Accumulation Of L-valine Research Articles

Metabolic profiles in drought-tolerant wheat with enhanced abscisic acid sensitivity.

Global warming has led to the expansion of arid lands and more frequent droughts, which are the largest cause of global food production losses. In our previous study, we developed TaPYLox wheat overexpressing the plant hormone abscisic acid (ABA) receptor, which is important for the drought stress response in plants. TaPYLox showed resistance to drought stress and acquired water-saving traits that enable efficient grain production with less water use. In this study, we used TaPYLox to identify ABA-dependent and -independent metabolites in response to drought stress. We compared the variation of metabolites in wheat under well-watered, ABA treatment, and drought stress conditions using the ABA-sensitive TaPYLox line and control lines. The results showed that tagatose and L-serine were ABA-dependently regulated metabolites, because their stress-induced accumulation was increased by ABA treatment in TaPYLox. In contrast, L-valine, L-leucine, and DL-isoleucine, which are classified as branched chain amino acids, were not increased by ABA treatment in TaPYLox, suggesting that they are metabolites regulated in an ABA-independent manner. Interestingly, the accumulation of L-valine, L-leucine, and DL-isoleucine was suppressed in drought-tolerant TaPYLox under drought stress, suggesting that drought-tolerant wheat might be low in these amino acids. 3-dehydroshikimic acid and α-ketoglutaric acid were decreased by drought stress in an ABA-independent manner. In this study, we have succeeded in identifying metabolites that are regulated by drought stress in an ABA-dependent and -independent manner. The findings of this study should be useful for future breeding of drought-tolerant wheat.

Mutagenesis Reveals That the OsPPa6 Gene Is Required for Enhancing the Alkaline Tolerance in Rice.

Alkaline stress (AS) is one of the abiotic stressful factors limiting plant’s growth and development. Inorganic pyrophosphatase is usually involved in a variety of biological processes in plant in response to the abiotic stresses. Here, to clarify the responsive regulation of inorganic pyrophosphatase in rice under AS, the mutagenesis of the OsPPa6 gene encoding an inorganic pyrophosphatase in rice cv. Kitaake (Oryza sativa L. ssp. japonica) was performed by the CRISPR/Cas9 system. Two homozygous independent mutants with cas9-free were obtained by continuously screening. qPCR reveals that the OsPPa6 gene was significantly induced by AS, and the mutagenesis of the OsPPa6 gene apparently delayed rice’s growth and development, especially under AS. Measurements demonstrate that the contents of pyrophosphate in the mutants were higher than those in the wild type under AS, however, the accumulation of inorganic phosphate, ATP, chlorophyll, sucrose, and starch in the mutants were decreased significantly, and the mutagenesis of the OsPPa6 gene remarkably lowered the net photosynthetic rate of rice mutants, thus reducing the contents of soluble sugar and proline, but remarkably increasing MDA, osmotic potentials and Na+/K+ ratio in the mutants under AS. Metabonomics measurement shows that the mutants obviously down-regulated the accumulation of phosphorylcholine, choline, anthranilic acid, apigenin, coniferol and dodecanoic acid, but up-regulated the accumulation of L-valine, alpha-ketoglutarate, phenylpyruvate and L-phenylalanine under AS. This study suggests that the OsPPa6 gene is an important osmotic regulatory factor in rice, and the gene-editing of CRISPR/Cas9-guided is an effective method evaluating the responsive regulation of the stress-induced gene, and simultaneously provides a scientific support for the application of the gene encoding a soluble inorganic pyrophosphatase in molecular breeding.

Improvement of l-Leucine Production in Corynebacterium glutamicum by Altering the Redox Flux.

The production of l-leucine was improved by the disruption of ltbR encoding transcriptional regulator and overexpression of the key genes (leuAilvBNCE) of the l-leucine biosynthesis pathway in Corynebacterium glutamicum XQ-9. In order to improve l-leucine production, we rationally engineered C. glutamicum to enhance l-leucine production, by improving the redox flux. On the basis of this, we manipulated the redox state of the cells by mutating the coenzyme-binding domains of acetohydroxyacid isomeroreductase encoded by ilvC, inserting NAD-specific leucine dehydrogenase, encoded by leuDH from Lysinibacillus sphaericus, and glutamate dehydrogenase encoded by rocG from Bacillus subtilis, instead of endogenous branched-chain amino acid transaminase and glutamate dehydrogenase, respectively. The yield of l-leucine reached 22.62 ± 0.17 g·L−1 by strain ΔLtbR-acetohydroxyacid isomeroreductase (AHAIR)M/ABNCME, and the concentrations of the by-products (l-valine and l-alanine) increased, compared to the strain ΔLtbR/ABNCE. Strain ΔLtbR-AHAIRMLeuDH/ABNCMLDH accumulated 22.87±0.31 g·L−1 l-leucine, but showed a drastically low l-valine accumulation (from 8.06 ± 0.35 g·L−1 to 2.72 ± 0.11 g·L−1), in comparison to strain ΔLtbR-AHAIRM/ABNCME, which indicated that LeuDH has much specificity for l-leucine synthesis but not for l-valine synthesis. Subsequently, the resultant strain ΔLtbR-AHAIRMLeuDHRocG/ABNCMLDH accumulated 23.31 ± 0.24 g·L−1 l-leucine with a glucose conversion efficiency of 0.191 g·g−1.

Application of leucine dehydrogenase Bcd from Bacillus subtilis for l-valine synthesis in Escherichia coli under microaerobic conditions

Microaerobic cultivation conditions have been shown experimentally and theoretically to improve the performance of a number of bioproduction systems. However, under these conditions, the production of l-valine by Escherichia coli is decreased mainly because of a redox cofactor imbalance and a decreased l-glutamate supply. The synthesis of one mole of l-valine from one mole of glucose generates two moles of NADH via glycolysis but consumes a total of two moles of NADPH, one in the ketol-acid reductoisomerase (KARI) reaction and the other in the regeneration of l-glutamate as an amino group donor for the branched-chain amino acid aminotransferase (BCAT) reaction. The improvement of l-valine synthesis under oxygen deprivation may be due to solving these problems. Increased l-valine synthesis under oxygen deprivation conditions was previously shown in Corynebacterium glutamicum (Hasegawa et al., 2012). In this study, we have proposed the use of NADH-dependent leucine dehydrogenase (LeuDH; EC 1.4.1.9) Bcd from B. subtilis instead of the native NADPH-dependent pathway including aminotransferase encoded by ilvE to improve l-valine production in E. coli under microaerobic conditions. We have created l-valine-producing strains on the base of the aminotransferase B-deficient strain V1 (B-7 ΔilvBN ΔilvIH ΔilvGME::PL-ilvBNN17KDA) by introducing one chromosomal copy of the bcd gene or the ilvE gene. Evaluation of the l-valine production by the obtained strains under microaerobic and aerobic conditions revealed that leucine dehydrogenase Bcd had a higher potential for l-valine production under microaerobic conditions. The Bcd-possessing strain exhibited 2.2-fold higher l-valine accumulation (up to 9.1 g/L) and 2.0-fold higher yield (up to 35.3%) under microaerobic conditions than the IlvE-possessing strain. The obtained results could be interpreted as follows: an altering of redox cofactor balance in the l-valine biosynthesis pathway increased the production and yield by E. coli cells under microaerobic conditions. Thus, the effective synthesis of l-valine by means of “valine fermentation” was shown in E. coli. This methodology has the advantages of being an economical and environmentally friendly process.

L-Serine overproduction with minimization of by-product synthesis by engineered Corynebacterium glutamicum.

The direct fermentative production of L-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low L-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing L-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both L-serine production level (26.23g/L) and its productivity (0.27g/L/h). Compared to the parent strain, the by-products L-alanine and L-valine accumulation in the resulting strain were reduced by 87% (from 9.80 to 1.23g/L) and 60% (from 6.54 to 2.63g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards L-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as L-serine productivity. With addition of 2g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72%) and the L-serine productivity (67%). In fed-batch fermentation, 42.62g/L of L-serine accumulation was achieved with a productivity of 0.44g/L/h and yield of 0.21g/g sucrose, which was the highest production of L-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve L-serine productivity.

Mathematical modeling of the production of cyclosporin A by Tolypocladium inflatum: effect of L-valine.

A mathematical model was developed to describe the kinetics of submerged fungal growth, consumption of nutrients, and production of the immunosuppressant drug, cyclosporin A (CyA). Special emphasis was placed on the experimentally observed stimulatory effect of L-valine on CyA production. The proposed model was based on kinetic information and emerging mechanistic data on CyA biosynthesis. It was assumed that L-valine acts as both precursor and inducer of the CyA-synthesizing multienzyme (CyA synthetase), and an unmethylated intermediate of CyA was postulated in the biosynthetic process. The model consisted of two parts: one describing cell growth and substrate consumption and the other addressing the kinetics of internal properties such as endogenous valine, biosynthetic enzyme, CyA intermediate, and CyA. The success of this extensive model was confirmed by its ability to simulate adequately the kinetic profiles of both external and internal variables. For instance, the model correctly predicted the time course of intracellular L-valine accumulation. In addition, both the optimal level and timing of exogenous L-valine addition could be predicted for maximum drug production. This study suggests rational new ways of improving the fungal production process of this important immunosuppressant.

Stabilization of an unstable L-leucine producing strain of Corynebacterium glutamicum by enhancement of L-valine biosynthesis.

The L-leucine productivity of an L-leucine producer, Corynebacterium glutamicum H-1204, was unstable due to reversion, which was assumed to be caused by the depletion of intracellular L-valine. The genetic enhancement of L-valine biosynthesis was attempted for stabilization using L-valine accumulation as an index. Strain AB-47, derived as an α-aminobutyrate resistant mutant, accumulated L-valine as a by-product besides L-leucine. The L-leucine productivity of strain AB-47 was remarkably stabilized under conditions under which that of parental strain H-12Q4 became unstable. These results indicate that the balanced biosynthesis of metabolites is important for the breeding of a stable amino acid producer.

Accumulation of Amino Acids by Saccharomyces cerevisiae Y185 with Phospholipids Enriched in Different Fatty-acyl Residues: a Statistical Analysis of Data

Saccharomyces cerevisiae Y185, grown anaerobically in media containing ergosterol and palmitoleic, oleic or linoleic acids, synthesized phospholipids extensively enriched in the exogenously supplied fatty acid. A study was made of the effect of solute concentration on rates of accumulation of nine amino acids by organisms enriched in different fatty-acyl residues. Data were fitted using computer-aided statistical analysis to three equations to derive kinetic constants for accumulation. Analysis of data for two of the amino acids, namely l-threonine and l-histidine, showed different kinetics in organisms enriched in different fatty-acyl residues. Woolf-Hofstee plots for accumulation of l-threonine, as well as l-serine, showed abrupt changes in curvature at low concentrations with differently enriched organisms. Data for accumulation of both amino acids gave a significant fit to the model describing accumulation by one transport system without diffusion. Data for accumulation of l-histidine as well as l-aspartic acid best fitted a model describing accumulation by one transport system and diffusion. Values for K T and the diffusion constant, but not V max, differed only for accumulation of L-histidine in organisms with different fatty-acyl enrichments. A third model, describing accumulation by two separable transport systems, best fitted data for accumulation of l-glutamic acid and L-methionine. Data for accumulation of l-leucine, l-isoleucine and l-valine could not be fitted to any of the models. Woolf-Hofstee plots for accumulation of l-leucine and l-isoleucine by organisms enriched in oleyl or linoleyl residues were superimposable, although similar plots for accumulation of l-valine differed in shape.

Uptake of amino acids by mucosa of rat colon in vitro.

1 Rings of mucosa from rat colon accumulated l-[14C5]valine against a concentration gradient. The accumulation of l-valine was inhibited to some extent by dinitrophenol and cyanide, and more markedly by glycine and l-methionine. l-Lysine was ineffective as an inhibitor. 2 Using everted sacs of colonic mucosa, l-valine, l-citrulline and l-histidine permeated the mucosa from the mucosal fluid to the serosal fluid down a concentration gradient. In experiments with a single amino acid in both the serosal and the mucosal fluids there was always a net loss of amino acid from the serosal fluid. 3 These results are discussed in relation to both tissue accumulation and intestinal absorption of amino acids.

THE FERMENTATIVE PRODUCTION OF L-VALINE BY BACTERIA

1. More than ten strains of bacteria which are capable of producing large amounts of valine were selected under a screening program. The isolates from natural sources we classified as Paracolobacterum coliforme, P. intermedium, Escherichia coli, and Aerobacter aerogenes.2. The production and accumulation of L-valine in P. coliforme and Brevibacterum ammoniagenes have been investigated rather in detail, showing the formation of up to 0.23 mole of valine from one mole of glucose.3. The biosynthetic pathway of valine revealed in the valine-producing bacteria is similar to the known pathway of other microorganisms. It was made clear by experimental results that the enzymes responsible to the condensation of pyruvate in the valine-producing cells are active at pH 5.5 7.5 and different from the orthodox biosynthetic enzyme which is active at pH 8, as two enzymes are known in the reaction of acetolactate formation.4. One of the mechanisms for the efficient production and accumulation of valine is ascribed to the action of an active acetolactate-forming enzyme which is present at least in the valine-producing cells and whose action is not suppressed by valine itself.