, and the stress hormone abscisic acid (ABA) are coordinated in this trade-off. Here, we show that tryptophan synthase β subunit 1 (TSB1) is involved in the coordination of Trp and ABA, thereby affecting plant growth and abiotic stress responses. Plants experiencing high salinity or drought display reduced TSB1 expression, resulting in decreased Trp and auxin accumulation and thus reduced growth. In comparison with the wild type, amiR-TSB1 lines and TSB1 mutants exhibited repressed growth under non-stress conditions but had enhanced ABA accumulation and stress tolerance when subjected to salt or drought stress. Furthermore, we found that TSB1 interacts with and inhibits β-glucosidase 1 (BG1), which hydrolyses glucose-conjugated ABA into active ABA. Mutation of BG1 in the amiR-TSB1 lines compromised their increased ABA accumulation and enhanced stress tolerance. Moreover, stress-induced H 2 O 2 disrupted the interaction between TSB1 and BG1 by sulfenylating cysteine-308 of TSB1, relieving the TSB1-mediated inhibition of BG1 activity. Taken together, we revealed that TSB1 serves as a key coordinator of plant growth and stress responses by balancing Trp and ABA homeostasis., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

Indole is produced in nature by diverse organisms and exhibits a characteristic odor described as animal, fecal, and floral. In addition, it contributes to the flavor in foods, and it is applied in the fragrance and flavor industry. In nature, indole is synthesized either from tryptophan by bacterial tryptophanases (TNAs) or from indole-3-glycerol phosphate (IGP) by plant indole-3-glycerol phosphate lyases (IGLs). While it is widely accepted that the tryptophan synthase α-subunit (TSA) has intrinsically low IGL activity in the absence of the tryptophan synthase β-subunit, in this study, we show that Corynebacterium glutamicum TSA functions as a bona fide IGL and can support fermentative indole production in strains providing IGP. By bioprospecting additional bacterial TSAs and plant IGLs that function as bona fide IGLs were identified. Capturing indole in an overlay enabled indole production to titers of about 0.7 g L -1 in fermentations using C. glutamicum strains expressing either the endogenous TSA gene or the IGL gene from wheat.

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5'-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and β-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3'-phosphate and l-Ser to l-Trp. In this process, the roles played by β-site residues proximal to the PLP cofactor have not yet been fully established. βGln114 is one such residue. To explore the roles played by βQ114, we conducted a detailed investigation of the βQ114A mutation on the structure and function of tryptophan synthase. Initial steady-state kinetic and static ultraviolet-visible spectroscopic analyses showed the Q to A mutation impairs catalytic activity and alters the stabilities of intermediates in the β-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and βQ114A mutant enzymes. These comparisons establish that the protein structural changes are limited to the Gln to Ala replacement, the loss of hydrogen bonds among the side chains of βGln114, βAsn145, and βArg148, and the inclusion of waters in the cavity created by substitution of the smaller Ala side chain. Because the conformations of the open and closed allosteric states are not changed by the mutation, we hypothesize that the altered properties arise from the lost hydrogen bonds that alter the relative stabilities of the open (β T state) and closed (β R state) conformations of the β-subunit and consequently alter the distribution of intermediates along the β-subunit catalytic path.

L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high-activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and k cat /K m of the mutant enzyme (V231A/K382G) is 4.36 mM -1 ∙s -1 . The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intracellular growth and pathogenesis of Chlamydia species is controlled by the availability of tryptophan, yet the complete biosynthetic pathway for l-Trp is absent among members of the genus. Some representatives, however, preserve genes encoding tryptophan synthase, TrpAB - a bifunctional enzyme catalyzing the last two steps in l-Trp synthesis. TrpA (subunit α) converts indole-3-glycerol phosphate into indole and glyceraldehyde-3-phosphate (α reaction). The former compound is subsequently used by TrpB (subunit β) to produce l-Trp in the presence of l-Ser and a pyridoxal 5'-phosphate cofactor (β reaction). Previous studies have indicated that in Chlamydia, TrpA has lost its catalytic activity yet remains associated with TrpB to support the β reaction. Here, we provide detailed analysis of the TrpAB from C. trachomatis D/UW-3/CX, confirming that accumulation of mutations in the active site of TrpA renders it enzymatically inactive, despite the conservation of the catalytic residues. We also show that TrpA remains a functional component of the TrpAB complex, increasing the activity of TrpB by four-fold. The side chain of non-conserved βArg267 functions as cation effector, potentially rendering the enzyme less susceptible to the solvent ion composition. The observed structural and functional changes detected herein were placed in a broader evolutionary and genomic context, allowing identification of these mutations in relation to their trp gene contexts in which they occur. Moreover, in agreement with the in vitro data, partial relaxation of purifying selection for TrpA, but not for TrpB, was detected, reinforcing a partial loss of TrpA functions during the course of evolution., (© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The obligate intracellular pathogen Chlamydia trachomatis ( Ct ) is the leading cause of bacterial sexually transmitted infections and blindness globally. To date, Ct urogenital strains are considered tryptophan prototrophs, utilizing indole for tryptophan synthesis within a closed-conformation tetramer comprised of two α (TrpA)- and two β (TrpB)-subunits. In contrast, ocular strains are auxotrophs due to mutations in TrpA, relying on host tryptophan pools for survival. It has been speculated that there is strong selective pressure for urogenital strains to maintain a functional operon. Here, we performed genetic, phylogenetic, and novel functional modeling analyses of 595 geographically diverse Ct ocular, urethral, vaginal, and rectal strains with complete operon sequences. We found that ocular and urogenital, but not lymphogranuloma venereum, TrpA-coding sequences were under positive selection. However, vaginal and urethral strains exhibited greater nucleotide diversity and a higher ratio of nonsynonymous to synonymous substitutions [Pi(a)/Pi(s)] than ocular strains, suggesting a more rapid evolution of beneficial mutations. We also identified nonsynonymous amino acid changes for an ocular isolate with a urogenital backbone in the intergenic region between TrpR and TrpB at the exact binding site for YtgR-the only known iron-dependent transcription factor in Chlamydia -indicating that selective pressure has disabled the response to fluctuating iron levels. In silico effects on protein stability, ligand-binding affinity, and tryptophan repressor (TrpR) affinity for single-stranded DNA (ssDNA) measured by calculating free energy changes (ΔΔ G ) between Ct reference and mutant tryptophan operon proteins were also analyzed. We found that tryptophan synthase function was likely suboptimal compared to other bacterial tryptophan prototrophs and that a diversity of urogenital strain mutations rendered the synthase nonfunctional or inefficient. The novel mutations identified here affected active sites in an orthosteric manner but also hindered α- and β-subunit allosteric interactions from distant sites, reducing efficiency of the tryptophan synthase. Importantly, strains with mutant proteins were inclined toward energy conservation by exhibiting an altered affinity for their respective ligands compared to reference strains, indicating greater fitness. This is not surprising as l-tryptophan is one of the most energetically costly amino acids to synthesize. Mutations in the tryptophan repressor gene ( trp R) among urogenital strains were similarly detrimental to function. Our findings indicate that urogenital strains are evolving more rapidly than previously recognized with mutations that impact tryptophan operon function in a manner that is energetically beneficial, providing a novel host-pathogen evolutionary mechanism for intracellular survival. IMPORTANCE Chlamydia trachomatis ( Ct ) is a major global public health concern causing sexually transmitted and ocular infections affecting over 130 million and 260 million people, respectively. Sequelae include infertility, preterm birth, ectopic pregnancy, and blindness. Ct relies on available host tryptophan pools and/or substrates to synthesize tryptophan to survive. Urogenital strains synthesize tryptophan from indole using their intact tryptophan synthase (TS). Ocular strains contain a trp A frameshift mutation that encodes a truncated TrpA with loss of TS function. We found that TS function is likely suboptimal compared to other tryptophan prototrophs and that urogenital stains contain diverse mutations that render TS nonfunctional/inefficient, evolve more rapidly than previously recognized, and impact operon function in a manner that is energetically beneficial, providing an alternative host-pathogen evolutionary mechanism for intracellular survival. Our research has broad scientific appeal since our approach can be applied to other bacteria that may explain evolution/survival in host-pathogen interactions., (Copyright © 2021 Bommana et al.)

Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this working state represents a four to one ratio of the enzyme bound with the indole-3-glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde-3-phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate- and products-bound states to the working state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in the working state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in the working state. As the enzyme transitions from the substrate-bound to the products-bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit., (© 2020 The Protein Society.)

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB's primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.

The clustered regularly interspaced short palindromic repeats (CRISPR) - Cas associated protein 9 (Cas9) system is very precise, efficient and relatively simple in creating genetic modifications at a predetermined locus in the genome. Genome editing with Cas9 ribonucleoproteins (RNPs) has reduced cytotoxic effects, off-target cleavage and increased on-target activity and the editing efficiencies. The unicellular alga Chlamydomonas reinhardtii is an emerging model for studying the production of high-value products for industrial applications. Development of C. reinhardtii as an industrial biotechnology host can be achieved more efficiently through genetic modifications using genome editing tools. We made an attempt to target MAA7 gene that encodes the tryptophan synthase β-Subunit using CRISPR-Cas9 RNPs to demonstrate knock-out and knock-in through homology-dependent repair template at the target site. In this study, we have demonstrated targeted gene knock-out in C. reinhardtii using programmed RNPs. Targeted editing of MAA7 gene was confirmed by sequencing the clones that were resistant to 5-Fluoroindole (5-FI). Non-homologous end joining (NHEJ) repair mechanism led to insertion, deletion, and/or base substitution in the Cas9 cleavage vicinity, encoding non-functional MAA7 protein product (knock-out), conferring resistance to 5-FI. Here, we report an efficient protocol for developing knock-out mutants in Chlamydomonas using CRISPR-Cas9 RNPs. The high potential efficiency of editing may also eliminate the need to select mutants by phenotype. These research findings would be more likely applied to other green algae for developing green cell factories to produce high-value molecules.

Structural studies with tryptophan synthase (TS) bienzyme complex (α2β2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated α- and isolated β-subunit allowed the purification of high amounts of pure subunits and α2β2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme β-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α2β2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α2β2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the α2β2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant α2β2 StTS complexes.

Tryptophan synthase (TS) is a heterotetrameric αββα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the β-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a αββα complex with indole channeling taking place. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis allosterically increases the activity of ncTrpB. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes., Competing Interests: The authors declare no competing interest.

We previously engineered the β-subunit of tryptophan synthase (TrpB), which catalyzes the condensation of l-serine and indole to l-tryptophan, to synthesize a range of noncanonical amino acids from l-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C-C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N-C bonds on N 1 of the substrate. Protecting N 1 encouraged evolution toward C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pf quat with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereocenter at the γ-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the γ-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L E. coli culture, TrpB Pf quat offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.

The αββα tryptophan synthase (TS), which is part of primary metabolism, is a paradigm for allosteric communication in multienzyme complexes. In particular, the intrinsically low catalytic activity of the α-subunit TrpA is stimulated several hundredfold through the interaction with the β-subunit TrpB1. The BX1 protein from Zea mays (zmBX1), which is part of secondary metabolism, catalyzes the same reaction as that of its homologue TrpA, but with high activity in the absence of an interaction partner. The intrinsic activity of TrpA can be significantly increased through the exchange of several active-site loop residues, which mimic the corresponding loop in zmBX1. The subsequent identification of activating amino acids in the generated "stand-alone" TrpA contributes to an understanding of allostery in TS. Moreover, findings suggest an evolutionary trajectory that describes the transition from a primary metabolic enzyme regulated by an interaction partner to a self-reliant, stand-alone, secondary metabolic enzyme., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The goal of this study was to investigate the relationship between the denitrification process and carbon metabolism in a full-scale tannery wastewater treatment plant bioaugmented with the microbial consortium BM-S-1. The metagenomic analysis of the microbial community showed that Brachymonas denitrificans, a known denitrifier, was present at a high level in the treatment stages of buffering (B), primary aeration (PA), and sludge digestion (SD). The occurrences of the amino acid-degrading enzymes alpha ketoglutarate dehydrogenase (α-KGDH) and tryptophan synthase were highly correlated with the presence of denitrification genes, such as napA, narG, nosZ and norB. The occurrence of glutamate dehydrogenase (GDH) was also highly paralleled with the occurrence of denitrification genes such as napA, narG, and norZ. The denitrification genes (nosZ, narG, napA, norB and nrfA) and amino acid degradation enzymes (tryptophan synthase, α-KGDH and pyridoxal phosphate dependent enzymes) were observed at high levels in B. This indicates that degradation of amino acids and denitrification of nitrate may potentially occur in B. The high concentrations of the fatty acid degradation enzyme groups (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and β-ketothiolase) were observed together with the denitrification genes, such as napA, narG and nosZ. Phospholipase/carboxylesterase, enoyl-CoA hydratase/isomerase, acyl-CoA dehydrogenase, phenylacetate degradation enzyme and 3-hydroxyacyl-CoA dehydrogenase 2 were also dominant in B. All these results clearly indicate that the denitrification pathways are potentially linked to the degradation pathways of amino acids and fatty acids whose degradation products go through the TCA cycle, generating the NADH that is used as electron donors for denitrification.

Clinical persistence of Chlamydia trachomatis ( Ct ) sexually transmitted infections (STIs) is a major public health concern. In vitro persistence is known to develop through interferon gamma (IFN-γ) induction of indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan, an essential amino acid for Ct replication. The organism can recover from persistence by synthesizing tryptophan from indole, a substrate for the enzyme tryptophan synthase. The majority of Ct strains, except for reference strain B/TW-5/OT, contain an operon comprised of α and β subunits that encode TrpA and TrpB, respectively, and form a functional αββα tetramer. However, trpA mutations in ocular Ct strains, which are responsible for the blinding eye disease known as trachoma, abrogate tryptophan synthesis from indole. We examined serial urogenital samples from a woman who had recurrent Ct infections over 4 years despite antibiotic treatment. The Ct isolates from each infection episode were genome sequenced and analyzed for phenotypic, structural, and functional characteristics. All isolates contained identical mutations in trpA and developed aberrant bodies within intracellular inclusions, visualized by transmission electron microscopy, even when supplemented with indole following IFN-γ treatment. Each isolate displayed an altered αββα structure, could not synthesize tryptophan from indole, and had significantly lower trpBA expression but higher intracellular tryptophan levels compared with those of reference Ct strain F/IC-Cal3. Our data indicate that emergent mutations in the tryptophan operon, which were previously thought to be restricted only to ocular Ct strains, likely resulted in in vivo persistence in the described patient and represents a novel host-pathogen adaptive strategy for survival. IMPORTANCE Chlamydia trachomatis ( Ct ) is the most common sexually transmitted bacterium with more than 131 million cases occurring annually worldwide. Ct infections are often asymptomatic, persisting for many years despite treatment. In vitro recovery from persistence occurs when indole is utilized by the organism's tryptophan synthase to synthesize tryptophan, an essential amino acid for replication. Ocular but not urogenital Ct strains contain mutations in the synthase that abrogate tryptophan synthesis. Here, we discovered that the genomes of serial isolates from a woman with recurrent, treated Ct STIs over many years were identical with a novel synthase mutation. This likely allowed long-term in vivo persistence where active infection resumed only when tryptophan became available. Our findings indicate an emerging adaptive host-pathogen evolutionary strategy for survival in the urogenital tract that will prompt the field to further explore chlamydial persistence, evaluate the genetics of mutant Ct strains and fitness within the host, and their implications for disease pathogenesis., (Copyright © 2019 Somboonna et al.)

Chlamydia trachomatis is the leading cause of preventable blindness and the most common bacterial sexually transmitted infection. Different strains are associated with ocular or urogenital infections, and a proposed mechanism that may explain this tissue tropism is the active tryptophan biosynthesis pathway encoded by the genomic trpRBA operon in urogenital strains. Here we describe genetic complementation studies that are essential to confirm the role of tryptophan synthase in the context of an ocular C. trachomatis genomic background. Ocular strain A2497 was transformed with the (urogenital) pSW2::GFP shuttle vector showing that there is no strain tropism barrier to this plasmid vector; moreover, transformation had no detrimental effect on the growth kinetics of A2497, which is important given the low transformation efficiency of C. trachomatis . A derivative of the pSW2::GFP vector was used to deliver the active tryptophan biosynthesis genes from a urogenital strain of C. trachomatis (Soton D1) to A2497 with the aim of complementing the truncated trpA gene common to most ocular strains. After confirmation of intact TrpA protein expression in the transformed A2497, the resulting transformants were cultivated in tryptophan-depleted medium with and without indole or tryptophan, showing that complementation of the truncated trpA gene by the intact and functional urogenital trpRBA operon was sufficient to bestow an indole rescuable phenotype upon A2497. This study proves that pSW2::GFP derived vectors do not conform to the cross-strain transformation barrier reported for other chlamydia shuttle vectors, suggesting these as a universal vector for transformation of all C. trachomatis strains. This vector promiscuity enabled us to test the indole rescue hypothesis by transforming ocular strain A2497 with the functional urogenital trpRBA operon, which complemented the non-functional tryptophan synthase. These data confirm that the trpRBA operon is necessary and sufficient for chlamydia to survive in tryptophan-limited environments such as the female urogenital tract.

Tryptophan synthase (TrpS) is a heterotetrameric αββα enzyme that exhibits complex substrate channeling and allosteric mechanisms and is a model system in enzymology. In this work, we characterize proposed early and late evolutionary states of TrpS and show that they have distinct quaternary structures caused by insertions-deletions of sequence segments (indels) in the β-subunit. Remarkably, indole hydrophobic channels that connect α and β active sites have re-emerged in both TrpS types, yet they follow different paths through the β-subunit fold. Also, both TrpS geometries activate the α-subunit through the rearrangement of loops flanking the active site. Our results link evolutionary sequence changes in the enzyme subunits with channeling and allostery in the TrpS enzymes. The findings demonstrate that indels allow protein quaternary architectures to escape "minima" in the evolutionary landscape, thereby overcoming the conservational constraints imposed by existing functional interfaces and being free to morph into new mechanistic enzymes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Noncanonical amino acids (ncAAs) with dual stereocenters at the α and β positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive β-branched ncAAs, their availability is limited due to the inefficiency of the multistep methods used to prepare them. Herein we report a stereoselective biocatalytic synthesis of β-branched tryptophan analogues using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB 7E6 . PfTrpB 7E6 is the first biocatalyst to synthesize bulky β-branched tryptophan analogues in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of PfTrpB 7E6 was explored through X-ray crystallography and UV/Vis spectroscopy, which revealed that a combination of active-site and remote mutations increase the abundance and persistence of a key reactive intermediate. PfTrpB 7E6 provides an operationally simple and environmentally benign platform for the preparation of β-branched tryptophan building blocks., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of noncanonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of the intermediates shifts to favor covalently bound Trp adducts, which have increased thermodynamic stability. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High-resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a subdomain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.

Thaxtomins are diketopiperazine phytotoxins produced by Streptomyces scabies and other actinobacterial plant pathogens that inhibit cellulose biosynthesis in plants. Due to their potent bioactivity and novel mode of action there has been considerable interest in developing thaxtomins as herbicides for crop protection. To address the need for more stable derivatives, we have developed a new approach for structural diversification of thaxtomins. Genes encoding the thaxtomin NRPS from S. scabies, along with genes encoding a promiscuous tryptophan synthase (TrpS) from Salmonella typhimurium, were assembled in a heterologous host Streptomyces albus. Upon feeding indole derivatives to the engineered S. albus strain, tryptophan intermediates with alternative substituents are biosynthesized and incorporated by the NRPS to deliver a series of thaxtomins with different functionalities in place of the nitro group. The approach described herein, demonstrates how genes from different pathways and different bacterial origins can be combined in a heterologous host to create a de novo biosynthetic pathway to "non-natural" product target compounds., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

β-Methyltryptophans (β-mTrp) are precursors in the biosynthesis of bioactive natural products and are used in the synthesis of peptidomimetic-based therapeutics. Currently β-mTrp is produced by inefficient multistep synthetic methods. Here we demonstrate how an engineered variant of tryptophan synthase from Salmonella (StTrpS) can catalyse the efficient condensation of l-threonine and various indoles to generate β-mTrp and derivatives in a single step. Although l-serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provided a variant (βL166V) that can better accommodate l-Thr as a substrate. The condensation of l-Thr and indole proceeds with retention of configuration at both α- and β-positions to give (2S,3S)-β-mTrp. The integration of StTrpS (βL166V) with l-amino acid oxidase, halogenase enzymes and palladium chemocatalysts provides access to further d-configured and regioselectively halogenated or arylated β-mTrp derivatives., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The natural course of sexually transmitted infections caused by Chlamydia trachomatis varies between individuals. In addition to parasite and host effects, the vaginal microbiota might play a key role in the outcome of C. trachomatis infections. Interferon-gamma (IFN-γ), known for its anti-chlamydial properties, activates the expression of indoleamine 2,3-dioxygenase (IDO1) in epithelial cells, an enzyme that catabolizes the amino acid L- tryptophan into N-formylkynurenine, depleting the host cell's pool of tryptophan. Although C. trachomatis is a tryptophan auxotroph, urogenital strains (but not ocular strains) have been shown in vitro to have the ability to produce tryptophan from indole using the tryptophan synthase (trpBA) gene. It has been suggested that indole producing bacteria from the vaginal microbiota could influence the outcome of Chlamydia infection., Results: We used two in vitro models (treatment with IFN-γ or direct limitation of tryptophan), to study the effects of direct rescue by the addition of exogenous indole, or by the addition of culture supernatant from indole-positive versus indole-negative Prevotella strains, on the growth and infectivity of C. trachomatis. We found that only supernatants from the indole-positive strains, P. intermedia and P. nigrescens, were able to rescue tryptophan-starved C. trachomatis. In addition, we analyzed vaginal secretion samples to determine physiological indole concentrations. In spite of the complexity of vaginal secretions, we demonstrated that for some vaginal specimens with higher indole levels, there was a link to higher recovery of the Chlamydia under tryptophan-starved conditions, lending preliminary support to the critical role of the IFN-γ-tryptophan-indole axis in vivo., Conclusions: Our data provide evidence for the ability of both exogenous indole as well as supernatant from indole producing bacteria such as Prevotella, to rescue genital C. trachomatis from tryptophan starvation. This adds weight to the hypothesis that the vaginal microbiota (particularly from women with lower levels of lactobacilli and higher levels of indole producing anaerobes) may be intrinsically linked to the outcome of chlamydial infections in some women.

Key Message: Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

Several transformation strains of Coprinopsis cinerea carry the defective tryptophan synthase allele trp1-1,1-6 which can be complemented by introduction of the trp1 (+) wild-type gene. Regularly in C. cinerea, single-trp1 (+)-vector transformations yield about half the numbers of clones than cotransformations with a non-trp1 (+)-plasmid done in parallel. The effect is also observed with the orthologous Schizophyllum commune trpB (+) gene shown here to function as a selection marker in C. cinerea. Parts of single-trp1 (+) - or single-trpB (+) -vector transformants are apparently lost. This paradoxical phenomenon relates to de-regulation of aromatic amino acid biosynthesis pathways. Adding tryptophan precursors to protoplast regeneration agar or feeding with other aromatic amino acids increases loss of single-trp1 (+)-vector transformants and also sets off loss of clones in cotransformation with a non-trp1 (+)-plasmid. Feedback control by tryptophan and cross-pathway control by tyrosine and phenylalanine are both active in the process. We deduce from the observations that more cotransformants than single-vector transformants are obtained by in average less disturbance of the tryptophan biosynthesis pathway. DNA in C. cinerea transformation usually integrates into the genome at multiple ectopic places. Integration events for a single vector per nucleus should statistically be 2-fold higher in single-vector transformations than in cotransformations in which the two different molecules compete for the same potential integration sites. Integration of more trp1 (+) copies into the genome might more likely lead to sudden tryptophan overproduction with subsequent rigid shut-down of the pathway. Blocking ectopic DNA integration in a Δku70 mutant abolished the effect of doubling clone numbers in cotransformations due to preferred single trp1 (+) integration by homologous recombination at its native genomic site.

Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Naturally occurring enzyme homologues often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homologue. A small set of mutations of the tryptophan synthase β-subunit (TrpB) from Pyrococcus furiosus, which mimics the activation afforded by binding of the α-subunit, was demonstrated to have a similar activating effect in different TrpB homologues with as little as 57 % sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism: mimicry of α-subunit binding. From these enzymes, we identified a new TrpB catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted tryptophans. This demonstrates that allosteric activation can be recapitulated throughout a protein family to explore natural sequence diversity for desirable biocatalytic transformations., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Indole is a characteristic volatile constituent in oolong tea. Our previous study indicated that indole was mostly accumulated at the turn over stage of oolong tea manufacturing process. However, formation of indole in tea leaves remains unknown. In this study, one tryptophan synthase α-subunit (TSA) and three tryptophan synthase β-subunits (TSBs) from tea leaves were isolated, cloned, sequenced, and functionally characterized. Combination of CsTSA and CsTSB2 recombinant protein produced in Escherichia coli exhibited the ability of transformation from indole-3-glycerol phosphate to indole. CsTSB2 was highly expressed during the turn over process of oolong tea. Continuous mechanical damage, simulating the turn over process, significantly enhanced the expression level of CsTSB2 and amount of indole. These suggested that accumulation of indole in oolong tea was due to the activation of CsTSB2 by continuous wounding stress from the turn over process. Black teas contain much less indole, although wounding stress is also involved in the manufacturing process. Stable isotope labeling indicated that tea leaf cell disruption from the rolling process of black tea did not lead to the conversion of indole, but terminated the synthesis of indole. Our study provided evidence concerning formation of indole in tea leaves for the first time.

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms.

During synthetic mutualistic interactions between the microalga Chlorella sorokiniana and the plant growth-promoting bacterium (PGPB) Azospirillum brasilense, mutual exchange of resources involved in producing and releasing the phytohormone indole-3-acetic acid (IAA) by the bacterium, using tryptophan and thiamine released by the microalga, were measured. Although increased activities of tryptophan synthase in C. sorokiniana and indole pyruvate decarboxylase (IPDC) in A. brasilense were observed, we could not detect tryptophan or IAA in the culture medium when both organisms were co-immobilized. This indicates that no extra tryptophan or IAA is produced, apart from the quantities required to sustain the interaction. Over-expression of the ipdC gene occurs at different incubation times: after 48 h, when A. brasilense was immobilized alone and grown in exudates of C. sorokiniana and at 96 h, when A. brasilense was co-immobilized with the microalga. When A. brasilense was cultured in exudates of C. sorokiniana, increased expression of the ipdC gene, corresponding increase in activity of IPDC encoded by the ipdC gene, and increase in IAA production were measured during the first 48 h of incubation. IAA production and release by A. brasilense was found only when tryptophan and thiamine were present in a synthetic growth medium (SGM). The absence of thiamine in SGM yielded no detectable IAA. In summary, this study demonstrates that C. sorokiniana can exude sufficient tryptophan and thiamine to allow IAA production by a PGPB during their interaction. Thiamine is essential for IAA production by A. brasilense and these three metabolites are part of a communication between the two microorganisms., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The phytohormone auxin regulates nearly all aspects of plant growth and development. Tremendous achievements have been made in elucidating the tryptophan (Trp)-dependent auxin biosynthetic pathway; however, the genetic evidence, key components, and functions of the Trp-independent pathway remain elusive. Here we report that the Arabidopsis indole synthase mutant is defective in the long-anticipated Trp-independent auxin biosynthetic pathway and that auxin synthesized through this spatially and temporally regulated pathway contributes significantly to the establishment of the apical-basal axis, which profoundly affects the early embryogenesis in Arabidopsis. These discoveries pave an avenue for elucidating the Trp-independent auxin biosynthetic pathway and its functions in regulating plant growth and development.

The rapid increase of the number of sequenced genomes asks for the functional annotation of the encoded enzymes. We used a combined computational-structural approach to determine the function of the TrpB2 subgroup of the tryptophan synthase β chain/β chain-like TrpB1-TrpB2 family (IPR023026). The results showed that TrpB2 enzymes are O-phospho-l-serine dependent tryptophan synthases, whereas TrpB1 enzymes catalyze the l-serine dependent synthesis of tryptophan. We found a single residue being responsible for the different substrate specificities of TrpB1 and TrpB2 and confirmed this finding by mutagenesis studies and crystallographic analysis of a TrpB2 enzyme with bound O-phospho-l-serine.

The last two steps of l-tryptophan (Trp) biosynthesis are catalyzed by Trp synthase, a heterotetramer composed of TrpA and TrpB. TrpB catalyzes the condensation of indole, synthesized by TrpA, and serine to Trp. In the hyperthermophilic archaeon Thermococcus kodakarensis, trpA and trpB (trpB1) are located adjacently in the trpCDEGFB1A operon. Interestingly, several organisms possess a second trpB gene (trpB2) encoding TrpB2, located outside of the trp operon in T. kodakarensis. Until now, the physiological function of trpB2 has not been examined genetically. In the present study, we report the biochemical and physiological analyses of TrpB2 from T. kodakarensis. Kinetic analysis indicated that TrpB2 catalyzed the TrpB reaction but did not interact with TrpA as in the case of TrpB1. When growth phenotypes were examined for gene disruption strains, the double-deletion mutant (ΔtrpB1ΔtrpB2) displayed Trp auxotrophy, whereas individual single mutants (ΔtrpB1 and ΔtrpB2 strains) did not. It has been proposed previously that, in Thermotoga maritima, TrpB2 provides an alternate route to generate Trp from serine and free indole (indole salvage). To accurately examine the capacity of TrpB1 and TrpB2 in Trp synthesis via indole salvage, we constructed ΔtrpEB1 and ΔtrpEB2 strains using strain KUW1 (ΔpyrFΔtrpE) as a host, eliminating the route for endogenous indole synthesis. Indole complemented the Trp auxotrophies of ΔtrpEB1 (ΔpyrFΔtrpEΔtrpB1) and ΔtrpEB2 (ΔpyrFΔtrpEΔtrpB2) to similar levels. The results indicate that TrpB1 and TrpB2 both contribute to Trp biosynthesis in T. kodakarensis and can utilize free indole, and that indole salvage does not necessarily rely on TrpB2 to a greater extent., (© 2014 FEBS.)

Fifteen different classically generated and mapped mutations at the tryptophan synthetase locus in Neurospora crassa have been characterized to the level of the primary sequence of the gene. This sequence analysis has demonstrated that intragenic recombination is accurate to order mutations within one open reading frame. While classic genetic analysis correctly ordered the mutations, the position of mutations characterized by gene sequence analysis was more accurate. A leaky mutation was found to have a wild-type primary sequence. The presence of unique polymorphisms in the primary sequence of the trp-3 gene from strain 861 confirms that it has a unique history relative to the other strains studied. Most strains that were previously shown to be immunologically nonreactive with antibody preparations raised against tryptophan synthetase protein were shown to have nonsense mutations. This work defines 14 alleles of the N. crassa trp-3 gene.

The α-subunit of tryptophan synthase (αTS) catalyzes the conversion of indole-3-glycerol phosphate to d-glyceraldehyde-3-phosphate and indole. We propose that allosteric networks intrinsic to αTS are modulated by the binding of the β-subunit to regulate αTS function. Understanding these long-range amino acid networks in αTS thus gives insight into the coordination of the two active sites within TS. In this study, we have used Ala residues as probes for structural and dynamic changes of αTS throughout its catalytic cycle, in the absence of the β-subunit. Projection analysis of the chemical shift changes by site-specific amino acid substitutions and ligand titrations indicates that αTS has three important conformational states: ligand-free, glyceraldehyde-3-phosphate-bound(like), and the active states. The amino acid networks within these conformations are different, as suggested by chemical shift correlation analysis. In particular, there are long-range connections, only in the active state, between Ala47, which reports on structural and dynamic changes associated with the general acid/base Glu49, and residues within the β2α2 loop, which contains the catalytically important Asp60 residue. These long-range interactions are likely important for coordinating chemical catalysis. In the free state, but not in the active state, there are connections between the β2α2 and β6α6 loops that likely help to coordinate substrate binding. Changes in the allosteric networks are also accompanied by protein dynamic changes. During catalytic turnover, the protein becomes more rigid on the millisecond timescale and the active-site dynamics are driven to a faster nanosecond timescale., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lipopolysaccharides (LPS), as lipoglycan microbe-associated molecular pattern molecules, trigger activation of signal transduction pathways involved in defence that generate an enhanced defensive capacity in plants. The transcriptional regulation of the genes for tryptophan synthase B, TSB1, and the cytochrome P450 monooxygenases CYP79B2 and CYP71B15, involved in the camalexin biosynthetic pathway, were investigated in response to LPS treatment. GUS-reporter assays for CYP71B15 and CYP79B2 gene promoter activation were performed on transgenic plants and showed positive histochemical staining in response to LPS treatment, indicating activation of the promoters. Quantitative PCR revealed that transcripts of TSB1, CYP79B2 and CYP71B15 exhibited differential, transient up-regulation. TSB1 transcript levels were up-regulated between 6 and 9 h after LPS-induction, while CYP71B15 and CYP79B2 both exhibited maxima at 12 h. To obtain information on the gene-to-metabolite network, the effect of the transcriptome changes on the metabolome was correlated to camalexin production. Increases in camalexin concentration were quantified by ultra pressure liquid chromatography-mass spectrometry and both absorbance spectra and elemental composition confirmed its identity. The concentrations increased from 0.03 to 3.7 μg g(-1) fresh weight over a 24-h time period, thus indicating that the up-regulation of the biosynthetic pathway in response to LPS was accompanied by a time-dependent increase in camalexin concentration. Metabolomic analysis through principal component analysis-derived scores plots revealed clusters of sample replicates for 0, 6, 12, 18 and 24 h while loadings plots for LPS data identified camalexin as a biomarker that clearly demonstrated the variability between samples.

Phosphoribosylanthranilate (PRA) isomerase (TrpF) and tryptophan synthase α-subunit (TrpA) are (βα)(8)-barrel enzymes that are involved in the biosynthesis of tryptophan. They contain a conserved phosphate binding site, which indicates a common evolutionary origin. In order to experimentally back this hypothesis, we have established TrpF activity on the scaffold of TrpA from Salmonella typhimurium using protein engineering. Based on the superposition of crystal structures with bound ligands, two residues in the active site of TrpA were replaced with catalytic residues from TrpF using site-directed mutagenesis. This TrpA variant as well as wild-type TrpA were each subjected to random mutagenesis using error-prone PCR. The two resulting trpA gene libraries were used to transform an auxotrophic Escherichia coli trpF deletion strain, and TrpA variants with PRA isomerisation activity were isolated by in vivo complementation. The amino acid substitutions of the selected TrpA variants were recombined by DNA shuffling, again followed by complementation in vivo. Several TrpA variants were produced in E. coli and purified, and their catalytic TrpF activities were determined in vitro by steady-state enzyme kinetics. Our results support that TrpA and TrpF have evolved by gene duplication and diversification from each other or a common predecessor, and provide insights into the minimum requirements for the catalysis of PRA isomerisation.

Background: The benzoxazinoids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA), are key defense compounds present in major agricultural crops such as maize and wheat. Their biosynthesis involves nine enzymes thought to form a linear pathway leading to the storage of DI(M)BOA as glucoside conjugates. Seven of the genes (Bx1-Bx6 and Bx8) form a cluster at the tip of the short arm of maize chromosome 4 that includes four P450 genes (Bx2-5) belonging to the same CYP71C subfamily. The origin of this cluster is unknown., Results: We show that the pathway appeared following several duplications of the TSA gene (α-subunit of tryptophan synthase) and of a Bx2-like ancestral CYP71C gene and the recruitment of Bx8 before the radiation of Poaceae. The origins of Bx6 and Bx7 remain unclear. We demonstrate that the Bx2-like CYP71C ancestor was not committed to the benzoxazinoid pathway and that after duplications the Bx2-Bx5 genes were under positive selection on a few sites and underwent functional divergence, leading to the current specific biochemical properties of the enzymes. The absence of synteny between available Poaceae genomes involving the Bx gene regions is in contrast with the conserved synteny in the TSA gene region., Conclusions: These results demonstrate that rearrangements following duplications of an IGL/TSA gene and of a CYP71C gene probably resulted in the clustering of the new copies (Bx1 and Bx2) at the tip of a chromosome in an ancestor of grasses. Clustering favored cosegregation and tip chromosomal location favored gene rearrangements that allowed the further recruitment of genes to the pathway. These events, a founding event and elongation events, may have been the key to the subsequent evolution of the benzoxazinoid biosynthetic cluster.

Mismatch repair (MMR) is a major DNA repair pathway in cells from all branches of life that removes replication errors in a strand-specific manner, such that mismatched nucleotides are preferentially removed from the newly replicated strand of DNA. Here we demonstrate a role for MMR in helping create new phenotypes in nondividing cells. We show that mispairs in yeast that escape MMR during replication can later be subject to MMR activity in a replication strand-independent manner in nondividing cells, resulting in either fully wild-type or mutant DNA sequence. In one case, this activity is responsible for what appears to be adaptive mutation. This replication strand-independent MMR activity could contribute to the formation of tumors arising in nondividing cells and could also contribute to mutagenesis observed during somatic hypermutation of Ig genes.