The role of oligomerization in the optimization of cyclohexadienyl dehydratase conformational dynamics and catalytic activity.

The emergence of oligomers is common during the evolution and diversification of protein families, yet the selective advantage of oligomerization is often cryptic or unclear. Oligomerization can involve the formation of isologous head-to-head interfaces (e.g., in symmetrical dimers) or heterologous head-to-tail interfaces (e.g., in cyclic complexes), the latter of which is less well studied and understood. In this work, we retrace the emergence of the trimeric form of cyclohexadienyl dehydratase from Pseudomonas aeruginosa (PaCDT) by introducing residues that form the PaCDT trimer-interfaces into AncCDT-5 (a monomeric reconstructed ancestor of PaCDT). We find that single interface mutations can switch the oligomeric state of the variants and that trimerization corresponds with a reduction in the KM value of the enzyme from a promiscuous level to the physiologically relevant range. In addition, we find that removal of a C-terminal extension present in PaCDT leads to a variant with reduced catalytic activity, indicating that the C-terminal region has a role in tuning enzymatic activity. We show that these observations can be rationalized at the structural and dynamic levels, with trimerization and C-terminal extension leading to reduced sampling of non-catalytic conformational substates in molecular dynamics simulations. Overall, this work provides insight into how neutral sampling of distinct oligomeric states along an evolutionary trajectory can facilitate the evolution and optimization of enzyme function.

The Komagataeibacter europaeus GqqA is the prototype of a novel bifunctional N-Acyl-homoserine lactone acylase with prephenate dehydratase activity.

Previously, we reported the isolation of a quorum quenching protein (QQ), designated GqqA, from Komagataeibacter europaeus CECT 8546 that is highly homologous to prephenate dehydratases (PDT) (Valera et al. in Microb Cell Fact 15, 88. https://doi.org/10.1186/s12934-016-0482-y , 2016). GqqA strongly interfered with N-acyl-homoserine lactone (AHL) quorum sensing signals from Gram-negative bacteria and affected biofilm formation in its native host strain Komagataeibacter europaeus. Here we present and discuss data identifying GqqA as a novel acylase. ESI-MS-MS data showed unambiguously that GqqA hydrolyzes the amide bond of the acyl side-chain of AHL molecules, but not the lactone ring. Consistent with this observation the protein sequence does not carry a conserved Zn2+ binding motif, known to be essential for metal-dependent lactonases, but in fact harboring the typical periplasmatic binding protein domain (PBP domain), acting as catalytic domain. We report structural details for the native structure at 2.5 Å resolution and for a truncated GqqA structure at 1.7 Å. The structures obtained highlight that GqqA acts as a dimer and complementary docking studies indicate that the lactone ring of the substrate binds within a cleft of the PBP domain and interacts with polar residues Y16, S17 and T174. The biochemical and phylogenetic analyses imply that GqqA represents the first member of a novel type of QQ family enzymes.

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme.

Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron-electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.

Pathway Engineering for Phenethylamine Production in Escherichia coli.

In this study, the metabolic pathway of phenethylamine synthesis was reconstructed by chromosomal integration and overexpression of the Enterococcus faecium pdc gene encoding phenylalanine decarboxylase in Escherichia coli. The genes encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroG), shikimate kinase II (aroL), chorismate mutase/prephenate dehydratase (pheA), and tyrosine aminotransferase (tyrB) in the phenethylamine synthetic pathway were sequentially chromosomally overexpressed. The phosphotransferase system was replaced by deleting the ptsH-ptsI-crr genes and chromosomally overexpressing the genes encoding galactose permease (galP) and glucokinase (glk). In addition, the zwf gene encoding glucose-6-phosphate dehydrogenase in the pentose phosphate pathway was chromosomally overexpressed, generating the final engineered E. coli strain AUD9. The AUD9 strain produced 2.65 g L-1 phenethylamine with a yield of 0.27 g of phenethylamine g-1 glucose in batch fermentation; fed-batch fermentation of AUD9 produced 38.82 g L-1 phenethylamine with a productivity of 1.08 g L-1 h-1 phenethylamine, demonstrating its potential for industrial fermentative production of phenethylamine.

Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants.

In addition to being a vital component of proteins, phenylalanine is also a precursor of numerous aromatic primary and secondary metabolites with broad physiological functions. In plants phenylalanine is synthesized predominantly via the arogenate pathway in plastids. Here, we describe the structure, molecular players and subcellular localization of a microbial-like phenylpyruvate pathway for phenylalanine biosynthesis in plants. Using a reverse genetic approach and metabolic flux analysis, we provide evidence that the cytosolic chorismate mutase is responsible for directing carbon flux towards cytosolic phenylalanine production via the phenylpyruvate pathway. We also show that an alternative transcription start site of a known plastidial enzyme produces a functional cytosolic prephenate dehydratase that catalyzes the conversion of prephenate to phenylpyruvate, the intermediate step between chorismate mutase and phenylpyruvate aminotransferase. Thus, our results complete elucidation of phenylalanine biosynthesis via phenylpyruvate in plants, showing that this pathway splits from the known plastidial arogenate pathway at chorismate, instead of prephenate as previously thought, and the complete pathway is localized in the cytosol.

Gut microbiome in ADHD and its relation to neural reward anticipation.

BACKGROUND: Microorganisms in the human intestine (i.e. the gut microbiome) have an increasingly recognized impact on human health, including brain functioning. Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder associated with abnormalities in dopamine neurotransmission and deficits in reward processing and its underlying neuro-circuitry including the ventral striatum. The microbiome might contribute to ADHD etiology via the gut-brain axis. In this pilot study, we investigated potential differences in the microbiome between ADHD cases and undiagnosed controls, as well as its relation to neural reward processing. METHODS: We used 16S rRNA marker gene sequencing (16S) to identify bacterial taxa and their predicted gene functions in 19 ADHD and 77 control participants. Using functional magnetic resonance imaging (fMRI), we interrogated the effect of observed microbiome differences in neural reward responses in a subset of 28 participants, independent of diagnosis. RESULTS: For the first time, we describe gut microbial makeup of adolescents and adults diagnosed with ADHD. We found that the relative abundance of several bacterial taxa differed between cases and controls, albeit marginally significant. A nominal increase in the Bifidobacterium genus was observed in ADHD cases. In a hypothesis-driven approach, we found that the observed increase was linked to significantly enhanced 16S-based predicted bacterial gene functionality encoding cyclohexadienyl dehydratase in cases relative to controls. This enzyme is involved in the synthesis of phenylalanine, a precursor of dopamine. Increased relative abundance of this functionality was significantly associated with decreased ventral striatal fMRI responses during reward anticipation, independent of ADHD diagnosis and age. CONCLUSIONS: Our results show increases in gut microbiome predicted function of dopamine precursor synthesis between ADHD cases and controls. This increase in microbiome function relates to decreased neural responses to reward anticipation. Decreased neural reward anticipation constitutes one of the hallmarks of ADHD.

Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome.

The A1 subunits of Shiga toxin 1 (Stx1A1) and Shiga toxin 2 (Stx2A1) interact with the conserved C termini of ribosomal-stalk P-proteins to remove a specific adenine from the sarcin/ricin loop. We previously showed that Stx2A1 has higher affinity for the ribosome and higher catalytic activity than Stx1A1. To determine if conserved arginines at the distal face of the active site contribute to the higher affinity of Stx2A1 for the ribosome, we mutated Arg172, Arg176, and Arg179 in both toxins. We show that Arg172 and Arg176 are more important than Arg179 for the depurination activity and toxicity of Stx1A1 and Stx2A1. Mutation of a single arginine reduced the depurination activity of Stx1A1 more than that of Stx2A1. In contrast, mutation of at least two arginines was necessary to reduce depurination by Stx2A1 to a level similar to that of Stx1A1. R176A and R172A/R176A mutations eliminated interaction of Stx1A1 and Stx2A1 with ribosomes and with the stalk, while mutation of Arg170 at the active site reduced the binding affinity of Stx1A1 and Stx2A1 for the ribosome, but not for the stalk. These results demonstrate that conserved arginines at the distal face of the active site are critical for interactions of Stx1A1 and Stx2A1 with the stalk, while a conserved arginine at the active site is critical for non-stalk-specific interactions with the ribosome. Arginine mutations at either site reduced ribosome interactions of Stx1A1 and Stx2A1 similarly, indicating that conserved arginines are critical for ribosome interactions but do not contribute to the higher affinity of Stx2A1 for the ribosome.

Characterization of two key enzymes for aromatic amino acid biosynthesis in symbiotic archaea.

Biosynthesis of L-tyrosine (L-Tyr) and L-phenylalanine (L-Phe) is directed by the interplay of three enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which can be either converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD) or to phenylpyruvate by prephenate dehydratase (PDT). This work reports the first characterization of a trifunctional PD-CM-PDT from the smallest hyperthermophilic archaeon Nanoarchaeum equitans and a bifunctional CM-PD from its host, the crenarchaeon Ignicoccus hospitalis. Hexa-histidine tagged proteins were expressed in Escherichia coli and purified by affinity chromatography. Specific activities determined for the trifunctional enzyme were 21, 80, and 30 U/mg for CM, PD, and PDT, respectively, and 47 and 21 U/mg for bifunctional CM and PD, respectively. Unlike most PDs, these two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP(+) to NAD(+) as a cofactor. Both the enzymes were highly thermally stable and exhibited maximal activity at 90 °C. N. equitans PDT was feedback inhibited by L-Phe (Ki = 0.8 µM) in a non-competitive fashion consistent with L-Phe's combination at a site separate from that of prephenate. Our results suggest that PD from the unique symbiotic archaeal pair encompass a distinct subfamily of prephenate dehydrogenases with regard to their regulation and co-substrate specificity.

Identification of a small protein domain present in all plant lineages that confers high prephenate dehydratase activity.

l-Phenylalanine serves as a building block for the biosynthesis of proteins, but also as a precursor for a wide range of plant-derived compounds essential for plants and animals. Plants can synthesize Phe within the plastids using arogenate as a precursor; however, an alternative pathway using phenylpyruvate as an intermediate, described for most microorganisms, has recently been proposed. The functionality of this pathway requires the existence of enzymes with prephenate dehydratase (PDT) activity (EC 4.2.1.51) in plants. Using phylogenetic studies, functional complementation assays in yeast and biochemical analysis, we have identified the enzymes displaying PDT activity in Pinus pinaster. Through sequence alignment comparisons and site-directed mutagenesis we have identified a 22-amino acid region conferring PDT activity (PAC domain) and a single Ala314 residue critical to trigger this activity. Our results demonstrate that all plant clades include PAC domain-containing ADTs, suggesting that the PDT activity, and thus the ability to synthesize Phe using phenylpyruvate as an intermediate, has been preserved throughout the evolution of plants. Moreover, this pathway together with the arogenate pathway gives plants a broad and versatile capacity to synthesize Phe and its derived compounds. PAC domain-containing enzymes are also present in green and red algae, and glaucophytes, the three emerging clades following the primary endosymbiont event resulting in the acquisition of plastids in eukaryotes. The evolutionary prokaryotic origin of this domain is discussed.

Chemical proteomics of host-pathogen interaction.

In less than two decades, activity-based protein profiling (ABPP) has expanded to become the de facto tool for the study of small molecule-protein interactions in a proteomic environment. In this issue, Na et al. (2015) present another ABPP method, which they called reactive probe-based chemical proteomics, to study host-pathogen interaction and subsequently identify the protein PheA as a potential key effector during the pathogen infection process.

Unbiased proteomic profiling strategy for discovery of bacterial effector proteins reveals that Salmonella protein PheA is a host cell cycle regulator.

Salmonella utilizes a type III secretion system to inject bacterial effector proteins into the host cell cytosol. Once in the cytosol, these effectors hijack various biochemical pathways to regulate virulence. Despite the importance of effector proteins, especially for understanding host-pathogen interactions, a potentially large number of effectors are yet to be identified. Here, we demonstrate that unbiased chemical proteomic profiling using off-the-shelf fluorescent probes leads to the discovery of a host cell cycle regulator encoded in the Salmonella genome. Our profiling combined with bioinformatic analysis implicates 29 Salmonella as potential effectors. We follow up on the top candidate, chorismate mutase-P/prehenate dehydratase, PheA, and present evidence that PheA is an effector that mimics E2F7 transcription factor of the host cell and promotes G1/S cell cycle arrest. This validates our strategy and opens opportunities for effector identification in the future.

A synthetic adenylation-domain-based tRNA-aminoacylation catalyst.

The incorporation of non-proteinogenic amino acids represents a major challenge for the creation of functionalized proteins. The ribosomal pathway is limited to the 20-22 proteinogenic amino acids while nonribosomal peptide synthetases (NRPSs) are able to select from hundreds of different monomers. Introduced herein is a fusion-protein-based design for synthetic tRNA-aminoacylation catalysts based on combining NRPS adenylation domains and a small eukaryotic tRNA-binding domain (Arc1p-C). Using rational design, guided by structural insights and molecular modeling, the adenylation domain PheA was fused with Arc1p-C using flexible linkers and achieved tRNA-aminoacylation with both proteinogenic and non-proteinogenic amino acids. The resulting aminoacyl-tRNAs were functionally validated and the catalysts showed broad substrate specificity towards the acceptor tRNA. Our strategy shows how functional tRNA-aminoacylation catalysts can be created for bridging the ribosomal and nonribosomal worlds. This opens up new avenues for the aminoacylation of tRNAs with functional non-proteinogenic amino acids.

Enhanced production of L-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH.

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving L-phenylalanine (L-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of L-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of L-Phe. In contrast, overexpression of aroF (wt) or pheA (fbr) from E. coli significantly increased L-Phe production. Co-overexpression of aroF (wt) and pheA (fbr) improved the titer of L-Phe to 4.46 ± 0.06 g l⁻¹. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of L-Phe production. Co-overexpression of the mutated pheA (fbr) and the wild-type gene aroH resulted in the production of L-Phe up to 4.64 ± 0.09 g l⁻¹. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.

Metabolic engineering of Escherichia coli for improving L-3,4-dihydroxyphenylalanine (L-DOPA) synthesis from glucose.

L-3,4-dihydroxyphenylalanine (L-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of L-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of L-tyrosine (L-Tyr), an L-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on L-Tyr production of PTS inactivation (PTS(-) gluc(+) phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of L-Tyr production (q( L-Tyr)), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q( L-Tyr) in the PTS(+) and the PTS(-) gluc(+) strains, respectively. An 8.6-fold increase in L-Tyr yield from glucose was observed in the PTS(-) gluc(+) tyrR (-) strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for L-Tyr production caused the synthesis of L-DOPA. One of such strains, having the PTS(-) gluc(+) tyrR (-) phenotype, displayed the best production parameters in minimal medium, with a specific rate of L-DOPA production of 13.6 mg/g/h, L-DOPA yield from glucose of 51.7 mg/g and a final L-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of L-DOPA in 50 h.

13C isotope effect on the reaction catalyzed by prephenate dehydratase.

The (13)C isotope effect for the conversion of prephenate to phenylpyruvate by the enzyme prephenate dehydratase from Methanocaldococcus jannaschii is 1.0334+/-0.0006. The size of this isotope effect suggests that the reaction is concerted. From the X-ray structure of a related enzyme, it appears that the only residue capable of acting as the general acid needed for removal of the hydroxyl group is threonine-172, which is contained in a conserved TRF motif. The more favorable entropy of activation for the enzyme-catalyzed process (25 eu larger than for the acid-catalyzed reaction) has been explained by a preorganized microenvironment that obviates the need for extensive solvent reorganization. This is consistent with forced planarity of the ring and side chain, which would place the leaving carboxyl and hydroxyl out of plane. Such distortion of the substrate may be a major contributor to catalysis.

Expression of a bacterial bi-functional chorismate mutase/prephenate dehydratase modulates primary and secondary metabolism associated with aromatic amino acids in Arabidopsis.

Plants can synthesize the aromatic amino acid Phe via arogenate, but it is still not known whether they also use an alternative route for Phe biosynthesis via phenylpyruvate, like many micro-organisms. To examine this possibility, we expressed a bacterial bi-functional PheA (chorismate mutase/prephenate dehydratase) gene in Arabidopsis thaliana that converts chorismate via prephenate into phenylpyruvate. The PheA-expressing plants showed a large increase in the level of Phe, implying that they can convert phenylpyruvate into Phe. In addition, PheA expression rendered the plants more sensitive than wild-type plants to the Trp biosynthesis inhibitor 5-methyl-Trp, implying that Phe biosynthesis competes with Trp biosynthesis from their common precursor chorismate. Surprisingly, GC-MS, LC-MS and microarray analyses showed that this increase in Phe accumulation only had a very minor effect on the levels of other primary metabolites as well as on the transcriptome profile, implying little regulatory cross-interaction between the aromatic amino acid biosynthesis network and the bulk of the Arabidopsis transcriptome and primary metabolism. However, the levels of a number of secondary metabolites derived from all three aromatic amino acids (Phe, Trp and Tyr) were altered in the PheA plants, implying regulatory cross-interactions between the flux of aromatic amino acid biosynthesis from chorismate and their further metabolism into various secondary metabolites. Taken together, our results provide insights into the regulatory mechanisms of aromatic amino acid biosynthesis and their interaction with central primary metabolism, as well as the regulatory interface between primary and secondary metabolism.

Engineered native pathways for high kaempferol and caffeoylquinate production in potato.

Flavonols and caffeoylquinates represent important groups of phenolic antioxidants with health-promoting activities. The genetic potential of potato (Solanum tuberosum) to produce high levels of these dietary compounds has not been realized in currently available commodity varieties. In this article, it is demonstrated that tuber-specific expression of the native and slightly modified MYB transcription factor gene StMtf1(M) activates the phenylpropanoid biosynthetic pathway. Compared with untransformed controls, transgenic tubers contained fourfold increased levels of caffeoylquinates, including chlorogenic acid (CGA) (1.80 mg/g dry weight), whilst also accumulating various flavonols and anthocyanins. Subsequent impairment of anthocyanin biosynthesis through silencing of the flavonoid-3',5'-hydroxylase (F3'5'h) gene resulted in the accumulation of kaempferol-rut (KAR) to levels that were approximately 100-fold higher than in controls (0.12 mg/g dry weight). The biochemical changes were associated with increased expression of both the CGA biosynthetic hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (Hqt) gene and the upstream chorismate mutase (Cm) and prephenate dehydratase (Pdh) genes. Field trials indicated that transgenic lines produced similar tuber yields to the original potato variety Bintje. Processed products of these lines retained most of their phenylpropanoids and were indistinguishable from untransformed controls in texture and taste.

Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan.

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size.

Structures of open (R) and close (T) states of prephenate dehydratase (PDT)--implication of allosteric regulation by L-phenylalanine.

The enzyme prephenate dehydratase (PDT) converts prephenate to phenylpyruvate in L-phenylalanine biosynthesis. PDT is allosterically regulated by L-Phe and other amino acids. We report the first crystal structures of PDT from Staphylococcus aureus in a relaxed (R) state and PDT from Chlorobium tepidum in a tense (T) state. The two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. Both enzymes are tetramers (dimer of dimers) in crystal and solution while a PDT dimer can be regarded as a basic catalytic unit. The N-terminal PDT domain consists of two similar subdomains with a cleft in between, which hosts the highly conserved active site. In one PDT dimer two clefts are aligned to form an extended active site across the dimer interface. Similarly at the interface two ACT regulatory domains create two highly conserved pockets. Upon binding of the L-Phe inside the pockets, PDT transits from an open to a closed conformation.

Metabolic engineering of Escherichia coli to enhance phenylalanine production.

The global regulatory system of Escherichia coli, carbon storage regulator (Csr), was engineered to increase the intracellular concentration of phosphoenolpyruvate. We examined the effects of csrA and csrD mutations and csrB overexpression on phenylalanine production in E. coli NST37 (NST). Overexpression of csrB led to significantly greater phenylalanine production than csrA and csrD mutations (2.33 vs 1.67 and 1.61 g l(-1), respectively; P < 0.01). Furthermore, the overexpression of csrB was confirmed by the observed increase in csrB transcription level. We also determined the effect of overexpressing transketolase A (TktA) or glucose-6-phosphate dehydrogenase (Zwf) in NST and the csrA mutant of NST (NSTCSRA) on phenylalanine production. The NSTCSRA strain overexpressing TktA (NSTCSRA [pTktA]) produced significantly more phenylalanine than that of Zwf (2.39 vs 1.61 g l(-1); P > 0.01). Furthermore, we examined the effect of overexpressing TktA, 3-deoxy-D: -arabino-heptulosonate-7-phosphate synthase (AroF(FR)), and chorismate mutase/prephenate dehydratase (PheA(FR)) together in NSTCSRA (NSTCSRA [pTkaFpA]). It is interesting to note that NSTCSRA [pTkaFpA] produced significantly less phenylalanine than both NSTCSRA [pTktA] and NST overexpressing csrB (NST [pCsrB]) (1.84 vs 2.39 and 2.33 g l(-1), respectively; P < 0.01). Thus, csrB overexpression or csrA mutation in combination with tktA overexpression was more effective than previous approaches that targeted the glycolytic or aromatic pathway enzymes for enhancing phenylalanine production.