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We determined the enzymatic characteristics of an industrially important biocatalyst, a-ketoglutarate-dependent L-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic L-amino acids, as well as L-isoleucine, and produced (S)-3-hydroxy-L-allo-isoleucine, 4-hydroxy-L-leucine, (S)-4-hydroxy-L-norvaline, 4-hydroxy-L-norleucine, and 5-hydroxy-L-norleucine. The IDO reaction product of L-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing L-amino acids and generated L-methionine sulfoxide and L-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. [ABSTRACT FROM AUTHOR]

Transcriptome data for a xylitol-producing recombinant Escherichia coli were obtained and used to tune up its productivity. Structural genes of NADPH-dependent D-xyloSe reductase and D-xylose permease were inserted into an Escherichia coli chromosome to construct a recombinant strain producing xylitol from n-xylose for use as a model system for NADPH-dependent bioconversion. Transcriptome analysis of xylitol-producing and nonproducing conditions for the recombinant revealed that xylitol production down-regulated 56 genes. These genes were then selected as candidate factors for suppression of the NADPH supply and were disrupted to validate their functions. Of the gene disruptants, that resulting from the deletion of yhbC showed the best bioconversion rate. Also, the deletion accelerated cell growth during log phase. The features of the mutant could be maintained in jar fermenter-scale production of xylitol. Thus, our novel molecular host strain breeding method using transcriptome analysis was fully effective and could be applied to improving various industrial strains. [ABSTRACT FROM AUTHOR]