Human gut-associated Bifidobacterium species salvage exogenous indole, a uremic toxin precursor, to synthesize indole-3-lactic acid via tryptophan.

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 ß 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 (2-13C)-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 ß 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.

Fungi Tryptophan Synthases: What Is the Role of the Linker Connecting the α and ß Structural Domains in Hemileia vastatrix TRPS? A Molecular Dynamics Investigation.

Tryptophan synthase (TRPS) is a complex enzyme responsible for tryptophan biosynthesis. It occurs in bacteria, plants, and fungi as an αßßα 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 ß 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 ß one. Such behavior was completely disrupted in the simulation without the linker. In light of these results and the αß 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.

A Molecular Dynamics Simulation Study of the Effects of ßGln114 Mutation on the Dynamic Behavior of the Catalytic Site of the Tryptophan Synthase.

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 ß subunits that typically form an α2ß2 tetramer. The enzyme's activity is regulated by the conformational switching of its α and ß 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 ßGln114 residue on the catalytic and allosteric sites of TS. Extensive molecular dynamics simulations were conducted on E(PLP), E(AEX1), E(A-A), and E(C3) forms of TS using the WT, ßQ114A, and ßQ114N versions. The results show that both the ßQ114A and ßQ114N mutations increase protein backbone root mean square deviation fluctuations, destabilizing all TS forms. Conformational and hydrogen bond analyses suggest the significance of ßGln114 drifting away from cofactor/intermediates and forming hydrogen bonds with water molecules necessary for l-Trp biosynthesis. The ßQ114A mutation creates a gap between ßAla114 and cofactor/intermediates, hindering hydrogen bond formation due to short side chains and disrupting ß-sites. Conversely, the ßQ114N mutation positions ßAsn114 closer to cofactor/intermediates, forming hydrogen bonds with O3 of cofactors/intermediates and nearby water molecules, potentially disrupting the l-Trp biosynthetic mechanism.

Improving the Activity of Tryptophan Synthetase via a Nucleic Acid Scaffold.

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.

CusProSe: a customizable protein annotation software with an application to the prediction of fungal secondary metabolism genes.

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.

Similarities of P1-Like Phage Plasmids and Their Role in the Dissemination of blaCTX-M-55.

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 blaCTX-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 IS1380 and IS5 and containing a blaCTX-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 ß-lactamase blaCTX-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 blaCTX-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 role of tryptophan in Chlamydia trachomatis persistence.

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.

Evidence from Co-expression Analysis for the Involvement of Amidase and INS in the Tryptophan-Independent Pathway of IAA Synthesis in Arabidopsis.

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.

Fermentative Indole Production via Bacterial Tryptophan Synthase Alpha Subunit and Plant Indole-3-Glycerol Phosphate Lyase Enzymes.

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 ß-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.

Coordination of plant growth and abiotic stress responses by tryptophan synthase ß subunit 1 through modulation of tryptophan and ABA homeostasis in Arabidopsis.

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 ß 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 ß-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 H2O2 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.

Mutation of ßGln114 to Ala Alters the Stabilities of Allosteric States in Tryptophan Synthase Catalysis.

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 ß-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 ß-site residues proximal to the PLP cofactor have not yet been fully established. ßGln114 is one such residue. To explore the roles played by ßQ114, we conducted a detailed investigation of the ßQ114A 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 ß-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and ßQ114A 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 ßGln114, ßAsn145, and ßArg148, 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 (ßT state) and closed (ßR state) conformations of the ß-subunit and consequently alter the distribution of intermediates along the ß-subunit catalytic path.

Catalytically impaired TrpA subunit of tryptophan synthase from Chlamydia trachomatis is an allosteric regulator of TrpB.

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 ß) to produce l-Trp in the presence of l-Ser and a pyridoxal 5'-phosphate cofactor (ß reaction). Previous studies have indicated that in Chlamydia, TrpA has lost its catalytic activity yet remains associated with TrpB to support the ß 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 ßArg267 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.

Tryptophan Operon Diversity Reveals Evolutionary Trends among Geographically Disparate Chlamydia trachomatis Ocular and Urogenital Strains Affecting Tryptophan Repressor and Synthase Function.

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 ß (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 (ΔΔG) 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 ß-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 (trpR) 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.IMPORTANCEChlamydia 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 trpA 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.

Distinct conformational dynamics and allosteric networks in alpha tryptophan synthase during active catalysis.

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.

Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities.

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 ß-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.

CRISPR based targeted genome editing of Chlamydomonas reinhardtii using programmed Cas9-gRNA ribonucleoprotein.

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 ß-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.

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase.

Structural studies with tryptophan synthase (TS) bienzyme complex (α2ß2 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 ß-subunit allowed the purification of high amounts of pure subunits and α2ß2 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 ß-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α2ß2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α2ß2 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 α2ß2 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 α2ß2 StTS complexes.

Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction.

Tryptophan synthase (TS) is a heterotetrameric αßßα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the ß-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 αßßα 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.

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation.

We previously engineered the ß-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 N1 of the substrate. Protecting N1 encouraged evolution toward C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pfquat 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 Pfquat offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.

Metagenomic analysis of relationships between the denitrification process and carbon metabolism in a bioaugmented full-scale tannery wastewater treatment plant.

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 ß-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.