Biosynthesis of l-tyrosine O-sulphate from the methyl and ethyl esters of l-tyrosine

1. Rat-liver supernatant preparations are capable of achieving the biological sulphation of l-tyrosine methyl ester, the reaction proceeding maximally at a substrate concentration of 30 mm and at pH 7·0. 2. Two sulphated products are formed, one of which has been identified as l-tyrosine O-sulphate. On the basis of indirect evidence the other product can be assumed to be l-tyrosine O-sulphate methyl ester. 3. An enzyme present in rat-liver supernatant preparations is capable of converting l-tyrosine O-sulphate methyl ester into l-tyrosine O-sulphate. This enzyme is inhibited by l-tyrosine methyl ester. 4. l-Tyrosine ethyl ester also yields two sulphated products when used as an acceptor in the liver sulphating system. One of these has been identified chromatographically as l-tyrosine O-sulphate and the other may be presumed to be l-tyrosine O-sulphate ethyl ester.     

Spontaneous Mutation Rates in Mammalian Cells: Effect of Differential Growth Rates and Phenotypic Lag

We calculated a spontaneous rate of 26-37 x 10(-6) mutations per cell division for L5178Y MOLY (mouse lymphoma) cells at the thymidine kinase locus (tk+/(-)----tk-/-) using a procedure that isolated and segregated cells during expression. This rate was 50 times higher than when cells expressed the mutant phenotype in suspension. The higher mutation rates obtained with the in situ procedure suggest that many of the mutants, whether expressed or unexpressed, grew more slowly than wild-type cells prior to selection with trifluorothymidine (TFT), implying that the slow growth phenotype is expressed earlier than the TFTr (TFT-resistant) phenotype. The loss of mutants was not restricted to cells forming small colonies; the mutation rate for cells forming large colonies was more than ten times higher using the in situ procedure. In this new procedure, the cells expressed spontaneous mutations while growing in semisolid medium for up to 3 days without TFT. Mutants were then selected in situ by adding an overlay of TFT and the visible colonies were analyzed after 11 days. Cells with spontaneous mutations at the tk locus required approximately 30 hr for the more rapidly expressing cells with new mutations to be detected. Most of the TFTr colonies selected after 60 hr of growth in semisolid medium represented independent mutations that had accumulated during the first 30 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biotechnological production of l-tyrosine and derived compounds

The aromatic amino acid l-tyrosine is a compound with multiple applications in the food, pharmaceutical, cosmetic and chemical industries. This review summarizes the current knowledge on the metabolic pathways involved in the synthesis of this amino acid and the strategies employed to develop and improve microbial production strains. Common strategies for l-tyrosine overproduction include the elimination of negative feedback control in key pathway enzymes and increasing the pool of the aromatic precursors phosphoenolpyruvate and erythrose-4-phosphate. Following these approaches, production strains have been generated that allow the synthesis of l-tyrosine with a yield from glucose corresponding to 80% of the theoretical maximum. Recent developments in the utilization of l-tyrosine as a substrate for microbial and enzymatic conversion into valuable products are also presented and discussed. For example, the production of the aromatic polymer melanin has been reported by the bioconversion of l-tyrosine using an Escherichia coli strain expressing a gene encoding the enzyme tyrosinase from Rhizobium etli. Metabolic engineering by expressing genes encoding the enzyme p-hydroxyphenylacetate 3-hydroxylase in an E. coli strain modified for l-tyrosine production from glucose results in the capacity to synthesize l-3,4-dihydroxyphenylalanine, a compound employed for treating Parkinson's disease.     

Engineering of l-tyrosine oxidation in Escherichia coli and microbial production of hydroxytyrosol

The hydroxylation of tyrosine is an important reaction in the biosynthesis of many natural products. The use of bacteria for this reaction has not been very successful due to either the over-oxidation to ortho-quinone when using tyrosinases from bacteria or plants, or the lack of the native cofactor, tetrahydrobiopterin (BH4), needed for the activity of tyrosine hydroxylases (TH). Here, we demonstrate that an Escherichia coli cofactor, tetrahydromonapterin (MH4), can be used as an alternative cofactor for TH in presence of the BH4 regeneration pathway, and tyrosine hydroxylation is performed without over-oxidation. We used this platform for biosynthesis of one of the most powerful antioxidants, hydroxytyrosol. An endogenous aromatic aldehyde oxidase was identified and knocked out to prevent formation of the side product, and this resulted in nearly exclusive production of hydroxytyrosol in engineered E. coli. Finally, hydroxytyrosol production from a simple sugar as a sole carbon source was demonstrated.     

Ribosomal RNA Synthesis in Sea Urchin Embryos: Differential Rates of Accumulation in Chemically-induced Animalized and Vegetalized Larvae

Animalization was induced with evans blue and with Zn⁺⁺ in embryos of Arbacia punctulata and of Lytechinus variegatus , respectively. Li⁺ induced vegetalization in A. punctulata embryos. While animalization did not affect the rate of cleavage, vegetalized embryos exhibited a reduction in cell number at post-morula stages. Mid-gastrulae and corresponding experimental embryos each were labeled for 4 hr with uridine-[5-³H] and with L-[³H-methyl]-methionine. The rate of uptake of each exogenous RNA precursor was similar in control and in experimental embryos. Purified RNA preparations were fractionated by electrophoresis on 2.4% acrylamide+0.5 % agarose gels. Comparison of rates of incorporation of each RNA precursor into 26s and 18s RNAs indicated that on a per cell basis evans blue- and Zn⁺⁺-animalized embryos showed a reduction (0.53–0.56) and Li⁺-vegetalized embryos an enhancement (1.41—1.53) in the rate of accumulation of newly made 26s and 18s RNAs compared to controls (1.00). These results suggest that chemically-induced animalized and vegetalized embryos provide useful tools for studying possible differential gene expression in different embryonic germ layers of the developing sea urchin embryo.     

Differential Rates of Digestion of Bacteria by Freshwater and Marine Phagotrophic Protozoa

Differential decreases over time of two bacterial species, Escherichia coli and Enterococcus faecalis, in a freshwater and a marine ecosystem were observed and explained by a differential rate of digestion of these bacteria by phagotrophic flagellates and ciliates. For this purpose, fluorescence-labeled bacteria (FLB) were used and prepared from the two species cited above. The number of FLB was observed for 5 days in fresh and marine waters in the presence or absence (0.2-μm-pore-size-filtered water) of natural microbiota. These experiments showed a longer persistence of Enterococcus faecalis FLB as opposed to Escherichia coli FLB in the presence of natural microbiota. Removal of FLB was due to protozoan grazing because no decrease of FLB number was observed in the absence of natural microbiota. In short-term (about 40 min) ingestion experiments, we found similar clearance rates of Escherichia coli and Enterococcus faecalis FLB by assemblages of flagellates from the freshwater and the marine ecosystem and by cultured assemblages of ciliates from the marine ecosystem. Clearance rates of Enterococcus faecalis FLB were greater than those of Escherichia coli FLB for assemblages of ciliates from the freshwater ecosystem. Comparison of rates of ingestion and digestion of FLB by protozoa showed that Escherichia coli FLB were digested and ingested at similar rates. However, Enterococcus faecalis FLB were digested slower than they were ingested. These results suggest that a longer persistence of Enterococcus faecalis as opposed to Escherichia coli can be explained by a differential digestion by flagellates and ciliates in aquatic ecosystems. Moreover, rates of ingestion and digestion were strongly correlated for both FLB types.     

The l - and d-threonine dehydratases accompanying l-tyrosine phenol-lyase: selective decomposition of d-threonine in racemic mixture

Low-purity preparations from Escherichia intermedia A-21 and Citrobacter freundii 62 cells producing tyrosine phenol-lyase [l-tyrosine phenol-lyase (deaminating), EC 4.1.99.2] catalyse the decomposition of both threonine enantiomers to α-ketobutyric acid. Reactions with l-threonine and d-threonine are effected by two independent enzymes different from tyrosine phenol-lyase. The enzyme which acts on l-threonine has properties characteristic of biosynthetic threonine dehydratase [l-threonine hydro-lyase (deaminating), EC 4.2.1.16]. l-Isoleucine and dl-allothreonine are inhibitors of this enzyme, permitting a selective inhibition of biosynthetic threonine dehydratase and use of the preparations to act selectively on d-threonine in the racemate.     

Allelochemic function for a primary metabolite: the case of l-tyrosine hyper-production in Inga umbellifera (Fabaceae)

Young leaves of tropical forest trees experience far higher herbivory pressure than mature leaves of the same species. Selection on young leaves has led to diverse forms of defense chemical expression. Though most allelochemicals are secondary metabolites, allelochemic function for a primary metabolite remains a possibility. We recently observed this phenomenon in the young leaves of Inga umbellifera, which accumulate the protein amino acid l-tyrosine to very high levels. We isolated l-tyrosine from young leaves of trees in Panama and characterized it using spectroscopic and chemical means. We chromatographically quantified leaf l-tyrosine levels across a range of developmental stages, showing that it was present in the youngest leaves and that its concentration increased throughout the period of expansion, reaching an average maximum of ca 10% of leaf dry mass in late-stage young leaves. This chemical phenotype was seen to be highly leaf-age specific: Free tyrosine was only present in mature leaves at very low levels. In bioassays with larvae of the noctuid moth H. virescens, l-tyrosine proved to be a potent growth inhibitor when added to artificial diet at 10% of dry mass. This suggests that a rarely observed defense strategy occurs in young I. umbellifera leaves, a hyper-produced primary metabolite functioning as an allelochemical.     

Construction of a heat-inducible Escherichia coli strain for efficient de novo biosynthesis of l-tyrosine

l-Tyrosine is an important amino acid widely used in food, agriculture, and pharmaceutical industries. However, the industrial application was severely constrained due to low production. To obtain the Escherichia coli mutant producing l-tyrosine in abundance, the heat-inducible expression vector carrying the two feedback resistance enzymes (3-deoxy-7-phosphoheptulonate synthase encoded by aroG^fbr and chorismate mutase/prephenate dehydrogenase encoded by tyrA^fbr) were introduced into the phenylalanine-producing E. coli strain to enable it to synthesize l-tyrosine directly from glucose. Furthermore, the CRISPR-Cas9 technology was applied to eliminate l-phenylalanine and l-tryptophan pathways for their competition for the carbon flux. The global regulatory protein TyrR, which mediates the biosynthesis and transportation of aromatic amino acids, was also deleted to increase l-tyrosine production. Among the recombinant strains, the pheA/tyrR double-gene deletion strain had the highest yield of 5.84 g/L on shake flasks. The feeding strategies were then optimized in a 3-L fermentor. The pheA/tyrR double-gene deletion strain with the heat-inducible expression plasmid pAP-aroG^fbr-tyrA^fbr was able to produce 55.54 g/L l-tyrosine by fed-batch fermentation; the substrate conversion rate was 0.25 g/g. The recombinant strains constructed in this study could be an industrial platform for the microbial production of l-tyrosine directly from glucose.     

The Evolution of Range Sizes in Mammals and Squamates: Heritability and Differential Evolutionary Rates for Low- and High-Latitude Limits

Species geographical ranges are at the core of many areas in evolutionary biology, yet empirical studies on the evolution of geographical ranges have been limited. Here, we integrate information on the phylogenetic relationships and geographical distribution of 3097 species of mammal (Artiodactyla, Carnivora, Chiroptera, Marsupialia, and Primates) and squamate (Anguimorpha, Gekkota, Iguania, Lacertoidea, Scincoidea, and Serpentes) to assess the degree of evolutionary “heritability” (i.e., phylogenetic autocorrelation) in range sizes and the extent to which range limits at higher and lower latitudes share similar evolutionary rates. Phylogenetic autocorrelation was highly variable among clades in the case of range size, but invariably high for range latitudinal centroid and range limits. Moreover, rates of evolution of high-latitude limits were 1.6–4 times faster than low-latitude limits. These results are consistent with previous experimental studies showing that heat tolerance is conserved across lineages, whereas tolerance to cold temperatures is more labile. The distinct evolutionary rates of low- and high-latitude limits has important implications for our understanding of the evolution of geographical ranges, as well as to understand how they could be affected by predicted anthropogenic climate changes.     

Crystallization and characterization of Smaug: a novel RNA-binding motif

During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interaction with a specific element in its 3(')UTR. The repression occurs in the bulk cytoplasm of the embryo; Nanos is, however, successfully translated in the specialized cytoplasm of the posterior pole. This generates a gradient of Nanos emanating from the posterior pole that is essential for organizing proper abdominal segmentation. To understand the structural basis of RNA binding and translational control, we have crystallized a domain of Drosophila Smaug that binds RNA. The crystals belong to the space group R3 with unit cell dimensions of a=b=129.3A, c=33.1A, alpha=beta=90 degrees, gamma=120 degrees and diffract to 1.80A with synchrotron radiation. Initial characterization of this domain suggests that it encodes a novel RNA-binding motif.