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.

[Reliability of the measurement of stature in subjects with severe motor and intellectual disabilities].

OBJECTIVE: The objective of this study was to examine the reliability of the measurement of stature in individuals with severe motor and intellectual disabilities. METHODS: Using a stratified sampling method 12 subjects (mean age of 28.7 +/- 11.5), were selected from 73 subjects with severe motor and intellectual disabilities (mean age of 37.1 +/- 15.0). Each subjects'stature was measured using two measurement methods. One measured the tibia length (TL-method) and, the other measured the whole body by measuring three sections. (Division-method). 3 examiners measured all subjects using the TL-method and Division-method two times repeatedly. In addition, one examiner measured all subjects within two weeks following the initial measurement. Intra-rater reliability was calculated from single and two times measurements using each method. The Inter-rater reliability was calculated using measurement results from 2 or 3 examiners in both single and two times measurements. The correlation between values measured by the TL-method and the Division-method was calculated using Spearman rank correlation coefficient. RESULTS: The TL-method and Division-method had good intra-rater reliability (ICC>0.90, 95%CI>0.80) and good inter-rater reliability (ICC>0.90, 95%CI>0.70) in both measurement methods. For both measurement methods, inter-rater reliability was more preferable when 3 examiners measured two times repeatedly (ICC>0.90, 95%CI>0.90). There was good correlation between values measured by TL-method and the Division-method (r=0.83). CONCLUSIONS: Both TL-method and Division-method had good reliability. However, the TL-method could be considered a more useful measurement method as it can be completed more easily and in a short period of time.

Association of genetic polymorphisms with hepatotoxicity in patients with childhood acute lymphoblastic leukemia or lymphoma.

The objective of this study was to identify novel pharmacogenetic determinants of treatment-related hepatotoxicity during the maintenance phase in children with acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL). Although the authors first determined whether genotypes of drug-metabolizing enzymes and transporters--glutathione S-transferase (GST) genes, GSTM1 positive/null, GSTT1 positive/null and GSTP1 A313G, methylenetetrahydrofolate reductase (MTHFR) C677T, reduced folate carrier 1 (RFC1) G80A, and breast cancer resistant protein (BCRP) C421A--were associated with hepatotoxicity for 24 patients, no significant difference was detected for genotype and allelic frequencies between the patients with and those without severe treatment-related hepatotoxicity. Therefore, the authors explored potential candidate polymorphisms associated with hepatotoxicity using the Illumina Infinium HumanHap300, encompassing more than 318,000 tag single-nucleotide polymorphisms (SNPs), for 8 of 24 patients with or without severe hepatotoxicity. Genome-wide genotyping uncovered a total of 28 candidate SNPs. rs1966862, in Rho GTPase-activating protein 24 (ARHGAP24), was the most significant of the candidates, and the genotypes of rs13424027 (PARD3B), rs1156304 (KCNIP4), rs10255262 (SLC13A1), rs7403531 (RASGRP1), and rs381423 (unidentified gene) were also significantly associated with severe hepatotoxicity. This study suggested rs1966862 (ARHGAP24) and the other SNPs to be predictive factors for drug-induced hepatotoxicity during the maintenance phase in pediatric patients with ALL or LBL.

Genetic polymorphisms associated with adverse events and elimination of methotrexate in childhood acute lymphoblastic leukemia and malignant lymphoma.

Methotrexate is administered in high doses to treat childhood acute lymphoblastic leukemia and malignant lymphoma. Hepatotoxicity and bone marrow suppression often limit its use, however. The objective of this study was to determine the genetic polymorphisms associated with the hepatotoxicity and elimination of methotrexate. Genetic polymorphisms of glutathione S-transferase (GST) genes including GSTT1 positive/null, GSTM1 positive/null, and GSTP1 A313G, and genes for reduced folate carrier 1 G80A (RFC1 G80A), methylenetetrahydrofolate reductase C677T (MTHFR C677T), and breast cancer resistant protein C421A (BCRP C421A) were determined for 26 patients by the polymerase chain reaction (PCR) method or by direct sequencing. A high frequency of hepatotoxicity (P = 0.035) was observed for patients with GSTM1 positive and RFC1 AA(80), and serum concentrations of methotrexate 48 h after the start of infusion were higher for patients with the TT(677) genotype of MTHFR (P = 0.028). In conclusion, GSTM1 positive/null and RFC1 G80A polymorphisms could be predictors for hepatotoxicity, and the MTHFR C677T polymorphism is associated with elimination of methotrexate.