High-Level Production of Lysine in the Yeast Saccharomyces cerevisiae by Rational Design of Homocitrate Synthase.

Homocitrate synthase (HCS) catalyzes the aldol condensation of 2-oxoglutarate (2-OG) and acetyl coenzyme A (AcCoA) to form homocitrate, which is the first enzyme of the lysine biosynthetic pathway in the yeast Saccharomyces cerevisiae. The HCS activity is tightly regulated via feedback inhibition by the end product lysine. Here, we designed a feedback inhibition-insensitive HCS of S. cerevisiae (ScLys20) for high-level production of lysine in yeast cells. In silico docking of the substrate 2-OG and the inhibitor lysine to ScLys20 predicted that the substitution of serine with glutamate at position 385 would be more suitable for desensitization of the lysine feedback inhibition than the substitution from serine to phenylalanine in the already known Ser385Phe variant. Enzymatic analysis revealed that the Ser385Glu variant is far more insensitive to feedback inhibition than the Ser385Phe variant. We also found that the lysine contents in yeast cells expressing the Ser385Glu variant were 4.62- and 1.47-fold higher than those of cells expressing the wild-type HCS and Ser385Phe variant, respectively, due to the extreme desensitization to feedback inhibition. In this study, we obtained highly feedback inhibition-insensitive HCS using in silico docking and enzymatic analysis. Our results indicate that the rational engineering of HCS for feedback inhibition desensitization by lysine could be useful for constructing new yeast strains with higher lysine productivity. IMPORTANCE A traditional method for screening toxic analogue-resistant mutants has been established for the breeding of microbes that produce high levels of amino acids, including lysine. However, another efficient strategy is required to further improve their productivity. Homocitrate synthase (HCS) catalyzes the first step of lysine biosynthesis in the yeast Saccharomyces cerevisiae, and its activity is subject to feedback inhibition by lysine. Here, in silico design of a key enzyme that regulates the biosynthesis of lysine was utilized to increase the productivity of lysine. We designed HCS for the high-level production of lysine in yeast cells by in silico docking simulation. The engineered HCS exhibited much less sensitivity to lysine and conferred higher production of lysine than the already known variant obtained by traditional breeding. The combination of in silico design and experimental analysis of a key enzyme will contribute to advances in metabolic engineering for the construction of industrial microorganisms.

Downregulation of the broad-specificity amino acid permease Agp1 mediated by the ubiquitin ligase Rsp5 and the arrestin-like protein Bul1 in yeast.

Most of plasma membrane transporters are downregulated by ubiquitination-dependent endocytosis to avoid the excess uptake of their substrates. In Saccharomyces cerevisiae, ubiquitination of transporters is mediated by the HECT-type ubiquitin ligase Rsp5. We report here a mechanism underlying the substrate-induced endocytosis of the broad-specificity amino acid permease Agp1. First, we found that Agp1 underwent ubiquitination and endocytosis in response to the addition of excess asparagine, which is a substrate of Agp1. Moreover, the substrate-induced internalization of Agp1 was dependent on the ubiquitination activity of Rsp5. Since Rsp5 requires α-arrestin family proteins as adaptors to bind with substrates, we next developed a method of genetic screening to identify adaptor proteins for Agp1 endocytosis. This screening and biochemical analysis revealed that Bul1, but not its paralogue Bul2, was essential for the substrate-induced endocytosis of Agp1. Our results support that the substrate-induced endocytosis of Agp1 requires Rsp5 and Bul1.

Crystal Systems and Lattice Parameters of CH3NH3Pb(I1-xBrx)3 Determined Using Single Crystals: Validity of Vegard's Law.

Metal halide perovskites are promising materials for light absorbers in solar cell applications. Use of the Br/I system enables us to control band gap energy and improves the efficiency of solar cells. Precise knowledge of lattice parameters and band gap energies as functions of compositions are crucially important for developing the devices using those materials. In this study, we have determined lattice parameters and band gap energies of CH3NH3Pb(I1-xBrx)3, one of the most intensively studied mix-halide perovskites, as functions of Br content x. We measured accurate Br contents and lattice parameters of CH3NH3Pb(I1-xBrx)3 (0 ≤ x ≤ 1) using single-crystalline samples by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) measurements, respectively. The CH3NH3Pb(I1-xBrx)3 crystal system is tetragonal for x ≤ 0.06 and cubic for x ≥ 0.08 at 300 K. Lattice parameters of CH3NH3Pb(I1-xBrx)3 strictly follow Vegard's law; i.e., they are linearly dependent on x. We give linear expressions of x of lattice parameters for the tetragonal and cubic phases of CH3NH3Pb(I1-xBrx)3 at 300 K. We have shown that these expressions can be used for determining the Br contents of CH3NH3Pb(I1-xBrx)3 polycrystalline thin-film samples based on XRD measurements and, in addition, demonstrated that XPS measurements on polycrystalline samples may be erroneous because of impure ingredients in the samples. Furthermore, we determined band gap energies of CH3NH3Pb(I1-xBrx)3 (0 ≤ x ≤ 1) at room temperature using absorption spectra of polycrystalline thin films taking account of excitonic effects.

Self-Organized Superlattice and Phase Coexistence inside Thin Film Organometal Halide Perovskite.

Organometal halide perovskites have attracted widespread attention as the most favorable prospective material for photovoltaic technology because of their high photoinduced charge separation and carrier transport performance. However, the microstructural aspects within the organometal halide perovskite are still unknown, even though it belongs to a crystal system. Here direct observation of the microstructure of the thin film organometal halide perovskite using transmission electron microscopy is reported. Unlike previous reports claiming each phase of the organometal halide perovskite solely exists at a given temperature range, it is identified that the tetragonal and cubic phases coexist at room temperature, and it is confirmed that superlattices composed of a mixture of tetragonal and cubic phases are self-organized without a compositional change. The organometal halide perovskite self-adjusts the configuration of phases and automatically organizes a buffer layer at boundaries by introducing a superlattice. This report shows the fundamental crystallographic information for the organometal halide perovskite and demonstrates new possibilities as promising materials for various applications.

Design of zigzag folded inversion-stacked AlGaAs waveguides for ultra-compact wavelength converters.

We have proposed and designed zigzag-folded U-shaped waveguides to realize highly efficient wavelength conversion with an extremely small footprint. Extreme high efficiencies are achieved with a combination of modal phase matching and 4¯ quasi phase matching in inversion-stacked AlGaAs/Alox waveguides. Numerical simulations reveal that the conversion efficiency of second harmonic generation pumped at 1.55 µm as high as 12000 %W-1 can be achieved in an 8.0-mm-long AlGaAs/Alox waveguide that is folded up into a small domain of 0.8×0.6 mm2 area. A phase matching signal bandwidth of difference frequency generation, 29 nm, covers 83 % of the telecommunication C band.

Optical properties of disilane-bridged donor-acceptor architectures: strong effect of substituents on fluorescence and nonlinear optical properties.

A series of disilane-bridged donor-acceptor architectures 1-9 containing strong electron-donating and -withdrawing substituents were designed and synthesized in acceptable yields. The substituents substantially affected the fluorescence and nonlinear optical properties of the compounds. In the solid state, the compounds showed purple-blue fluorescence (λ(em) = 360-420 nm) with high quantum yields (up to 0.81). Compound 3, which had p-N,N-dimethylamino and o-cyano substituents, exhibited optical second harmonic generation (activity 2.9 times that of urea, calculated molecular hyperpolarizability ß = 1.6 × 10(-30) esu) in the powder state. Density functional theory calculations for the ground and excited states indicated that both the locally excited state and the intramolecular charge transfer excited state make important contributions to the luminescence behavior.