aroA Gene Research Articles

Characterization of Schu S4 aro mutants as live attenuated tularemia vaccine candidates

ABSTRACT There is a need for development of an effective vaccine against Francisella tularensis, as this potential bioweapon has a high mortality rate and low infectious dose when delivered via the aerosol route. Moreover, this Tier 1 agent has a history of weaponization. We engineered targeted mutations in the Type A strain F. tularensis subspecies tularensis Schu S4 in aro genes encoding critical enzymes in aromatic amino acid biosynthesis. F. tularensis Schu S4ΔaroC, Schu S4ΔaroD, and Schu S4ΔaroCΔaroD mutant strains were attenuated for intracellular growth in vitro and for virulence in vivo and, conferred protection against pulmonary wild-type (WT) F. tularensis Schu S4 challenge in the C57BL/6 mouse model. F. tularensis Schu S4ΔaroD was identified as the most promising vaccine candidate, demonstrating protection against high-dose intranasal challenge; it protected against 1,000 CFU Schu S4, the highest level of protection tested to date. It also provided complete protection against challenge with 92 CFU of a F. tularensis subspecies holarctica strain (Type B). Mice responded to vaccination with Schu S4ΔaroD with systemic IgM and IgG2c, as well as the production of a functional T cell response as measured in the splenocyte-macrophage co-culture assay. This vaccine was further characterized for dissemination, histopathology, and cytokine/chemokine gene induction at defined time points following intranasal vaccination which confirmed its attenuation compared to WT Schu S4. Cytokine, chemokine, and antibody induction patterns compared to wild-type Schu S4 distinguish protective vs. pathogenic responses to F. tularensis and elucidate correlates of protection associated with vaccination against this agent.

Flux Balance Analysis for Media Optimization and Genetic Targets to Improve Heterologous Siderophore Production.

SummarySiderophores are small molecule metal chelators secreted in sparse quantities by their native microbial hosts but can be engineered for enhanced production from heterologous hosts like Escherichia coli. These molecules have been proved to be capable of binding heavy metals of commercial and/or environmental interest. In this work, we incorporated, as needed, the appropriate pathways required to produce several siderophores (anguibactin, vibriobactin, bacillibactin, pyoverdine, and enterobactin) into the base E. coli K-12 MG1655 metabolic network model to computationally predict, via flux balance analysis methodologies, gene knockout targets, gene over-expression targets, and media modifications capable of improving siderophore reaction flux. E. coli metabolism proved supportive for the underlying production mechanisms of various siderophores. Within such a framework, the gene deletion and over-expression targets identified, coupled with complementary insights from medium optimization predictions, portend experimental implementation to both enable and improve heterologous siderophore production. Successful production of siderophores would then spur novel metal-binding applications.

Prioritisation of potential drug targets against Bartonella bacilliformis by an integrative in-silico approach.

BACKGROUND Carrion’s disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens.OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis.METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery.FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion’s disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/).MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.

Safety of the live Escherichia coli vaccine Poulvac® E. coli in layer parent stock in a field trial

The safety of the live Escherichia coli vaccine Poulvac® E. coli was tested with a flock (10,000) of layer parents aged 30 weeks. Three and 7 days after vaccination, 60 whole unbroken eggs, the egg white and yolk of 60 eggs and 60 cloacal swabs were enriched in MacConkey broth. At both sampling times, 6 out of 60 whole eggs were found positive for coliform bacteria. None of the enriched samples of yolk + egg white were positive for coliform bacteria. Three and seven days after vaccination 44 and 37, respectively out of 60 swabs were positive for coliform bacteria in MacConkey broth. All coliform isolates collected from whole eggs and cloacal swabs were tested in parallel for growth on minimal agar and blood agar to identify the vaccine strain. Some isolates showed reduced growth on minimal agar compared to blood agar and they were tested further with a PCR for the aroA gene mutation and all were found with the wild type version of the gene. Only two isolates did not grow on minimal agar but grew on blood agar and they were tested both with PCR and PFGE. They also showed the wild type version of the aroA gene and their PFGE profile was different from the vaccine strain of Poulvac® E. coli. In conclusion, the Poulvac® E. coli vaccine strain of E. coli was not identified at the detection limit of one CFU on one egg or in the content of one egg or from a cloacal swab of one hen with at least 95 % probability on flock level. The use of the vaccine is safe for hens in lay with lack of survival of the vaccine strain and lack of negative effects on the hens including egg production.

Construction and in vitro characterisation of aroA defective (aroAΔ) mutant Salmonella Infantis.

Poultry vaccine programs are important for control of Salmonella infections. Although there are vaccines for Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Typhi, there are no vaccines for Salmonella Infantis which has an increased rate in the world. In this study, it was aimed to generate aroA gene deleted mutant bacteria for the constitution of S. Infantis vaccine prototype and the in vitro characterisation of this bacterium. S. Infantis auxotrophic mutant which has a block at any step of chorismate pathway has been constituted for the first time in the world and it was determined that this bacterium gets susceptibility against some antibiotics and antimicrobial substances. It was also observed that the adhesion and invasion rate of mutant strain tenfold decreased in comparison with the field strain in cell culture assay. It is understood from the in vitro evaluation of this mutant strain that it can be used as a vaccine candidate in further vaccine development studies.

Genome characterization of an extensively drug-resistant Streptococcus pneumoniae serotype 11A strain.

In this study, the whole genome sequences of two Streptococcus pneumoniae clinical isolates from South Korea were determined and compared. They were found to be the same serotype (11 A) and multilocus sequence typing analysis showed that they are single-locus variants (SLVs; ST8279 and ST166) of each other, differing at one allele (aroE). However, the ST8279 strain is extensively drug-resistant (XDR) whereas the ST166 strain is not. The genome of the XDR strain is very similar in structure to that of two previously reported genomes, AP200 (11 A:ST62) and 70585 (5:ST5803); however, some regions were inverted and there were some exogenous regions in the ST8279 strain. It was found that 6,502 single nucleotide polymorphisms are dispersed across the genome between the two serotype 11 A ST8279 and ST166 strains. Many of them are located in genes associated with antibiotic resistance. In addition, many amino acid differences were also identified in genes involved in DNA repair (mutL, uvrA and uvrC) and recombination (recU, recR and recA). On the basis of these results, it was inferred that the XDR strain did not evolve from its SLV via a single recombination event involving a large portion of the genome including the aroE gene. Rather, the strain likely evolved through many point mutations and recombination events involving small portions of the genome.

Simulations of Shikimate Dehydrogenase from Mycobacterium tuberculosis in Complex with 3-Dehydroshikimate and NADPH Suggest Strategies for MtbSDH Inhibition.

Shikimate dehydrogenase (SDH) from Mycobacterium tuberculosis ( MtbSDH), encoded by the aroE gene, is essential for viability of M. tuberculosis but absent from humans. Therefore, it is a potentially promising target for antituberculosis agent development. Molecular-level understanding of the interactions of MtbSDH with its 3-dehydroshikimate (DHS) substrate and NADPH cofactor will help in the design of novel and effective MtbSDH inhibitors. However, this is limited by the lack of relevant crystal structures for MtbSDH complexes. Here, molecular dynamics (MD) simulations were performed to generate these MtbSDH complexes and investigate interactions of MtbSDH with substrate and cofactor and the role of MtbSDH dynamics within these. The results indicate that, while structural rearrangements are not necessary for DHS binding, reorientation of individual side chains in the NADPH binding pocket is involved in ternary complex formation. The mechanistic roles for Lys69, Asp105, and Ala213 were investigated by generating Lys69Ala, Asp105Asn, and Ala213Leu mutants in silico and investigating their complexes with DHS and NADPH. Our results show that Lys69 plays a dual role, in positioning NADPH and in catalysis. Asp105 plays a crucial role in positioning both the ε-amino group of Lys69 and nicotinamide ring of NADPH for MtbSDH catalysis but makes no direct contribution to DHS binding. Ala213 is the selection key for NADPH binding with the nicotinamide ring in the proS, rather than proR, conformation in the MtbSDH complex. Our results identify three strategies for MtbSDH inhibition: prevention of MtbSDH binary and ternary complex formation by blocking DHS and NADPH binding (first and second strategies, respectively) and the prevention of MtbSDH complex formation with either DHS or NADPH by blocking both DHS and NADPH binding (third strategy). Further, based on this third strategy, we propose guidelines for the rational design of "hybrid" MtbSDH inhibitors able to bind in both the substrate (DHS) and cofactor (NADPH) pockets, providing a new avenue of exploration in the search for anti-TB therapeutics.

Biochemical Characterization of Aspergillus fumigatus AroH, a Putative Aromatic Amino Acid Aminotransferase.

The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. Aspergillus fumigatus is an opportunistic fungus normally present in the environment directly responsible for lethal invasive infections. Recent results suggest that the metabolic pathways related to amino acid metabolism can regulate the fungus-host interaction and that an important role is played by enzymes involved in the catabolism of L-tryptophan. In particular, in A. fumigatus L-tryptophan regulates Aro genes. Among them, AroH encodes a putative pyridoxal 5'-phosphate-dependent aminotransferase. Here we analyzed the biochemical features of recombinant purified AroH by spectroscopic and kinetic analyses corroborated by in silico studies. We found that the protein is dimeric and tightly binds the coenzyme forming a deprotonated internal aldimine in equilibrium with a protonated ketoenamine form. By setting up a new rapid assay method, we measured the kinetic parameters for the overall transamination of substrates and we demonstrated that AroH behaves as an aromatic amino acid aminotransferase, but also accepts L-kynurenine and α-aminoadipate as amino donors. Interestingly, computational approaches showed that the predicted overall fold and active site topology of the protein are similar to those of its yeast ortholog, albeit with some differences in the regions at the entrance of the active site, which could possibly influence substrate specificity. Should targeting fungal metabolic adaptation be of therapeutic value, the results of the present study may pave the way to the design of specific AroH modulators as potential novel agents at the host/fungus interface.

The role of yeast ARO8, ARO9 and ARO10 genes in the biosynthesis of 3-(methylthio)-1-propanol from L-methionine during fermentation in synthetic grape medium.

3-(methylthio)-1-propanol (methionol), produced by yeast as an end-product of L-methionine (L-Met) catabolism, imparts off-odours reminiscent of cauliflower and potato to wine. Saccharomyces cerevisiae ARO genes, including transaminases Aro8p and Aro9p, and decarboxylase Aro10p, catalyse two key steps forming methionol via the Ehrlich pathway. We compared methionol concentrations in wines fermented by single Δaro8, Δaro9 and Δaro10 deletants in lab strain BY4743 versus wine strain Zymaflore F15, and F15 double- and triple-aro deletants versus single-aro deletants, using headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry.Deletion of two or more aro genes increased growth lag phase, with the greatest delay exhibited by F15 Δaro8 Δaro9. The single Δaro8 deletion decreased methionol by 44% in BY4743 and 92% in F15, while the Δaro9 deletion increased methionol by 46% in F15 but not BY4743. Single deletion of Δaro10 had no effect on methionol.Unexpectedly, F15 Δaro8 Δaro9 and F15 Δaro8 Δaro9 Δaro10 produced more methionol than F15 Δaro8. In the absence of Aro8p and Aro9p, other transaminases may compensate or an alternative pathway may convert methanethiol to methionol. Our results confirm that Ehrlich pathway genes differ greatly between lab and wine yeast strains, impacting downstream products such as methionol.

Expression of a bacterial aroA gene confers tolerance to glyphosate in tobacco plants.

Glyphosate is a widely used herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-encoding aroA gene in the shikimate pathway. The discovery and cloning of the aroA gene with high resistance is central to breeding a transgenic glyphosate-resistant plant. A novel aroAPantoea gene from Pantoea G-1 was previously isolated and cloned. The aroA Pantoea enzyme was defined as a new class I EPSPS with glyphosate resistance. The aroA Pantoea gene was introduced into tobacco through Agrobacteriummediated transformation. The transgenic tobacco plants were confirmed by PCR, RT-PCR, and Southern blot. The analysis of glyphosate resistance also showed that the transgenic tobacco plants could survive at 15 mM glyphosate; the glyphosate resistance level of the transgenic plants is higher than the agricultural application level recommended by most manufacturers. Overall, this study shows that aroAPantoea can be used as a candidate gene for the development of genetically modified crops.

Diversity and enzymatic potentialities of Bacillus sp. strains isolated from a polluted freshwater ecosystem in Cuba.

Genotypic and phenotypic characterization of Bacillus spp. from polluted freshwater has been poorly addressed. The objective of this research was to determine the diversity and enzymatic potentialities of Bacillus spp. strains isolated from the Almendares River. Bacilli strains from a polluted river were characterized by considering the production of extracellular enzymes using API ZYM. 14 strains were selected and identified using 16S rRNA, gyrB and aroE genes. Genotypic diversity of the Bacillus spp. strains was evaluated using pulsed field gel electrophoresis. Furthermore, the presence of genetic determinants of potential virulence toxins of the Bacillus cereus group and proteinaceous crystal inclusions of Bacillus thuringiensis was determined. 10 strains were identified as B. thuringiensis, two as Bacillus megaterium, one as Bacillus pumilus and one as Bacillus subtilis. Most strains produced proteases, amylases, phosphatases, esterases, aminopeptidases and glucanases, which reflect the abundance of biopolymeric matter in Almendares River. Comparison of the typing results revealed a spatio-temporal distribution among B. thuringiensis strains along the river. The results of the present study highlight the genotypic and phenotypic diversity of Bacillus spp. strains from a polluted river, which contributes to the knowledge of genetic diversity of Bacilli from tropical polluted freshwater ecosystems.

Identification and analysis of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from glyphosate-resistant Ochrobactrum intermedium Sq20.

Glyphosate is a herbicide that acts by inhibition of the enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), involved in the catalysis of an essential step in the biosynthesis of aromatic amino acids. The objective of this study was the isolation of glyphosate-resistant bacterial strains and subsequent characterization of the gene(s) encoding glyphosate resistance in these isolates. Using an enrichment culture technique, a glyphosate-resistant bacterium, Ochrobactrum intermedium Sq20 was isolated from glyphosate-contaminated indigenous soil and characterized. An open reading frame (ORF) comprising of 1353 bp potentially encoding aroAO. intermediumSq20 was amplified from O. intermedium Sq20. It showed 97% homology with aroA genes from other Ochrobactrum spp. Physicochemical characterization revealed that aroAO. intermediumSq20 encodes a polypeptide of 450 amino acids with a calculated molecular mass of 48.9782 kDa and an isoelectric point of 5.21. Secondary structure prediction of AroAO. intermediumSq20 demonstrated a high percentage of random coils and α helices. Methodical optimization and validation of the protein structure helped to build a reliable protein model indicating the presence of 91.8% amino acid residues in most favoured regions. In addition, strain Sq20 was found to be capable of complete degradation of glyphosate at 500 mg L-1 initial concentration as the sole carbon and energy source within 4 days. A glyphosate-resistant bacterial strain O. intermedium Sq20 was discovered. Sequence analysis and structure modelling demonstrated that AroAO. intermediumSq20 closely resembles class II EPSPS and possesses high glyphosate resistance. This provides a good foundation for functional analysis of experimentally derived crystal structures. The cloning and characterization of AroAO. intermediumSq20 will further help in understanding its role at the molecular level and its potential use in the production of glyphosate-resistant transgenic crops. © 2017 Society of Chemical Industry.

Comparison of Protective Efficacy between Formalin‐killed and aroA Gene‐knockout Vibrio anguillarum Vaccines in Olive Flounder, Paralichthys olivaceus

AbstractThe aro genes in bacteria encode enzymes needed for the biosynthesis of aromatic amino acids, and mutant bacteria that are defective in the enzymes can replicate only a limited number in vertebrates owing to the lack or scarceness of chorismate, through which the mutant bacteria of the aro genes become attenuated. In the present study, the 5‐enolpyruvylshikimate‐3‐phosphate synthase (aroA) gene‐knockout Vibrio anguillarum (ΔaroA V. anguillarum) were generated by the allelic exchange method, and its vaccine potential was evaluated in the olive flounder, Paralichthys olivaceus, by comparing the protective efficacy of a formalin‐inactivated V. anguillarum. The LD50 (50% lethal dose) value of ΔaroA V. anguillarum was 1000 times higher than that of wild‐type V. anguillarum in olive flounder fingerlings, and the growth of ΔaroA V. anguillarum was significantly suppressed by coincubation with nonimmune olive flounder serum compared with that of wild‐type V. anguillarum. The survival rates and serum agglutination titers of fish immunized with ΔaroA V. anguillarum were significantly higher than those of fish immunized with the same amount of formalin‐inactivated V. anguillarum, suggesting that although the inactivated V. anguillarum vaccine can provide a high protection in olive flounder, the protective efficacy can be enhanced by immunization with an auxotrophic mutant ΔaroA V. anguillarum.

Differentiation between vaccinal and field strains of E. coli using phenotype and genotype characterization

Background: E. coli infection is considered as an important bacterial problem associated with significant economic losses and usually associated with a variety of disease conditions, including acute septicaemia, haemorrhagic enteritis, pericariditis, salpingitis and complicated air saculitis. These considerations suggest that control of E. coli by vaccination could be of great value especially live vaccine that based on defined mutations that impair and non-revering virulence. Material and Methods: Wild type O78 strain, Live O78 aroA gene deleted vaccine and Live O78 crp gene deleted vaccine were used to accomplish this study. Phenotypic characterization was adopted by studying the cultural, biochemical and serological properties. Also genotypic characterization was studied for 16S rRNA , aroA and crp genes. Results: Growth pattern on different media differed among the used strains as the wild type and Δ aroA mutant were nearly similar while the Δ crp mutant strain was greatly differed. Biochemically the difference between the wild-type and Δ aroA mutant was inability of mutant strain to produce arginine dihydrase (ADH) and fermentation of saccharose. On the other hand the Δ crp mutant failed to produce ADH and to ferment any of the carbohydrates except glucose. A successful amplification of the 16S rRNA gene at 585 bp was detected with the all tested strains while was 1206 bp with only wild type and Δ crp mutant strain when aroA gene primers were used. Regarding crp genes, the amplified products was at 1029 bp with the wild and Δ aroA mutant but not with Δ crp mutant strains. Conclusion: Findings of this study prove the use of methods based on molecular techniques like PCR to differentiate between different types of E. coli either wild or vaccinal mutant type strains. Also it may help in exclusion or proving the return back to virulence of the mutant vaccinal strains.

A Novel Naturally Occurring Class I 5-Enolpyruvylshikimate-3-Phosphate Synthase from Janibacter sp. Confers High Glyphosate Tolerance to Rice.

As glyphosate is a broad spectrum herbicide extensively used in agriculture worldwide, identification of new aroA genes with high level of glyphosate tolerance is essential for the development and breeding of transgenic glyphosate-tolerant crops. In this study, an aroA gene was cloned from a Janibacter sp. strain isolated from marine sediment (designated as aroAJ. sp). The purified aroAJ. sp enzyme has a Km value of 30 μM for PEP and 83 μM for S3P, and a significantly higher Ki value for glyphosate (373 μM) than aroAE. coli. AroAJ. sp is characterized as a novel and naturally occurring class I aroA enzyme with glyphosate tolerance. Furthermore, we show that aroAJ. sp can be used as an effective selectable marker in both japonica and indica rice cultivar. Transgenic rice lines were tested by herbicide bioassay and it was confirmed that they could tolerate up to 3360 g/ha glyphosate, a dosage four-fold that of the recommended agricultural application level. To our knowledge, it is the first report of a naturally occurring novel class I aroA gene which can be efficiently utilized to study and develop transgenic glyphosate-tolerant crops, and can facilitate a more economical and simplified weed control system.

Genotyping of coa and aroA Genes of Methicillin-Resistant Staphylococcus aureus Strains Isolated From Nasal Samples in Western Iran

Background:Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen frequently isolated in both hospital and community environments. Methicillin-resistant Staphylococcus aureus is considered a major nosocomial pathogen that causes severe morbidity and mortality.Objectives:The main objective of this study was to determine the genotypes of MRSA strains isolated from the nares of hospitalized and community patients in Kermanshah Hospital, western Iran, by PCR-restriction fragment length polymorphism (PCR-RFLP).Materials and Methods:Of 1387 patients, 1217 patients were screened for more than 48 hours after admission in hospital wards and 170 patients were screened in the hemodialysis unit of Kermanshah Hospital, which is the largest hospital in western Iran. S. aureus was identified by standard biochemical tests, including colonial morphology, production of coagulase, and DNase and the API20 Staph test. Methicillin-resistant Staphylococcus aureus was identified by the Oxacillin strip test.Results:In total, 258 S. aureus isolates were recovered from 1387 samples, of which 96 isolates were MRSA, 82 were hospital acquired, and 14 were community acquired. Digestion of the aro A gene revealed only one distinctive RFLP pattern in the 258 isolates.Conclusions:Methicillin-resistant Staphylococcus aureus is an increasingly common cause of nosocomial infections. Our results are in agreement with those of other studies reporting that a few specialized clones are responsible for most cases of MRSA nasal carriage. In this study, MRSA strains isolated from different wards of hospital were closely related when analyzed by coagulase gene typing. Identifying patients colonized with MRSA during hospitalization and rapidly typing them with these methods may facilitate detection of outbreaks and prevention of the spread of organisms in hospitals.

Steroid Receptors and Aromatase Gene Expression in Different Brain Areas of Copulating and Sexually Sluggish Male Rats.

Sexually sluggish (SS) males have been identified in several species of mammals including rats. These animals take more than 30 minutes to ejaculate; they do not ejaculate or do so inconsistently despite being tested repeatedly with sexually receptive females. Different brain areas and hormones play an important role in the control of male sexual behavior. Determine gene expression for the androgen receptor (AR), the estrogen receptor alpha (ERα), the progesterone receptor (PR), and the aromatase enzyme (ARO), in brain regions important in the control of male sexual behavior including the medial preoptic area (MPOA), the amygdala (AMG), the olfactory bulb (OB), and, as a control, the cortex (CTX) of copulating (C) and SS male rats. Males that ejaculated within 30 minutes in three tests with receptive females were included in the C group, while those males that ejaculated in one or none of the four tests were included in the SS group. RNA was isolated 1 week after the last test of sexual behavior, and cDNA was synthesized from the brain areas listed above. Expression of the AR, ERα, PR, and ARO genes was determined by quantitative polymerase chain reaction (qPCR). Cyclophilin A (CycA) and tyrosine 3-monooxygenase-tryptophan activation protein zeta (Ywhaz) were housekeeping genes used to determine relative gene expression with the 2(-ΔΔCt) method. The expression of mRNA for AR and ARO increased in the MPOA of SS males. ARO mRNA was increased in the AMG of SS males. In the OB, ERα mRNA was increased and AR mRNA reduced in SS males. These results indicate SS and C males show differences in gene expression within brain regions controlling sexual behavior.

Novel glyphosate-resistant aroA Gene from Paracoccus denitrificans in transgenic Arabidopsis

To date, only aroA variants derived from Agrobacterium tumefaciens CP4 have been used to generate the commercial glyphosate-resistant crops currently available in the market. Therefore, it is of interest to increase aroA gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In this study, a novel aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase from Paracoccus denitrificans was isolated using the genomic library construction and complementary screening. Furthermore, the transgenic Arabidopsis plants with the aroA gene from P. denitrificans were obtained to confirm the potential of the novel aroA gene in developing glyphosate-resistant crops.

Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum.

BackgroundThe shikimic acid (SA) pathway is a fundamental route to synthesize aromatic building blocks for cell growth and metabolic processes, as well as for fermentative production of various aromatic compounds. Genes encoding enzymes of SA pathway are not continuous on genome and they are differently regulated.ResultsIn this study, efforts were made to construct continuous genetic modules of SA pathway that are regulated by a same Ptac promoter. Firstly, aro genes [aroG (NCgl2098), aroB (NCgl1559), aroD (NCgl0408) and aroE (NCgl1567)] from Corynebacterium glutamicum and ribosome binding site (RBS) libraries that were tailored for the above genes were obtained, and the strength of each RBS in the 4 libraries was quantified. Secondly, 9 genetic modules were built up from the RBS libraries, a previously characterized ribozyme insulator (RiboJ) and transcriptional promoter (Ptac) and terminator, and aroG, aroB, aroD and aroE. The functionality and efficiency of the constructed genetic modules were evaluated in C. glutamicum by determination of SA synthesis. Results showed that C. glutamicum RES167ΔaroK carrying a genetic module produced 4.3 g/L of SA, which was 54 folds higher compared to that of strain RES167ΔaroK (80 mg/L, without the genetic module) during fermentation in 250-mL flasks. The same strain produced 7.4, and 11.3 g/L of SA during 5-L batch and fed-batch fermentations, respectively, which corresponding to SA molar yields of 0.39 and 0.24 per mole sucrose consumption.ConclusionThese results demonstrated that the constructed SA pathway modules are effective in increasing SA synthesis in C. glutamicum, and they might be useful for fermentative production of aromatic compounds derived from SA pathway.

Genome-wide identification and comparative analysis of EPSPS (aroA) genes in different plant species

Shikimate pathway produces aromatic amino acids such as phenylalanine, tyrosine and tryptophan, which is essential for plant life. EPSPS (5-enolpyruvylshikimate-3-phosphate synthase, EC 2.5.1.1.9) (aroA) that observed in plants and bacteria plays an important role in shikimate pathway as being the sixth crucial enzyme. In this study, genome-wide comparative analyses of EPSPS genes were performed in different plant species, including Glycine max, Medicago truncatula, Brachypodium disctahyon, Zea mays, Chlamydomonas reinhardtii (green alga), and Physcomitrella patens (moss). One EPSPS gene was identified in M. truncatula, B. distachyon, Z. mays, and C. reinhardtii while two EPSPS genes identified in G. max and P. patens. Based on nucleotide diversity analyses of EPSPS genes, nucleotide diversity was found to be π: 0.29 and θ: 0.34, respectively. Gene structure analysis revealed that 6 of 8 EPSPS genes (75 %) contained seven introns while the length of open reading frame (ORF) ranged from 1173 to 1611 bp. Two highly conserved motifs (LPGSKSLSNRILLLAAL and LFLGNAGTAMRPL) were detected in all plant species. All putative EPSPSs were predicted to be localized in chloroplast while 7 of 8 EPSPSs (87.5 %) contained N-glycosylation sites except for Chlamydomonas. Phylogenetic analyses showed that monocots, dicots, and moss were clustered in group A whereas green alga (C. reinhardtii) was located alone in group B. 3D structure analyses indicated that EPSP synthases contained N-terminal and C-terminal domains while electrostatic potential of EPSPSs were found to exhibit various patterns. In addition, ligand molecule glyphosate was docked with EPSPS of B. disctahyon and G. max. The result showed that there were strong favorable bonds between the proteins and the ligand with favorable conformation.