Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L⁻¹). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.     

Effect of Nanoencapsulated Vitamin B1 Derivative on Inhibition of Both Mycelial Growth and Spore Germination of Fusarium oxysporum f. sp. raphani

Nanoencapsulation of thiamine dilauryl sulfate (TDS), a vitamin B1 derivative, was proved to effectively inhibit the spore germination of Fusarium oxysporum f. sp. raphani (F. oxysporum), as well as mycelial growth. The average diameter of nanoparticles was measured as 136 nm by being encapsulated with an edible encapsulant, lecithin, whose encapsulation efficiency was about 55% in containing 200 ppm of TDS concentration: the 100 ppm TDS nanoparticle solution showed a mycelial growth inhibition rate of 59%. These results were about similar or even better than the cases of treating 100 ppm of dazomet, a positive antifungal control (64%). Moreover, kinetic analysis of inhibiting spore germination were estimated as 6.6% reduction of spore germination rates after 24 h treatment, which were 3.3% similar to the case of treating 100 ppm of a positive control (dazomet) for the same treatment time. It was also found that TDS itself could work as an antifungal agent by inhibiting both mycelial growth and spore germination, even though its efficacy was lower than those of nanoparticles. Nanoparticles especially played a more efficient role in limiting the spore germination, due to their easy penetration into hard cell membranes and long resident time on the surface of the spore shell walls. In this work, it was first demonstrated that the nanoparticle of TDS not a harmful chemical can control the growth of F. oxysporum by using a lower dosage than commercial herbicides, as well as the inhibiting mechanism of the TDS. However, field trials of the TDS nanoparticles encapsulated with lecithin should be further studied to be effectively used for field applications.     

A model of non‐isothermal degradation of nutrients,pigments and enzymes

Published isothermal degradation curves for chlorophyll A and thiamine in the range 100–150 °C and the inactivation curves of polyphenol oxidase (PPO) in the range 50–80 °C could be described by the model C(t)/C₀ = exp[?b(T)tⁿ] where C(t) and C₀ are the momentary and initial concentrations, respectively, b(T) a temperature dependent ‘rate parameter’ and n, a constant. This suggested that the temporal degradation/inactivation events of all three had a Weibull distribution with a practically constant shape factor. The temperature dependence of the ‘rate parameter’ could be described by the log logistic model, b(T) = log_e[1 + exp[k(T ? T_c)], where T_c is a marker of the temperature level where the degradation/inactivation occurs at a significant rate and k the steepness of the b(T) increase once this temperature range has been exceeded. These two models were combined to produce a non‐isothermal degradation/inactivation model, similar to one recently developed for microbial inactivation. It is based on the assumption that the local slope of the non‐isothermal decay curve, ie the momentary decay rate, is the slope of the isothermal curve at the momentary temperature at a time that corresponds to the momentary concentration of the still intact or active molecules. This model, in the form of a differential equation, was solved numerically to produce degradation/inactivation curves under temperature profiles that included heating and cooling and oscillating temperatures. Such simulations can be used to assess the impact of planned commercial heat processes on the stability of compounds of nutritional and quality concerns and the efficacy of methods to inactivate enzymes. Simulated decay curves on which a random noise was superimposed were used to demonstrate that the degradation/inactivation parameters, k and T_c, can be calculated directly from non‐isothermal decay curves, provided that the validity of the Weibullian and log logistic models and the constancy of the shape factor n could be assumed. Copyright © 2004 Society of Chemical Industry     

Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida

Pyruvate decarboxylase from Zymomonas mobilis (PDC) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are thiamine diphosphate-dependent enzymes that decarboxylate 2-keto acids. Although they share a common homotetrameric structure they have relatively low sequence similarity and different substrate spectra. PDC prefers short aliphatic substrates whereas BFD favours aromatic 2-keto acids. These preferences are also reflected in their carboligation reactions. PDC catalyses the conversion of benzaldehyde and acetaldehyde to (R)-phenylacetylcarbinol and predominantly (S)-acetoin, whereas (R)-benzoin and mainly (S)-2-hydroxypropiophenone are the products of BFD catalysis. Comparison of the X-ray structures of both enzymes identified two residues in each that were likely to be involved in determining substrate specificity. Site-directed mutagenesis was used to interchange these residues in both BFD and PDC. The substrate range and kinetic parameters for the decarboxylation reaction were studied for each variant. The most successful variants, PDCI472A and BFDA460I, catalysed the decarboxylation of benzoylformate and pyruvate, respectively, although both variants now preferred the long-chain aliphatic substrates, 2-ketopentanoic and 2-ketohexanoic acid. With respect to the carboligase activity, PDCI472A proved to be a real chimera between PDC and BFD whereas BFDA460I/F464I provided the most interesting result with an almost complete reversal of the stereochemistry of its 2-hydroxypropiophenone product.     

The effect of different salting procedures and qualities of raw material on some nutrients during processing and storage of salted-dried mackerel

Salted-dried mackerel ( Scomber scombrus ) was prepared using fresh fish or fish which had been stored in ice for 75h and four methods of salting, namely 15% brine, saturated brine, pickle and dry salting. Samples were analysed for available lysine, thiamine and riboflavin before salting, after salting, after drying and after 1 month's storage at 20°C. Final losses of each of these nutrients were lowest when the fish were pickle cured. Losses were also lower when fresher raw material was used. the use of good quality fish and a pickle salting method is therefore recommended for this type of product.     

Preferences for tastes paired with recovery from thiamine deficiency in rats: Appetitive conditioning or learned safety?

Conducted a study of 80 male Sprague-Dawley rats to investigate earlier findings that rats develop learned preferences for flavors paired with recovery from vitamin deficiencies. Results show that thiamine deficient Ss preferred flavors paired with recovery from deficiency to other familiar flavors, suggesting that part of the preference for flavors paired with recovery was the result of appetitive conditioning. Data are discussed in relation to "learned safety," specific hungers, illness-induced neophobia, and other phenomena in the taste-aversion literature. (16 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Mapping of gene controlling thiamine transport in Saccharomyces cerevisiae

A recessive mutation leading to complete loss of thiamine uptake in Saccharomyces cerevisiae was mapped on the left arm of chromosome VII, approximately 56cM centromere-distal to trp5. As the analysed locus is relatively distant from its centromere and from the markers used, its attachment to chromosome VII was confirmed by chromosome loss methods.     

Vitamins in brewing: presence and influence of thiamine and riboflavin on wort fermentation

Thiamine and riboflavin vitamers are present in a wide range of foods including beer. These vitamers play critical roles in a variety of enzymatic complexes and can promote and maintain metabolism. Currently, the presence and role of these vitamers in the malting and brewing industry have not been widely explored. This research investigated the effects of various fermentation conditions that may lead to the variations in the vitamin content in beer observed by previous researchers. The present research found that during fermentation, the thiamine content of wort is quickly utilized within the first 6 h of a standard fermentation and the uptake of this vitamin is not affected by increases in wort gravity. While no significant changes were observed in extracellular phosphorylated vitamers of thiamine, both free thiamine and thiamine diphosphate accumulated intracellularly during the wort fermentation. Meanwhile extracellular riboflavin vitamers were only poorly utilized during beer fermentations, however flavin mononucleotide rapidly accumulated intracellularly and more so under aerobic conditions. When yeast was exposed to an all‐malt high‐gravity wort, the thiamine or riboflavin utilization was not affected. However, thiamine utilization was reduced in adjunct‐driven high‐gravity worts. Notwithstanding the lowered thiamine uptake under high‐gravity conditions; there were some minor improvements in fermentation performance and yeast viability. The addition of thiamine to an all‐malt wort did appear to enhance yeast viability, both under normal and high‐gravity conditions. Copyright © 2016 The Institute of Brewing & Distilling     

Vitamins in brewing: effects of post‐fermentation treatments and exposure and maturation on the thiamine and riboflavin vitamer content of beer

Post‐fermentation processes and maturation are important steps in beer production as they help to shape the organoleptic properties and stabilize the final product. Brewers can use a variety of processing aids (e.g. isinglass, PVPP, etc.) and processes (e.g. pasteurization, bottle conditioning, etc.) to achieve a desired final product with a desirable shelf life; however, these processes can have detrimental effects on the vitamin content of the beer. This research found that heat treatments have a marked influence on the decrease in the thiamine diphosphate vitamer, while PVPP and silica treatments have a greater influence on the decrease in riboflavin vitamers. Refrigeration, filtration or centrifugation have no, or only very limited, influence on thiamine or riboflavin vitamers, while application of isinglass, bentonite, tannic acid and SO₂ causes a decrease in both thiamine and riboflavin vitamers. Storage of beer at refrigerated temperatures appears to provide protection against significant degradation of both thiamine and riboflavin vitamers; however, storage of filtered beer at elevated temperatures shows a decrease in thiamine diphosphate and riboflavin. Storage of bottle‐conditioned beer at elevated temperatures shows a marked decrease in yeast viability, accompanied by a decrease in thiamine diphosphate and free riboflavin, and a marked increase in free thiamine. These findings provide an insight into the reason why there is a significant variation in the vitamer content of beers, even within a single beer style. Copyright © 2016 The Institute of Brewing & Distilling     

GENETIC CONTROL OF THIAMINE EXCRETION AND OF ITS SUPPRESSION IN SACCHAROMYCES CEREVISIAE

Three non-linked loci were found to be involved in thiamine excretion in Saccharomyces cerevisiae, i.e. thi2 which is complementary with thi3 and thi4, the latter two being substitutable mutually but acting also together. Mutation thi3 was present already in strain Nα, which produced relatively high frequencies of excreters after UV treatment. Non-excreting ‘revertants’ of unstable excreters derived from another haploid strain contained recessive mutation thi2, so that mutation thi3 can be substituted also by the metabolic or physiological disorders ruling after UV treatment. Mutations thi2 were found also in all 43 analysed stable excreting mutants obtained by UV treatment of strain Nα which indicates that they are either inevitable for the excretion or are specifically selected by the selection method used. At least one suppressor locus of thiamine excretion was present in that strain of S. cerevisiae, which did not produce thiamine excreters after UV treatment in spite of its ability to synthetise thiamine.