Evaluation of in situ valine production by Bacillus subtilis in young pigs

Mutants of Bacillus subtilis can be developed to overproduce Val in vitro. It was hypothesized that addition of Bacillus subtilis mutants to pig diets can be a strategy to supply the animal with Val. The objective was to investigate the effect of Bacillus subtilis mutants on growth performance and blood amino acid (AA) concentrations when fed to piglets. Experiment 1 included 18 pigs (15.0±1.1 kg) fed one of three diets containing either 0.63 or 0.69 standardized ileal digestible (SID) Val: Lys, or 0.63 SID Val: Lys supplemented with a Bacillus subtilis mutant (mutant 1). Blood samples were obtained 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding and analyzed for AAs. In Experiment 2, 80 piglets (9.1±1.1 kg) were fed one of four diets containing 0.63 or 0.67 SID Val: Lys, or 0.63 SID Val: Lys supplemented with another Bacillus subtilis mutant (mutant 2) or its parent wild type. Average daily feed intake, daily weight gain and feed conversion ratio were measured on days 7, 14 and 21. On day 17, blood samples were taken and analyzed for AAs. On days 24 to 26, six pigs from each dietary treatment were fitted with a permanent jugular vein catheter, and blood samples were taken for AA analysis 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding. In experiment 1, Bacillus subtilis mutant 1 tended (P<0.10) to increase the plasma levels of Val at 2 and 3 h post-feeding, but this was not confirmed in Experiment 2. In Experiment 2, Bacillus subtilis mutant 2 and the wild type did not result in a growth performance different from the negative and positive controls. In conclusion, results obtained with the mutant strains of Bacillus subtilis were not better than results obtained with the wild-type strain, and for both strains, the results were not different than the negative control.     

The dissimilation of leucine, isoleucine and valine to volatile fatty acids by adult Fasciola hepatica

The dissimilation of leucine, isoleucine and valine to volatile fatty acids was determined in Fasciola hepatica and the degradation of (U−¹⁴C) branched amino acids to the volatile fatty acids end products demonstrated. F. hepatica was found to metabolize leucine, isoleucine and valine to isovaleric, 2-methylbutyric and isobutyric acid respectively. The rate of formation of isobutyrate, isovalerate and 2-methylbutyrate was found to be positively related to the rate of propionic acid production with air or nitrogen as the gas phase. However, under 95% O₂/5% CO₂ the formation of the branched chain acids was independent of propionic acid production. The production of isobutyrate, isovalerate and 2-methylbutyrate caused a simultaneous reduction in the rate of acetate formation. The role of propionate formation in regulating metabolism is discussed.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

Regulatory mutants of Streptomyces cinnamonensis producing monensin A

Abstract We prepared mutants of Streptomyces cinnamonensis resistant to amino acid analogues: 2-aminobutyrate, norvaline, norleucine, 2-amino-3-chlorobutyrate and ethionine. The regulatory mutants were studied as to their production of oligoketide antibiotics, monensins A and B, as dependent on the formation of valine which is a precursor of the butyrate building unit of monensin A. Strains resistant to both 2-amino-3-chlorobutyrate and norleucine exhibited an increased production of monensin A from 50% to 90–93% of total monensins.     

Valine inhibition of β-isopropylmalate dehydrogenase takes part in the regulation of leucine biosynthesis inCandida maltosa

The -isopropylmalate (IPM) dehydrogenase (EC 1.1.1.85) ofCandida maltosa, the third pathway-specific enzyme of leucine biosynthesis, was purified, some properties of the enzyme were studied and a novel regulatory pattern was found. The K_m values of the enzyme were estimated to be 0.42 mM for -IPM and 0.34 mM for NAD⁺. It is demonstrated that the enzyme can be regulated by L-valine. The inhibition was competitive with respect to -IPM (K_i=1.84 mM) and non-competitive with respect to NAD⁺ (K_i=5.67 mM). Exogenous addition of L-valine toC. maltosa cells increased the intracellular pool of some intermediates of leucine biosynthesis (-ketoisovalerate, -IPM, -IPM), but has hardly influence on the leucine pool.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The -isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by l-leucine and -ketoisocaproate. The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH. Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects. The maximal Hill coefficient was found to be two. At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate -ketoisovalerate. The inhibition was specifically relieved by the addition of valine or isoleucine. The anomalous effect of temperature on enzyme activity was diminished by leucine. The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine. The further addition of valine reversed this effect. The physiological relevance of the -ketoisocaproate-mediated inhibition is discussed.Abbreviations IPM -isopropylmalate - KIC -ketoisocaproate - KIV -ketoisovalerate - DTNB 5:5 Dithiobis-(2-nitrobenzoe acid)     

Structure-activity analysis of peptidic Chlamydia HtrA inhibitors

Chlamydia trachomatis high temperature requirement A (CtHtrA) is a serine protease that performs proteolytic and chaperone functions in pathogenic Chlamydiae; and is seen as a prospective drug target. This study details the strategies employed in optimizing the irreversible CtHtrA inhibitor JO146 [Boc-Val-Pro-Val^P(OPh)₂] for potency and selectivity. A series of adaptations both at the warhead and specificity residues P₁ and P₃ yielded 23 analogues, which were tested in human neutrophil elastase (HNE) and CtHtrA enzyme assays as well as Chlamydia cell culture assays. Trypsin and chymotrypsin inhibition assays were also conducted to measure off-target selectivity. Replacing the phosphonate moiety with α-ketobenzothiazole produced a reversible analogue with considerable CtHtrA inhibition and cell culture activity. Tertiary leucine at P₃ (8a) yielded approximately 33-fold increase in CtHtrA inhibitory activity, with an IC₅₀ = 0.68 ± 0.02 µM against HNE, while valine at P₁ retained the best anti-chlamydial activity. This study provides a pathway for obtaining clinically relevant inhibitors.     

外源氨基酸胁迫下植物体内游离氨基酸的测定

目的：通过检测外源氨基酸胁迫下植物体内的游离氨基酸含量,探讨氨基酸胁迫下植物生长受抑制的机制。方法：小麦胚接种在加入D-丙氨酸、D-丝氨酸和L-缬氨酸等3种外源氨基酸的培养基中,其发芽和生长会受到强烈抑制。取材胁迫植株和对照植株,提取并用HPLC方法检测游离氨基酸含量,分析主要氨基酸特征的改变。结果：作为处理的胁迫氨基酸在体内有数倍到数十倍的增高,其他氨基酸,尤其是同族及相近族氨基酸的量也出现较大改变,有增加也有减少,有些氨基酸甚至检测不出。结论：在外源氨基酸胁迫下,植物可直接吸收这些氨基酸,胁迫氨基酸在体内的积累。至少影响到了部分氨基酸的合成,使细胞内正常游离态氨基酸的数量增加或减少,这些变化引起代谢失调,进而引起生长抑制。     

Importance of NADPH supply for improved L‐valine formation in Corynebacterium glutamicum

Cofactor recycling is known to be crucial for amino acid synthesis. Hence, cofactor supply was now analyzed for L ‐valine to identify new targets for an improvement of production. The central carbon metabolism was analyzed by stoichiometric modeling to estimate the influence of cofactors and to quantify the theoretical yield of L ‐valine on glucose. Three different optimal routes for L ‐valine biosynthesis were identified by elementary mode (EM) analysis. The modes differed mainly in the manner of NADPH regeneration, substantiating that the cofactor supply may be crucial for efficient L ‐valine production. Although the isocitrate dehydrogenase as an NADPH source within the tricarboxylic acid cycle only enables an L ‐valine yield of Y_Val/Glc = 0.5 mol L ‐valine/mol glucose (mol Val/mol Glc), the pentose phosphate pathway seems to be the most promising NADPH source. Based on the theoretical calculation of EMs, the gene encoding phosphoglucoisomerase (PGI) was deleted to achieve this EM with a theoretical yield Y_Val/Glc = 0.86 mol Val/mol Glc during the production phase. The intracellular NADPH concentration was significantly increased in the PGI‐deficient mutant. L ‐Valine yield increased from 0.49 ± 0.13 to 0.67 ± 0.03 mol Val/mol Glc, and, concomitantly, the formation of by‐products such as pyruvate was reduced. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010