Heterologous production of ascofuranone and ilicicolin A in Aspergillus sojae.

Ascofuranone and its precursor, ilicicolin A, are secondary metabolites with various pharmacological activities that are produced by Acremonium egyptiacum. In particular, ascofuranone strongly inhibits trypanosome alternative oxidase and represents a potential drug candidate against African trypanosomiasis. However, difficulties associated with industrial production of ascofuranone by A. egyptiacum, specifically the co-production of ascochlorin, which inhibits mammalian respiratory chain complex III at low concentrations, has precluded its widespread application. Therefore, in this study, ascofuranone biosynthetic genes (ascA-E and H-J) were heterologously expressed in Aspergillus sojae, which produced very low-levels of endogenous secondary metabolites under conventional culture conditions. As a result, although we obtained transformants producing both ilicicolin A and ascofuranone, they were produced only when an adequate concentration of chloride ions was added to the medium. In addition, we succeeded in increasing the production of ilicicolin A, by enhancing the expression of the rate-determining enzyme AscD, using a multi-copy integration system. The heterologous expression approach described here afforded the production of both ascofuranone and ilicicolin A, allowing for their development as therapeutics.

Contribution of the retronasal odor of soy sauce to salt reduction.

The characteristic odor of soy sauce has been reported to enhance saltiness. However, soy sauce is used not only as a sauce that is added directly to food, but also as a seasoning. In addition, some of the aromatic compounds that contribute to the soy sauce odor change during cooking or heating. In the present study, the effects of the retronasal odor of uncooked and cooked soy sauce on the enhancement of saltiness and palatability of a low-salt solution were sensory evaluated. A probit analysis indicated that the saltiness-enhancing effect of the odor of 15% uncooked soy sauce was lost by heating. The odors of soy sauce boiled for 10 min (cooked SS) and the residue of soy sauce heated at 200 °C for 1 min improved the palatability of the low-salt solution. Gas chromatography (GC) analyses, namely, GC-olfactometry and GC-mass spectrometry, showed that one active candidate aromatic component of soy sauce contributing to saltiness enhancement was 3-methyl-1-butanol (3-Me-BuOH). The saltiness-enhancing effects of cooked SS could be restored by adding 3-Me-BuOH, as assessed by the sensory evaluation. These data demonstrated that 3-Me-BuOH contributes to saltiness enhancement. PRACTICAL APPLICATION: We found that the odor of cooked soy sauce could improve the palatability of low-salt food. Although the saltiness-enhancing effect provided by the odor of uncooked soy sauce was lost, the saltiness-enhancing effect of the odor of cooked soy sauce can be partially restored by adding 3-methyl butanol. Thus, not only the odor of unheated soy sauce but also the odor of heated soy sauce following addition of 3-methyl butanol may be useful for developing palatable salt-reduced food.

Crystallization and preliminary crystallographic analysis of bacterial fructosyl amino acid oxidase.

Bacterial fructosyl amino acid oxidase [fructosyl alpha-L-amino acid:oxygen oxidoreductase (defructosylating); EC 1.5.3] has been crystallized by the hanging-drop vapour-diffusion technique using sodium citrate as the precipitant. Two types of crystals were grown: one type are rhombic prismatic yellow crystals that belong to space group C2 with unit-cell parameters a = 101.08, b = 63.36, c = 83.07 A, beta = 108.80 degrees and diffract to at least 1.8 A resolution, while the second type are rod-like crystals that belong to space group P4(1)22 or P4(3)22 with unit-cell parameters a = b = 119.09, c = 164.66 A and diffract to 2.7 A resolution.

Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution.

We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45 degrees C, whereas the wild-type enzyme was not stable above 37 degrees C. The K(m) values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.

Cloning and expression of fructosyl-amino acid oxidase gene from Corynebacterium sp. 2-4-1 in Escherichia coli.

The gene encoding the fructosyl-amino acid oxidase (fructosyl-alpha-L-amino acid: oxygen oxidoreductase (defructosylating); EC 1.5.3) of Corynebacterium sp. 2-4-1 was cloned and expressed in Escherichia coli. The gene consists of 1,116 nucleotides and encodes a protein of 372 amino acids with a predicted molecular mass of 39,042. The open reading frame was confirmed as the gene of the fructosyl-amino acid oxidase by comparison with the N-terminal amino acid sequence of the purified fructosyl-amino acid oxidase from Corynebacterium sp. 2-4-1. The sequence of the AMP-binding motif, GXGXXG, was found in the deduced N-terminal region. The amino acid sequence of the fructosyl-amino acid oxidase showed no similarity to that of fungal fructosyl-amino acid oxidases. In addition, substrate specificities of this fructosyl-amino acid oxidase were different from those of other fructosyl-amino acid oxidases. The fructosyl-amino acid oxidase of Corynebacterium sp. 2-4-1 is an enzyme that has unique substrate specificity and primary structure in comparison with fungal fructosyl-amino acid oxidases.