Pathway engineering for the production of heterologous aromatic chemicals and their derivatives in Saccharomyces cerevisiae: bioconversion from glucose.

Saccharomyces cerevisiae has been extensively engineered for optimising its performance as a microbial cell factory to produce valuable aromatic compounds and their derivatives as bulk and fine chemicals. The production of heterologous aromatic molecules in yeast is achieved via engineering of the aromatic amino acid biosynthetic pathway. This pathway is connected to two pathways of the central carbon metabolism, and is highly regulated at the gene and protein level. These characteristics impose several challenges for tailoring it, and various modifications need to be applied in order to redirect the carbon flux towards the production of the desired compounds. This minireview addresses the metabolic engineering approaches targeting the central carbon metabolism, the shikimate pathway and the tyrosine and phenylalanine biosynthetic pathway of S. cerevisiae for biosynthesis of aromatic chemicals and their derivatives from glucose.

Recent Advances in Microbial Production of Aromatic Chemicals and Derivatives.

Along with the development of metabolic engineering and synthetic biology tools, various microbes are being used to produce aromatic chemicals. In microbes, aromatics are mainly produced via a common important precursor, chorismate, in the shikimate pathway. Natural or non-natural aromatics have been produced by engineering metabolic pathways involving chorismate. In the past decade, novel approaches have appeared to produce various aromatics or to increase their productivity, whereas previously, the targets were mainly aromatic amino acids and the strategy was deregulating feedback inhibition. In this review, we summarize recent studies of microbial production of aromatics based on metabolic engineering approaches. In addition, future perspectives and challenges in this research area are discussed.

An Overview of Compounds Derived from the Shikimate and Phenylpropanoid Pathways and Their Medicinal Importance.

OBJECTIVE: There has been a massive increase in the number of reports about the medicinal benefits of the consumption of phenylpropanoids derived from the plastidic shikimate pathway. These benefits include anti-retroviral, anti-hypertensive, anti-inflammatory, anti-aging and insulin-sensitizing activities, the reduction of the risk of a range of chronic diseases including cardiovascular disease, cancer and osteoporosis as well as inhibition of LDL (low-density lipoprotein) oxidation. In addition, chorismate-derived salicylate which was originally isolated from plants, albeit now under chemical production, is massively used for pain relief in the form of acetylsalycilic acid, namely aspirin. Chorismate also acts as precursor in the biosynthesis of folate and phylloquinone, i.e., vitamins B9 and K1, respectively. RESULTS: Cumulative evidence suggests that deficiencies of either of these vitamins in the diet can result in a wide range of diseases. In parallel to our enhanced comprehension of the dietary importance of shikimate-derived compounds, the advent of metabolomics and the development of next-generation sequencing technologies have dramatically accelerated advances in our understanding of the biosynthetic, decorative and degradation pathways underlying their metabolism. Furthermore, forward and reverse genetic approaches have begun to facilitate the metabolic engineering of plants for biofortification of these compounds. CONCLUSION: Here we review data about the bioactivities of these compounds and provide an overview of our current understanding of biosynthesis, molecular function and their in planta occurrence. Finally we discuss the future perspectives and the importance of further development of cross-disciplinary research efforts in this rapidly expanding research field.

Glyphosate's impact on vegetative growth in leafy spurge identifies molecular processes and hormone cross-talk associated with increased branching.

BACKGROUND: Leafy spurge (Euphorbia esula) is a perennial weed that is considered glyphosate tolerant, which is partially attributed to escape through establishment of new vegetative shoots from an abundance of underground adventitious buds. Leafy spurge plants treated with sub-lethal concentrations of foliar-applied glyphosate produce new vegetative shoots with reduced main stem elongation and increased branching. Processes associated with the glyphosate-induced phenotype were determined by RNAseq using aerial shoots derived from crown buds of glyphosate-treated and -untreated plants. Comparison between transcript abundance and accumulation of shikimate or phytohormones (abscisic acid, auxin, cytokinins, and gibberellins) from these same samples was also done to reveal correlations. RESULTS: Transcriptome assembly and analyses confirmed differential abundance among 12,918 transcripts (FDR ≤ 0.05) and highlighted numerous processes associated with shoot apical meristem maintenance and stem growth, which is consistent with the increased number of actively growing meristems in response to glyphosate. Foliar applied glyphosate increased shikimate abundance in crown buds prior to decapitation of aboveground shoots, which induces growth from these buds, indicating that 5-enolpyruvylshikimate 3-phosphate (EPSPS) the target site of glyphosate was inhibited. However, abundance of shikimate was similar in a subsequent generation of aerial shoots derived from crown buds of treated and untreated plants, suggesting EPSPS is no longer inhibited or abundance of shikimate initially observed in crown buds dissipated over time. Overall, auxins, gibberellins (precursors and catabolites of bioactive gibberellins), and cytokinins (precursors and bioactive cytokinins) were more abundant in the aboveground shoots derived from glyphosate-treated plants. CONCLUSION: Based on the overall data, we propose that the glyphosate-induced phenotype resulted from complex interactions involving shoot apical meristem maintenance, hormone biosynthesis and signaling (auxin, cytokinins, gibberellins, and strigolactones), cellular transport, and detoxification mechanisms.

In vitro production and purification of isochorismate using a two-enzyme cascade.

Combining the isochorismate synthase EntC and the chorismatase FkbO in a sequential enzyme cascade provides a useful system for the biocatalytic production and subsequent purification of isochorismate from an isochorismate/chorismate mixture. FkbO has a strict preference for chorismate - isochorismate is not accepted as a substrate - therefore the enzyme can be used to selectively hydrolyse chorismate, leading to the chiral building block 3,4-dihydroxycyclohexa-1,5-dienecarboxylate. This simplifies the final purification step, as isochorismate is much easier to separate from the chorismate hydrolysis products than from chorismate itself. The presented procedure starts with an optimised method for purifying chorismate from Escherichia coli culture supernatants, which is followed by conversion into isochorismate with the isochorismate synthase EntC, removal of the remaining chorismate by FkbO and a final purification step using an automated flash chromatography system. Isochorismate was isolated in up to 20% yield and >95% purity from chorismate, and has been characterised with respect to its degradation and suitability as a substrate in enzyme assays.

Insight into the transmission biology and species-specific functional capabilities of tsetse (Diptera: glossinidae) obligate symbiont Wigglesworthia.

UNLABELLED: Ancient endosymbionts have been associated with extreme genome structural stability with little differentiation in gene inventory between sister species. Tsetse flies (Diptera: Glossinidae) harbor an obligate endosymbiont, Wigglesworthia, which has coevolved with the Glossina radiation. We report on the ~720-kb Wigglesworthia genome and its associated plasmid from Glossina morsitans morsitans and compare them to those of the symbiont from Glossina brevipalpis. While there was overall high synteny between the two genomes, a large inversion was noted. Furthermore, symbiont transcriptional analyses demonstrated host tissue and development-specific gene expression supporting robust transcriptional regulation in Wigglesworthia, an unprecedented observation in other obligate mutualist endosymbionts. Expression and immunohistochemistry confirmed the role of flagella during the vertical transmission process from mother to intrauterine progeny. The expression of nutrient provisioning genes (thiC and hemH) suggests that Wigglesworthia may function in dietary supplementation tailored toward host development. Furthermore, despite extensive conservation, unique genes were identified within both symbiont genomes that may result in distinct metabolomes impacting host physiology. One of these differences involves the chorismate, phenylalanine, and folate biosynthetic pathways, which are uniquely present in Wigglesworthia morsitans. Interestingly, African trypanosomes are auxotrophs for phenylalanine and folate and salvage both exogenously. It is possible that W. morsitans contributes to the higher parasite susceptibility of its host species. IMPORTANCE: Genomic stasis has historically been associated with obligate endosymbionts and their sister species. Here we characterize the Wigglesworthia genome of the tsetse fly species Glossina morsitans and compare it to its sister genome within G. brevipalpis. The similarity and variation between the genomes enabled specific hypotheses regarding functional biology. Expression analyses indicate significant levels of transcriptional regulation and support development- and tissue-specific functional roles for the symbiosis previously not observed in obligate mutualist symbionts. Retention of the genetically expensive flagella within these small genomes was demonstrated to be significant in symbiont transmission and tailored to the unique tsetse fly reproductive biology. Distinctions in metabolomes were also observed. We speculate an additional role for Wigglesworthia symbiosis where infections with pathogenic trypanosomes may depend upon symbiont species-specific metabolic products and thus influence the vector competence traits of different tsetse fly host species.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase.

BACKGROUND: The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. RESULTS: In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. CONCLUSION: This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents.

Mutagenic analysis of an invariant aspartate residue in chorismate synthase supports its role as an active site base.

Chorismate synthase catalyzes the anti-1,4-elimination of the 3-phosphate and the C(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, the final product of the common shikimate pathway and a precursor for the biosynthesis of aromatic compounds. The enzyme has an absolute requirement for reduced FMN, which is thought to facilitate cleavage of C-O bonds by transiently donating an electron to the substrate. The crystal structure of the enzyme revealed that EPSP is bound near the flavin isoalloxazine ring with several invariant amino acid residues in contact with the substrate and/or cofactor. Here, we report the results of a mutagenesis study in which an invariant aspartate residue at position 367 of the Neurospora crassa chorismate synthase was replaced with alanine and asparagine. Both single mutant proteins (Asp367Ala and Asp367Asn) were comparable to the wild-type enzyme with respect to substrate and cofactor binding, indicating that Asp367 is not required for binding of either the flavin or the substrate. In sharp contrast to these results, the activity of both single mutant proteins was found to be 620 and 310 times lower for the Asp367Ala and Asp367Asn mutant proteins, respectively. This finding provides strong evidence that the carboxylate group of Asp367 plays a major role during the catalytic reaction. On the basis of the structure of the enzyme, our data provide the first experimental evidence that the carboxylate group of aspartate 367 participates in the deprotonation of N(5) of the reduced flavin cofactor, which in turn abstracts the C(6proR) hydrogen yielding chorismate as the product.

Arabidopsis isochorismate synthase functional in pathogen-induced salicylate biosynthesis exhibits properties consistent with a role in diverse stress responses.

Salicylic acid (SA) is a phytohormone best known for its role in plant defense. It is synthesized in response to diverse pathogens and responsible for the large scale transcriptional induction of defense-related genes and the establishment of systemic acquired resistance. Surprisingly, given its importance in plant defense, an understanding of the underlying enzymology is lacking. In Arabidopsis thaliana, the pathogen-induced accumulation of SA requires isochorismate synthase (AtICS1). Here, we show that AtICS1 is a plastid-localized, stromal protein using chloroplast import assays and immunolocalization. AtICS1 acts as a monofunctional isochorismate synthase (ICS), catalyzing the conversion of chorismate to isochorismate (IC) in a reaction that operates near equilibrium (K(eq) = 0.89). It does not convert chorismate directly to SA (via an IC intermediate) as does Yersinia enterocolitica Irp9. Using an irreversible coupled spectrophotometric assay, we found that AtICS1 exhibits an apparent K(m) of 41.5 mum and k(cat) = 38.7 min(-1) for chorismate. This affinity for chorismate would allow it to successfully compete with other pathogen-induced, chorismate-utilizing enzymes. Furthermore, the biochemical properties of AtICS1 indicate its activity is not regulated by light-dependent changes in stromal pH, Mg(2+), or redox and that it is remarkably active at 4 degrees C consistent with a role for SA in cold-tolerant growth. Finally, our analyses support plastidic synthesis of stress-induced SA with the requirement for one or more additional enzymes responsible for the conversion of IC to SA, because non-enzymatic conversion of IC to SA under physiological conditions was negligible.

The isolation and nucleotide sequence of the complex AROM locus of Aspergillus nidulans.

The AROM locus of A. nidulans, which governs five consecutive steps in pre-chorismate aromatic amino acid biosynthesis, has been cloned in a bacteriophage vector. The nucleotide sequence of the locus reveals a single, open reading-frame of 4,812 base-pairs, apparently without introns. An internal segment of the A. nidulans AROM sequence has extensive homology with the E. coli aroA gene that encodes the 5-enolpyruvylshikimate 3-phosphate synthase.

Purification of the arom multienzyme aggregate from Euglena gracilis.

The arom multienzyme complex that catalyzes steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway has been purified up to 2000-fold from Euglena gracilis. The native arom aggregate has a molecular weight of approx. 249 000 based on a sedimentation coefficient of 9.5 and Stokes radius of 60 angstrom. A comparison between the arom aggregates of Neurospora crassa and Euglena gracilis and the possible phylogenetic relationships between the organisms are discussed.