Nonaqueous fractionation and overexpression of fluorescent‐tagged enzymes reveals the subcellular sites of L‐theanine biosynthesis in tea

l ‐Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of l ‐theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing l ‐theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution of l ‐theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of l ‐theanine synthetase and also to determine the subcellular localization of fluorescent‐tagged proteins by confocal laser scanning microscopy. In tea root tissue, the cytosol was the main site of l ‐theanine biosynthesis, and cytosol‐located CsTSI was the key l ‐theanine synthase. In tea shoot tissue, l ‐theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key l ‐theanine synthases. In addition, l ‐theanine content and distribution were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.     

Engineered disulfide bonds improve thermostability and activity of L‐isoleucine hydroxylase for efficient 4‐HIL production in Bacillus subtilis 168

4‐Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l ‐Isoleucine hydroxylase specifically converts l‐ Ile to 4‐hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4‐hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l ‐isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half‐life of variant T181C was 4.03 h at 50°C, 10.27‐fold the half‐life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56‐fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole‐cell biotransformation, Bacillus subtilis 168/pMA5‐ido^T181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4‐hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4‐hydroxyisoleucine. Rational design of disulfide bond improved l ‐isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases.     

Microbial larvicides for mosquito control: Impact of long lasting formulations of Bacillus thuringiensis var. israelensis and Bacillus sphaericus on non‐target organisms in western Kenya highlands

The microbial larvicides Bacillus thuringiensis var. israelensis and Bacillus sphaericus have been used extensively for mosquito control and have been found to be effective and safe to non‐target organisms cohabiting with mosquito larvae. Recently developed long lasting microbial larvicides (LLML), although evading the previous challenge of short duration of activity, increase the risk of persistence of toxins in the treated larval habitats. This study monitored the impact of LLML FourStar^® and LL3 on non‐target organisms cohabiting with mosquito larvae in an operational study to control malaria vectors in western Kenya highlands. A total of 300 larval habitats were selected in three highland villages. The habitats were first monitored for 5 weeks to collect baseline data on non‐target organisms cohabiting with mosquito larvae and then randomized into two treatment arms (respective FourStar^® and LL3) and one control arm. Non‐target organisms were sampled weekly for 5 months after treatment to assess the impact of LLML intervention. Before treatment, the mean density of all non‐target organisms combined in the control, LL3 and FourStar^® treated habitats was 1.42, 1.39 and 1.49 individuals per habitat per sampling occasion, respectively. Following treatment, this density remained fairly unchanged for 21 weeks at which time it was 1.82, 2.11, and 2.05 for the respective control, LL3 and FourStar^® treated habitats. Statistical analysis revealed that LL3 and FourStar^® did not significantly alter abundance, richness or diversity of the 11 taxa studied, when comparing the intervention and control larval habitats. However, both FourStar^® and LL3 significantly reduced the density of malaria vectors. In conclusion, one round of label rate application of FourStar^® or LL3 in natural larval habitats did not alter richness, abundance or diversity of the monitored aquatic non‐target organisms cohabiting with mosquito larvae to an ecologically significant level.     

Aminooxy‐naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l‐tryptophan aminotransferase: structure–activity relationships

We previously reported l ‐α‐aminooxy‐phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure–activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole‐3‐acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2‐(aminooxy)‐3‐(naphthalen‐2‐yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin‐deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia‐lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them ‘pyruvamine’.     

Modular pathway engineering of key carbon‐precursor supply‐pathways for improved N‐acetylneuraminic acid production in Bacillus subtilis

N‐acetylneuraminic acid (NeuAc) is widely used as a nutraceutical for facilitating infant brain development, maintaining brain health, and enhancing immunity. Currently, NeuAc is mainly produced by extraction from egg yolk and milk, or via chemical synthesis. However, its low concentration in natural resources and its non‐ecofriendly chemical synthesis result in insufficient NeuAc production and environmental pollution, respectively. In this study, improved NeuAc production was attained via modular pathway engineering of the supply pathways of two key precursors—N‐acetylglucosamine (GlcNAc) and phosphoenolpyruvate (PEP)—and by balancing NeuAc biosynthesis and cell growth in engineered Bacillus subtilis. Specifically, we used a previously constructed GlcNAc‐producing B. subtilis as the initial host for NeuAc biosynthesis. First, we constructed a de novo NeuAc biosynthetic pathway utilizing glucose by coexpressing glucosamine‐6‐phosphate acetyl‐transferase (GNA1), N‐acetylglucosamine 2‐epimerase (AGE), and N‐acetylneuraminic acid synthase (NeuB), resulting in 0.33 g/l NeuAc production. Next, to balance the supply of the two key precursors for NeuAc biosynthesis, modular pathway engineering was performed. The optimal strategy for balancing the GlcNAc module and PEP supply module involved the use of an engineered, unique glucose and malate coutilization pathway in B. subtilis, supplied with both glucose (for the GlcNAc moiety) and malate (for the PEP moiety) at high strength. This led to 1.65 g/L NeuAc production, representing a 5.0‐fold improvement over the existing methods. Furthermore, to enhance the NeuAc yield on cell, glucose and malate coutilization pathways were engineered to balance NeuAc biosynthesis and cell growth via the blocking of glycolysis, the introduction of the Entner–Doudoroff pathway, and the overexpression of the malic enzyme YtsJ. NeuAc titer reached 2.18 g/L, with 0.38 g/g dry cell weight NeuAc yield on cell, which represented a 1.32‐fold and 2.64‐fold improvement over the existing methods, respectively. The strategy of modular pathway engineering of key carbon precursor supply pathways via engineering of the unique glucose‐malate coutilization pathway in B. subtilis should be generically applicable for engineering of B. subtilis for the production of other important biomolecules. Our study also provides a good starting point for further metabolic engineering to achieve industrial production of NeuAc by a Generally Regarded As Safe bacterial strain.     

Structural scaffold for eIF4E binding selectivity of 4E‐BP isoforms: crystal structure of eIF4E binding region of 4E‐BP2 and its comparison with that of 4E‐BP1

To clarify the higher eukaryotic initiation factor 4E (eIF4E) binding selectivity of 4E‐binding protein 2 (4E‐BP2) than of 4E‐BP1, as determined by Trp fluorescence analysis, the crystal structure of the eIF4E binding region of 4E‐BP2 in complex with m⁷GTP‐bound human eIF4E has been determined by X‐ray diffraction analysis and compared with that of 4E‐BP1. The crystal structure revealed that the Pro47‐Ser65 moiety of 4E‐BP2 adopts a L ‐shaped conformation involving extended and α‐helical structures and extends over the N‐terminal loop and two different helix regions of eIF4E through hydrogen bonds, and electrostatic and hydrophobic interactions; these features were similarly observed for 4E‐BP1. Although the pattern of the overall interaction of 4E‐BP2 with eIF4E was similar to that of 4E‐BP1, a notable difference was observed for the 60–63 sequence in relation to the conformation and binding selectivity of the 4E‐BP isoform, i.e. Met‐Glu‐Cys‐Arg for 4E‐BP1 and Leu‐Asp‐Arg‐Arg for 4E‐BP2. In this paper, we report that the structural scaffold of the eIF4E binding preference for 4E‐BP2 over 4E‐BP1 is based on the stacking of the Arg63 planar side chain on the Trp73 indole ring of eIF4E and the construction of a compact hydrophobic space around the Trp73 indole ring by the Leu59‐Leu60 sequence of 4E‐BP2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis,conformational study,and spectroscopic characterization of the cyclic Cα,α‐disubstituted glycine 9‐amino‐9‐fluorenecarboxylic acid

A series of terminally blocked peptides (to the pentamer level) from l ‐Ala and the cyclic C^α,α‐disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc‐Afc‐OMe and the dipeptide Boc‐Afc‐Gly‐OMe were determined in the crystal state by X‐ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT‐IR absorption and ¹H‐NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully‐extended (C₅) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C^α,α‐disubstituted Gly residues Ac₅c and Dϕg is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C^α,α‐disubstituted Gly residue is also reported. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.