Biocatalytic synthesis of 3,4,5,3′,5′-pentahydroxy-trans-stilbene from piceatannol by two-component flavin-dependent monooxygenase HpaBC

HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3′,5′-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100?mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9?mM (1.8?g L^?1) in 48?h.     

Activation studies with amines and amino acids of the β-carbonic anhydrase encoded by the Rv3273 gene from the pathogenic bacterium Mycobacterium tuberculosis

The activation of a β-class carbonic anhydrase (CAs, EC 4.2.1.1) from Mycobacterium tuberculosis, encoded by the gene Rv3273 (mtCA 3), was investigated using a panel of natural and non-natural amino acids and amines. mtCA 3 was effectively activated by D-DOPA, L-Trp, dopamine and serotonin, with K_As ranging between 8.98 and 12.1?µM. L-His and D-Tyr showed medium potency activating effects, with K_As in the range of 17.6–18.2?µM, whereas other amines and amino acids were relatively ineffective activators, with K_As in the range of 28.9–52.2?µM. As the physiological roles of the three mtCAs present in this pathogen are currently poorly understood and considering that inhibition of these enzymes has strong antibacterial effects, discovering molecules that modulate their enzymatic activity may lead to a better understanding of the factors related to the invasion and colonisation of the host during Mycobacterium tuberculosis infection.     

多巴脱羧酶及其与神经类疾病的关联性研究进展

多巴脱羧酶(dopa decarboxylase,DDC)又称作芳香族L-氨基酸脱羧酶,是儿茶酚胺生物合成途径中重要的酶之一,具有多种生物学功能。多巴脱羧酶可分别催化L-3,4-二羟基苯丙氨酸(L-多巴)和L-5-羟色氨酸合成两种神经递质多巴胺和五羟色胺。多巴胺和五羟色胺在脊椎动物和无脊椎动物的生殖、发育、行为和免疫应答过程中均具有重要作用。此外,它还与多种神经类疾病和社会行为有关。多巴脱羧酶一般以二聚体的形式存在于哺乳类和昆虫的多种神经和非神经组织中。本文从多巴脱羧酶的结构、催化机制、与神经类疾病及其攻击性社会行为的关联性研究进展等方面进行了综述。     

Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives

A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-β-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase pathway. Five analogues produced ≥62% HSD1 inhibition at 5?µM and, furthermore, three of them produced ≥68% inhibition at 1?µM. A docking-based structure-activity relationship analysis was done to determine the molecular basis of the inhibition and the cross-reactivity of the analogues was tested against oestrogen receptor, aromatase, cytochrome P450 1A2, and monoamine oxidases. Most of the analogues are only modestly active with 17-β-hydroxysteroid dehydrogenase 2 – a requirement for lowering effective oestradiol levels in vivo. Moreover, the analysis led to the synthesis and discovery of 3-imidazolecoumarin as a potent aromatase inhibitor. In short, coumarin core can be tailored with specific ring and polar moiety substitutions to block either the sulphatase pathway or the aromatase pathway for treating breast cancer and endometriosis.     

β-CA-specific inhibitor dithiocarbamate Fc14–584B: a novel antimycobacterial agent with potential to treat drug-resistant tuberculosis

Inhibition of novel biological pathways in Mycobacterium tuberculosis (Mtb) creates the potential for alternative approaches for treating drug-resistant tuberculosis. In vitro studies have shown that dithiocarbamate-derived β-carbonic anhydrase (β-CA) inhibitors Fc14–594?A and Fc14–584B effectively inhibit the activity of Mtb β-CA enzymes. We screened the dithiocarbamates for toxicity, and studied the in vivo inhibitory effect of the least toxic inhibitor on M. marinum in a zebrafish model. In our toxicity screening, Fc14–584B emerged as the least toxic and showed minimal toxicity in 5-day-old larvae at 300?µM concentration. In vitro inhibition of M. marinum showed that both compounds inhibited growth at a concentration of 75?µM. In vivo inhibition studies using 300?µM Fc14–584B showed significant (p?>?.05) impairment of bacterial growth in zebrafish larvae at 6?days post infection. Our studies highlight the therapeutic potential of Fc14–584B as a β-CA inhibitor against Mtb, and that dithiocarbamate compounds may be developed into potent anti-tuberculosis drugs.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

Establishing synthesis pathway-host compatibility via enzyme solubility

Current pathway synthesis tools identify possible pathways that can be added to a host to produce the desired target molecule through the exploration of abstract metabolic and reaction network space. However, not many of these tools explore gene-level information required to physically realize the identified synthesis pathways, and none explore enzyme-host compatibility. Developing tools that address this disconnect between abstract reactions/metabolic design space and physical genetic sequence design space will enable expedited experimental efforts that avoid exploring unprofitable synthesis pathways. This work describes a workflow, termed Probabilistic Pathway Assembly with Solubility Confidence Scores (ProPASS), which links synthesis pathway construction with the exploration of the physical design space as imposed by the availability of enzymes with predicted characterized activities within the host. Predicted protein solubility propensity scores are used as a confidence level to quantify the compatibility of each pathway enzyme with the host Escherichia coli (E. coli). This study also presents a database, termed Protein Solubility Database (ProSol DB), which provides solubility confidence scores in E. coli for 240,016 characterized enzymes obtained from UniProtKB/Swiss-Prot. The utility of ProPASS is demonstrated by generating genetic implementations of heterologous synthesis pathways in E. coli that target several commercially useful biomolecules.     

Cloning,expression, and characterization of catechol 1,2-dioxygenase from a phenol-degrading Candida tropicalis JH8 strain

The sequence cato encoding catechol 1,2-dioxygenase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The sequence cato contained an ORF of 858?bp encoding a polypeptide of 285?amino acid residues. The recombinant catechol 1,2-dioxygenase exists as a homodimer structure with a subunit molecular mass of 32 KD. Recombinant catechol 1,2-dioxygenase was unstable below pH 5.0 and stable from pH 7.0 to 9.0; its optimum pH was at 7.5. The optimum temperature for the enzyme was 30°C, and it possessed a thermophilic activity within a broad temperature range. Under the optimal conditions with catechol as substrate, the K_m and V_max of recombinant catechol 1,2-dioxygenase were 9.2?µM and 0.987?µM/min, respectively. This is the first article presenting cloning and expressing in E. coli of catechol 1,2-dioxygenase from C. tropicalis and characterization of the recombinant catechol 1,2-dioxygenase.     

Inhibitory effects of some phenolic compounds on the activities of carbonic anhydrase: from in vivo to ex vivo

Carbonic anhydrase (CA) inhibitors have been used for more than 60 years for therapeutic purposes in many diseases table such as in medications against antiglaucoma and as diuretics. Phenolic compounds are a new class of CA inhibitor. In our study, we tested the effects of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid on esterase and the CO₂-hydratase activities of CA I and II isozymes purified from in vivo to ex vivo. The K_i values of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid were 203.80, 1170.00 and 910.00?μM, respectively for hCA I and 75.25, 354.00 and 1510.00?μM, respectively for hCA II. Additionally, IC₅₀ values from in vivo studies were found to be in the range of 173.25–1360.0?μM for CA I and II, respectively, using CO₂-hydratase activity methods. These results demonstrated that phenolic compounds used in in vivo studies could be used in different biomedical applications to inhibit approximately 30% of the CO₂-hydratase activity of the total CA enzyme of rat erythrocytes.     

Enterohaemorrhagic Escherichia coli O157:H7 Shiga‐like toxin 1 is required for full pathogenicity and activation of the p38 mitogen‐activated protein kinase pathway in Caenorhabditis elegans

Enterohaemorrhagic Escherichia coli (EHEC) causes life‐threatening infections in humans as a consequence of the production of Shiga‐like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga‐like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen‐activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1‐dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.     

Identification and characterization of the ‘missing’ terminal enzyme for siroheme biosynthesis in α‐proteobacteria

It has recently been shown that the biosynthetic route for both the d₁‐haem cofactor of dissimilatory cd₁ nitrite reductases and haem, via the novel alternative‐haem‐synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d₁‐haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin–ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild‐type phenotype of an Escherichia coli mutant‐strain lacking sirohydrochlorin–ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α‐proteobacteria as the terminal enzyme of siroheme biosynthesis.     

Control of threonine pathway in E. coli. application to biotechnologies

Threonine is an essential amino acid for mammals and birds and an adequate supply is necessary for growth and maintenance. Its production has become the aim of metabolic bioengineering and genetic manipulations. We propose in this paper a rational approach for increasing threonine production in anE. coli strain based on metabolic control theory. We have derived a way to measure the control coefficients of threonine pathwayin vivo. The method consists in modelling the results of presteady-state experiments. Thein vivo concentrations and activities of the enzymes can then be measured and introduced into the model, so that thein vivo steady-state of the pathway can be evaluated. With such a model it is possible to calculate the theoretical values of the control coefficients of the threonine synthesis fluxin vivo.     

Secondary carbamate linker can facilitate the sustained release of dopamine from brain-targeted prodrug

To achieve the sustained release of dopamine in the brain for the symptomatic treatment of Parkinson’s disease, dopamine was conjugated to l-tyrosine, an l-type amino acid transporter 1 (LAT1)-targeting vector, using a secondary carbamate linker. The resulting prodrug, dopa-CBT, inhibited the uptake of the LAT1 substrate [¹⁴C]-l-leucine in LAT1-expressing MCF-7 cells with an IC₅₀ value of 28?µM, which was 3.5-times lower than that of the gold standard for dopamine replacement therapy, l-dopa (IC₅₀ ca. 100?µM). Despite its high affinity for LAT1, dopa-CBT was transported via LAT1 into MCF-7 cells 850-times more slowly (V_max?<?3?pmol/min/mg) than l-dopa (V_max 2.6?nmol/min/mg), most likely due to its large size compared to l-dopa. However, dopa-CBT was significantly more stable in 10% rat liver homogenate than l-dopa, releasing dopamine and l-tyrosine, an endogenous dopamine precursor, slowly, which indicates that it may serve as a dual carrier of dopamine across the blood-brain barrier selectively expressing LAT1.     

Comparison of the amine/amino acid activation profiles of the β- and γ-carbonic anhydrases from the pathogenic bacterium Burkholderia pseudomallei

The β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, BpsCAβ, that is responsible for the tropical disease melioidosis was investigated for its activation with natural and non-natural amino acids and amines. Previously, the γ-CA from this bacterium has been investigated with the same library of 19 amines/amino acids, which show very potent activating effects on both enzymes. The most effective BpsCAβ activators were L- and D-DOPA, L- and D-Trp, L-Tyr, 4-amino-L-Phe, histamine, dopamine, serotonin, 2-pyridyl-methylamine, 1-(2-aminoethyl)-piperazine and L-adrenaline with K_As of 0.9–27?nM. Less effective activators were D-His, L- and D-Phe, D-Tyr, 2-(2-aminoethyl)pyridine and 4-(2-aminoethyl)-morpholine with K_As of 73?nM–3.42?µM. The activation of CAs from bacteria, such as BpsCAγ/β, has not been considered previously for possible biomedical applications. It would be of interest to perform studies in which bacteria are cultivated in the presence of CA activators, which may contribute to understanding processes connected with the virulence and colonization of the host by pathogenic bacteria.     

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for β-carotene production in recombinant Escherichia coli harboring β-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of β-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and β-carotene synthesis genes produced high amount of β-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains – MG1655, DH5α, S17-1, XL1-Blue and BL21 – the DH5α was found to be the best β-carotene producer. Using glycerol as the carbon source for β-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5α harboring the whole MVA pathway and β-carotene synthesis genes produced β-carotene of 465 mg/L at glycerol concentration of 2% (w/v).     

Metabolic engineering of Saccharomyces cerevisiae for hydroxytyrosol overproduction directly from glucose

Hydroxytyrosol (HT) is one of the most powerful dietary antioxidants with numerous applications in different areas, including cosmetics, nutraceuticals and food. In the present work, heterologous hydroxylase complex HpaBC from Escherichia coli was integrated into the Saccharomyces cerevisiae genome in multiple copies. HT productivity was increased by redirecting the metabolic flux towards tyrosol synthesis to avoid exogenous tyrosol or tyrosine supplementation. After evaluating the potential of our selected strain as an HT producer from glucose, we adjusted the medium composition for HT production. The combination of the selected modifications in our engineered strain, combined with culture conditions optimization, resulted in a titre of approximately 375 mg l⁻¹ of HT obtained from shake-flask fermentation using a minimal synthetic-defined medium with 160 g l⁻¹ glucose as the sole carbon source. To the best of our knowledge, this is the highest HT concentration produced by an engineered S. cerevisiae strain.     

Fitness of Escherichia coli during Urinary Tract Infection Requires Gluconeogenesis and the TCA Cycle

Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium''s ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes.     

Coexpression of thecys E andcys M genes ofSalmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis

TheSalmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of theS. typhimurium andEscherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. Thein vitro andin vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. Thecys M gene ofS. typhimurium possessed a GUG initiation codon, like itsE. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteinein vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.     

Amine neuromediators,their precursors,and oxidation products in the culture of <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

The growth dynamics of the synthesis of monoamine neuromediators serotonin, norepinephrine, and dopamine in Escherichia coli K-12 was investigated for the first time using high performance liquid chromatography with electrodetection. Maximum (micromolar) concentrations of these compounds were detected in E. coli cells during the early growth phases; their intracellular content decreases after the transition to late growth phases. E. coli biomass contains (i) the substances DOPA and 5-hydroxytryptamine that serve in animal cells as neuromediator precursors and (ii) the products of their oxidative deamination. Presumably, the biosynthesis and degradation of monoamine neuromediators in bacterial cells involves enzyme systems analogous to those typical of animals. The culture fluid of E. coli contains micromolar concentrations of DOPA and nanomolar of serotonin, dopamine, and norepinephrine during the late growth phase. These concentrations are sufficient for animal/human receptors to bind them. This article deals with the potential biotechnological applications of the data obtained.