Histidine Biosynthesis Pathway Research Articles

Computational insights into the role of structurally diverse plant secondary metabolites as inhibitors against Imidazole Glycerol Phosphate Dehydratase of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is one of the major causes of death worldwide and there is a pressing need for the development of novel drug leads. The Imidazole Glycerol Phosphate Dehydratase (IGPD) of Mtb is one of the key enzymes in the histidine biosynthesis pathway and has been recognized as the potentially underexploited drug target for anti-tuberculosis treatment. In the present study, 6063 structurally diverse plant secondary metabolites (PSM) were screened for their efficiency in inhibiting the catalytic activity of IGPD through molecular docking. The top 150 PSMs with the lowest binding energy represent the chemical classes, including Tannins (34%), Flavonoid Glycosides (14%), Terpene Glycosides (10%), Steroid Lactones (9.3%), Flavonoids (6.6%), Steroidal Glycosides (4.6%), etc. Bismahanine, Ashwagandhanolide, and Daurisoline form stable IGPD-inhibitor complexes with binding free energies of −291.3 ± 16.5, −279.0 ± 25.0, and −279.8 ± 17.6 KJ/mol, respectively, as determined by molecular dynamics simulations. These PSM demonstrated strong H-bond interactions with the amino acid residues Ile279, Arg281, and Lys276 in the catalytic region of IGPD, as revealed by structural snapshots. On the basis of our findings, these three PSM could be considered as possible leads against IGPD and should be explored in vitro and in vivo. Communicated by Ramaswamy H. Sarma

Identification of novel inhibitors against Escherichia coli utilizing HisC as a target from histidine biosynthesis pathway

Escherichia coli is a gram-negative bacterial pathogen that poses a significant challenge both clinically and epidemiologically. Large numbers of multi-drug resistant E. coli have emerged in the last decade, because of the selection pressure generated by the inadequate use of antibiotics. Although research to combat antibiotic resistance has been going on extensively but still lags in the rate of development of newer antibiotics. Therefore, newer approaches are required to speed up the rate of discovery of antibiotics. Computational methods for screening of inhibitors have made a significant contribution to the discovery of novel antimicrobials. The present study utilized histidinol-phospho aminotransferase (HisC) as a target. HisC is an enzyme that plays a crucial role in the biosynthesis of histidine and its absence in mammals makes it an attractive drug target. A ZINC library of 5000 natural compounds was screened against HisC (PDB ID: 1FG7) using PyRx and the first 500 hits were selected for secondary screening after sorting the result on the basis of binding score. Fifteen compounds passed the secondary filter ADME and out of these five passed toxicity filters; the best among five hits was selected on the basis of its binding score and inhibition constants. Further, molecular dynamics simulations and free binding were computed of selected five compounds and two natural compounds ZINC402598829 and ZINC31157928 complexed with HisC were found as highly stable. Overall, our results indicated that these natural sources could be used as potential HisC inhibitors. Communicated by Ramaswamy H. Sarma

Histidine auxotroph mutant is defective for cell separation and allows the identification of crucial factors for cell division in Brucella abortus

The pathogenic bacterium Brucella abortus invades and multiplies inside host cells. To grow inside host cells, B. abortus requires a functional histidine biosynthesis pathway. Here, we show that a B. abortus histidine auxotroph mutant also displays an unexpected chaining phenotype. The intensity of this phenotype varies according to the culture medium and is exacerbated inside host cells. Chains of bacteria consist of contiguous peptidoglycan, and likely result from the defective cleavage of peptidoglycan at septa. Genetic suppression of the chaining phenotype unearthed two essential genes with a role in B. abortus cell division: dipM and cdlP. Loss of function of dipM and cdlP generates swelling at the division site. While DipM is strictly localized at the division site, CdlP is localized at the growth pole and the division site. Altogether, the unexpected chaining phenotype of a hisB mutant allowed the discovery of new crucial actors in cell division in B. abortus.

Discovery of the Lead Molecules Targeting the First Step of the Histidine Biosynthesis Pathway of Acinetobacter baumannii.

Acinetobacter baumannii is a multidrug-resistant, opportunistic, nosocomial pathogen for which a new line of treatments is desperately needed. We have targeted the enzyme of the first step of the histidine biosynthesis pathway, viz., ATP-phosphoribosyltransferase (ATP-PRT). The three-dimensional structure of ATP-PRT was predicted on the template of the known three-dimensional structure of ATP-PRT from Psychrobacter arcticus (PaATPPRT) using a homology modeling approach. High-throughput virtual screening (HTVS) of the antibacterial library of Life Chemicals Inc., Ontario, Canada was carried out followed by molecular dynamics simulations of the top hit compounds. In silico results were then biochemically validated using surface plasmon resonance spectroscopy. We found that two compounds, namely, F0843-0019 and F0608-0626, were binding with micromolar affinities to the ATP-phosphoribosyltransferase from Acinetobacter baumannii (AbATPPRT). Both of these compounds were binding in the same way as AMP in PaATPPRT, and the important residues of the active site, viz., Val4, Ser72, Thr76, Tyr77, Glu95, Lys134, Val136, and Tyr156, were also interacting via hydrogen bonds. The calculated binding energies of these compounds were -10.5 kcal/mol and -11.1 kcal/mol, respectively. These two compounds can be used as the potential lead molecules for designing antibacterial compounds in the future, and this information will help in drug discovery programs against Acinetobacter worldwide.

Expanding the toolbox: another auxotrophic marker for targeted gene integrations in Trichoderma reesei

BackgroundThe filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei.ResultsIn this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci.ConclusionWith the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.

Unveiling the novel characteristics of IGPD polymer and inhibitors binding affinities using 12-6-4 LJ-type nonbonded Mn2+ model

The metalloenzyme imidazole glycerophosphate dehydratase (IGPD) (EC 4.2.1.19) is essential in the histidine biosynthesis pathway in prokaryotes and eukaryotes, hence it has been identified as an attractive target with a majority of inhibitor studies focused on the development of broad-spectrum herbicides and bacteriostatic agent. However, due to the lack of work in structural biology, the reported inhibitors developed slowly with poor selectivity and diversity. To reveal the inhibitors binding affinities in ions centers of IGPD, we built point charge models based on a 12-6-4 type Lennard-Jones (LJ) potential, while combined DFT analyses, molecular docking and molecular dynamics (MD) simulations. This work introduced the interaction with surrounding solvent molecules and revealed a few undiscovered structural and kinetic characterizations of IGPD Mn2+ ions centers and ligand binding path, which revised some previous inexact assumptions inferred from crystal structures information. In addition, we also compared the dynamic binding characteristics of five IGPD inhibitors, and found that hydrogen bond interaction, spatial position and coordination stability with two Mn2+ ions can lead to the difference in potency. Overall, this work firstly unraveled the dynamic characteristics of IGPD polymer systems in explicit solvent and also provided a reference for the theoretical work of Mn2+-containing metalloenzyme, contributing for further target development.

Genetic investigation of purine nucleotide imbalance in Saccharomyces cerevisiae.

Because metabolism is a complex balanced process involving multiple enzymes, understanding how organisms compensate for transient or permanent metabolic imbalance is a challenging task that can be more easily achieved in simpler unicellular organisms. The metabolic balance results not only from the combination of individual enzymatic properties, regulation of enzyme abundance, but also from the architecture of the metabolic network offering multiple interconversion alternatives. Although metabolic networks are generally highly resilient to perturbations, metabolic imbalance resulting from enzymatic defect and specific environmental conditions can be designed experimentally and studied. Starting with a double amd1 aah1 mutant that severely and conditionally affects yeast growth, we carefully characterized the metabolic shuffle associated with this defect. We established that the GTP decrease resulting in an adenylic/guanylic nucleotide imbalance was responsible for the growth defect. Identification of several gene dosage suppressors revealed that TAT1, encoding an amino acid transporter, is a robust suppressor of the amd1 aah1 growth defect. We show that TAT1 suppression occurs through replenishment of the GTP pool in a process requiring the histidine biosynthesis pathway. Importantly, we establish that a tat1 mutant exhibits synthetic sickness when combined with an amd1 mutant and that both components of this synthetic phenotype can be suppressed by specific gene dosage suppressors. Together our data point to a strong phenotypic connection between amino acid uptake and GTP synthesis, a connection that could open perspectives for future treatment of related human defects, previously reported as etiologically highly conserved.

MIG1 as a positive regulator for the histidine biosynthesis pathway and as a global regulator in thermotolerant yeast Kluyveromyces marxianus

Kmmig1 as a disrupted mutant of MIG1 encoding a regulator for glucose repression in Kluyveromyces marxianus exhibits a histidine-auxotrophic phenotype. Genome-wide expression analysis revealed that only HIS4 in seven HIS genes for histidine biosynthesis was down-regulated in Kmmig1. Consistently, introduction of HIS4 into Kmmig1 suppressed the requirement of histidine. Considering the fact that His4 catalyzes four of ten steps in histidine biosynthesis, K. marxianus has evolved a novel and effective regulation mechanism via Mig1 for the control of histidine biosynthesis. Moreover, RNA-Seq analysis revealed that there were more than 1,000 differentially expressed genes in Kmmig1, suggesting that Mig1 is directly or indirectly involved in the regulation of their expression as a global regulator.

Comparative genomics reveals the distinct evolutionary trajectories of the robust and complex coral lineages

BackgroundDespite the biological and economic significance of scleractinian reef-building corals, the lack of large molecular datasets for a representative range of species limits understanding of many aspects of their biology. Within the Scleractinia, based on molecular evidence, it is generally recognised that there are two major clades, Complexa and Robusta, but the genomic bases of significant differences between them remain unclear.ResultsDraft genome assemblies and annotations were generated for three coral species: Galaxea fascicularis (Complexa), Fungia sp., and Goniastrea aspera (Robusta). Whilst phylogenetic analyses strongly support a deep split between Complexa and Robusta, synteny analyses reveal a high level of gene order conservation between all corals, but not between corals and sea anemones or between sea anemones. HOX-related gene clusters are, however, well preserved across all of these combinations. Differences between species are apparent in the distribution and numbers of protein domains and an apparent correlation between number of HSP20 proteins and stress tolerance. Uniquely amongst animals, a complete histidine biosynthesis pathway is present in robust corals but not in complex corals or sea anemones. This pathway appears to be ancestral, and its retention in the robust coral lineage has important implications for coral nutrition and symbiosis.ConclusionsThe availability of three new coral genomes enabled recognition of a de novo histidine biosynthesis pathway in robust corals which is only the second identified biosynthetic difference between corals. These datasets provide a platform for understanding many aspects of coral biology, particularly the interactions of corals with their endosymbionts.

Structural and functional studies of histidine biosynthesis in Acanthamoeba spp. demonstrates a novel molecular arrangement and target for antimicrobials.

Acanthamoeba is normally free-living, but sometimes facultative and occasionally opportunistic parasites. Current therapies are, by necessity, arduous and yet poorly effective due to their inabilities to kill cyst stages or in some cases to actually induce encystation. Acanthamoeba can therefore survive as cysts and cause disease recurrence. Herein, in pursuit of better therapies and to understand the biochemistry of this understudied organism, we characterize its histidine biosynthesis pathway and explore the potential of targeting this with antimicrobials. We demonstrate that Acanthamoeba is a histidine autotroph, but with the ability to scavenge preformed histidine. It is able to grow in defined media lacking this amino acid, but is inhibited by 3-amino-1,2,4-triazole (3AT) that targets Imidazoleglycerol-Phosphate Dehydratase (IGPD) the rate limiting step of histidine biosynthesis. The structure of Acanthamoeba IGPD has also been determined in complex with 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate [(R)-C348], a recently described novel inhibitor of Arabidopsis thaliana IGPD. This compound inhibited the growth of four Acanthamoeba species, having a 50% inhibitory concentration (IC50) ranging from 250–526 nM. This effect could be ablated by the addition of 1 mM exogenous free histidine, but importantly not by physiological concentrations found in mammalian tissues. The ability of 3AT and (R)-C348 to restrict the growth of four strains of Acanthamoeba spp. including a recently isolated clinical strain, while not inducing encystment, demonstrates the potential therapeutic utility of targeting the histidine biosynthesis pathway in Acanthamoeba.

Identification and structural characterization of a histidinol phosphate phosphatase from Mycobacterium tuberculosis

The absence of a histidine biosynthesis pathway in humans, coupled with histidine essentiality for survival of the important human pathogen Mycobacterium tuberculosis (Mtb), underscores the importance of the bacterial enzymes of this pathway as major antituberculosis drug targets. However, the identity of the mycobacterial enzyme that functions as the histidinol phosphate phosphatase (HolPase) of this pathway remains to be established. Here, we demonstrate that the enzyme encoded by the Rv3137 gene, belonging to the inositol monophosphatase (IMPase) family, functions as the Mtb HolPase and specifically dephosphorylates histidinol phosphate. The crystal structure of Rv3137 in apo form enabled us to dissect its distinct structural features. Furthermore, the holo-complex structure revealed that a unique cocatalytic multizinc-assisted mode of substrate binding and catalysis is the hallmark of Mtb HolPase. Interestingly, the enzyme-substrate complex structure unveiled that although monomers possess individual catalytic sites they share a common product-exit channel at the dimer interface. Furthermore, target-based screening against HolPase identified several small-molecule inhibitors of this enzyme. Taken together, our study unravels the missing enzyme link in the Mtb histidine biosynthesis pathway, augments our current mechanistic understanding of histidine production in Mtb, and has helped identify potential inhibitors of this bacterial pathway.

ATP phosphoribosyltransferase from symbiont Entomomyces delphacidicola invovled in histidine biosynthesis of Nilaparvata lugens (Stål).

Histidine is an essential amino acid assumed to be synthesized by an obligatory yeast-like symbiont (Entomomyces delphacidicola str. NLU) in Nilaparvata lugens, an important rice pest. The adenosine-triphosphate phosphoribosyltransferase (ATP-PRTase) facilities the committed first step of the histidine biosynthesis pathway. In the current study, a putative ATP-PRTase was cloned and verified to be of E. delphacidicola origin (EdePRTase). The expression of the gene was spatial and temporal universal with a profile that matched the distribution of the fungal symbiont. RNA interference aided the knockdown of the EdePRTase-suppressed EdePRTase expression by 32-48%. Hemolymph histidine level was also reduced followed by significant reduction of adult body weight. However, other performance characters including nymph development, survival, and adult sex ratio were not adversely affected by the knockdown. Furthermore, forced histidine exposure (through injection or feeding) significantly inhibited the EdePRTase mRNA levels at higher concentrations, but significantly increased EdePRTase expression levels at lower concentrations (feeding only). The significance of these findings support that the EdePRTase is from symbiont E. delphacidicola, and its involvement in histidine biosynthesis of N. lugens was discussed. The results provide a better understanding of EdePRTase and the encoded functional ATP-PRTase enzyme regulation in N. lugens and insects in general.

Two-step Ligand Binding in a (βα)8 Barrel Enzyme: SUBSTRATE-BOUND STRUCTURES SHED NEW LIGHT ON THE CATALYTIC CYCLE OF HisA

HisA is a (βα)8 barrel enzyme that catalyzes the Amadori rearrangement of N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N'-((5'-phosphoribulosyl) formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the histidine biosynthesis pathway, and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo-state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp-7 acts as the catalytic base, and Asp-176 acts as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen bond network including His-47, Asp-129, Thr-171, and Ser-202, all shown to be functionally important. The closed conformation structure is highly similar to the bifunctional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.

Transcriptomic analysis of three Veillonella spp. present in carious dentine and in the saliva of caries-free individuals.

Veillonella spp. are predominant bacteria found in all oral biofilms. In this study, a metatranscriptomic approach was used to investigate the gene expression levels of three oral Veillonella spp. (V. parvula, V. dispar and V. atypica) in whole stimulated saliva from caries-free volunteers and in carious lesions (n = 11 for each group). In the lesions the greatest proportion of reads were assigned to V. parvula and genes with the highest level of expression in carious samples were those coding for membrane transport systems. All three Veillonella spp. increased expression of genes involved in the catabolism of lactate and succinate, notably the alpha- and beta-subunits of L(+)-tartrate dehydratase (EC 4.2.1.32). There was also significantly increased expression of histidine biosynthesis pathway in V. parvula, suggesting higher intra-cellular levels of histidine that could provide intra-cellular buffering capacity and, therefore, assist survival in the acidic environment. Various other systems such as potassium uptake systems were also up regulated that may aid in the survival and proliferation of V. parvula in carious lesions.

Characterization of the inositol monophosphatase gene family in Arabidopsis.

Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca2+ signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.

Structures of native, substrate-bound and inhibited forms ofMycobacterium tuberculosisimidazoleglycerol-phosphate dehydratase

Imidazoleglycerol-phosphate dehydratase (IGPD; HisB), which catalyses the conversion of imidazoleglycerol-phosphate (IGP) to imidazoleacetol-phosphate in the histidine biosynthesis pathway, is absent in mammals. This feature makes it an attractive target for herbicide discovery. Here, the crystal structure of Mycobacterium tuberculosis (Mtb) IGPD is reported together with the first crystal structures of substrate-bound and inhibited (by 3-amino-1,2,4-triazole; ATZ) forms of IGPD from any organism. The overall tertiary structure of Mtb IGPD, a four-helix-bundle sandwiched between two four-stranded mixed β-sheets, resembles the three-dimensional structures of IPGD from other organisms; however, Mtb IGPD possesses a unique structural feature: the insertion of a one-turn 310-helix followed by a loop ten residues in length. The functional form of IGPD is 24-meric, exhibiting 432 point-group symmetry. The structure of the IGPD-IGP complex revealed that the imidazole ring of the IGP is firmly anchored between the two Mn atoms, that the rest of the substrate interacts through hydrogen bonds mainly with residues Glu21, Arg99, Glu180, Arg121 and Lys184 which protrude from three separate protomers and that the 24-mer assembly contains 24 catalytic centres. Both the structural and the kinetic data demonstrate that the inhibitor 3-amino-1,2,4-triazole inhibits IGPD competitively.

Structural basis for the rational design of new anti-Brucella agents: The crystal structure of the C366S mutant of l-histidinol dehydrogenase from Brucella suis

L-Histidinol dehydrogenase from Brucella suis (BsHDH) is an enzyme involved in the histidine biosynthesis pathway which is absent in mammals, thus representing a very interesting target for the development of anti-Brucella agents. In this paper we report the crystallographic structure of a mutated form of BsHDH both in its unbound form and in complex with a nanomolar inhibitor. These studies provide the first structural background for the rational design of potent HDH inhibitors, thus offering new hints for clinical applications.

HISN3 Mediates Adaptive Response of Chlamydomonas reinhardtii to Excess Nickel

Investigation of genes for heavy metal [e.g. nickel (Ni) and zinc (Zn)] absorption and detoxification in green algae is of great importance because some of the metals have become one of the major contaminants in the aquatic ecosystem. In plants, overload of heavy metals modifies many aspects of biological processes. However, the mechanisms by which heavy metals exert detrimental effects are not fully understood. The present study identified a biological role for HISN3 (the gene coding for phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleotide isomerase) in regulating the response of Chlamydomonas reinhardtii, a unicellular green alga, to Ni toxicity. In higher plants, HISN3 encodes an enzyme catalyzing the fourth step in the histidine biosynthesis pathway, but its functional importance is yet to be identified. Transgenic algae overexpressing HISN3 in C. reinhardtii showed high tolerance to excess Ni, with a 48.3-57.4% increase in cell population and moderate histidine accumulation compared with the wild type. HISN3 overexpression improved accumulation of Chl and photosynthesis efficiency, but suppressed Ni-induced generation of reactive oxygen species and lipid peroxides. Interestingly, more Ni and other metals [Zn, iron (Fe), copper (Cu), manganese (Mn) and magnesium (Mg)] were accumulated in HISN3-overexpressing cells than in the wild type. In contrast, RNA interference of HISN3 depressed Ni accumulation but caused cellular sensitivity to Ni. The elevated metal absorption in the HISN3-overexpressing algae implies that the metals can be removed from water media. Thus, our work presents an example for algae genetically designed to improve tolerance to metal toxicity and environmental restoration of metal-contaminated aquatic ecosystems.

Modification of histidine biosynthesis pathway genes and the impact on production of l-histidine in Corynebacterium glutamicum

Histidine biosynthesis in Corynebacterium glutamicum is regulated not only by feedback inhibition by the first enzyme in the pathway, but also by repression control of the synthesis of the histidine enzymes. C. glutamicum histidine genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. We constructed plasmid pK18hisDPtac to replace the native hisD promoter with the tac promoter, and overexpressed phosphoribosyl-ATP-pyrophosphohydrolase, encoded by hisE, and ATP-phosphoribosyltransferase, encoded by hisG. The L-histidine titer at 0.85 g l(-1) was 80 % greater in the transformed bacterium and production of byproducts, L-alanine and L-tryptophan, was significantly decreased. However, accumulation of glutamic acid increased by 58 % (2.8 g l(-1)). This study represents the first attempt to substitute the histidine biosynthesis pathway promoter in the chromosome with a stronger promoter to increase histidine production.

Cellular Plasticity Enables Adaptation to Unforeseen Cell-Cycle Rewiring Challenges

The fundamental dynamics of the cell cycle, underlying cell growth and reproduction, were previously found to be robust under a wide range of environmental and internal perturbations. This property was commonly attributed to its network structure, which enables the coordinated interactions among hundreds of proteins. Despite significant advances in deciphering the components and autonomous interactions of this network, understanding the interfaces of the cell cycle with other major cellular processes is still lacking. To gain insight into these interfaces, we used the process of genome-rewiring in yeast by placing an essential metabolic gene HIS3 from the histidine biosynthesis pathway, under the exclusive regulation of different cell-cycle promoters. In a medium lacking histidine and under partial inhibition of the HIS3p, the rewired cells encountered an unforeseen multitasking challenge; the cell-cycle regulatory genes were required to regulate the essential histidine-pathway gene in concert with the other metabolic demands, while simultaneously driving the cell cycle through its proper temporal phases. We show here that chemostat cell populations with rewired cell-cycle promoters adapted within a short time to accommodate the inhibition of HIS3p and stabilized a new phenotypic state. Furthermore, a significant fraction of the population was able to adapt and grow into mature colonies on plates under such inhibiting conditions. The adapted state was shown to be stably inherited across generations. These adaptation dynamics were accompanied by a non-specific and irreproducible genome-wide transcriptional response. Adaptation of the cell-cycle attests to its multitasking capabilities and flexible interface with cellular metabolic processes and requirements. Similar adaptation features were found in our previous work when rewiring HIS3 to the GAL system and switching cells from galactose to glucose. Thus, at the basis of cellular plasticity is the emergence of a yet-unknown general, non-specific mechanism allowing fast inherited adaptation to unforeseen challenges.