A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1 % of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8n5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in itro was estimated to be 0n95 h −" , and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate : (1) it was labelled with "%C, and therefore derived from the ["%C]dethiobiotin substrate ; (2) it was present only in reaction mixtures containing biotin synthase ; (3) it was not derived from ["%C]biotin ; (4) $&S from [$&S]cystine was

We isolated, characterised, and cloned an enantio-specific amidase from Klebsiella oxytoca and used it to resolve (R,S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide, giving (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid and (S)-3,3,3trifluoro-2-hydroxy-2-methylpropionamide. The (S)-amide could then be hydrolysed chemically to (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. The process can therefore be adapted to produce both (R)-and (S)-enantiomers of 3,3,3-trifluoro-2hydroxy-2-methylpropionic acid, or (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide. The biocatalytic step is part of a combined chemical and biocatalytic route that starts from ethyl trifluoroacetoacetate. The products typically have a purity of greater than 98% and ee values of essentially 100% after isolation. The process has been used to produce 100-g amounts of the (S)-acid, and successfully scaled up to produce 100-kg amounts of the (R)-acid, with the biotransformation carried out at the 1500-L scale.

We have previously described the isolation of the new bacterial species, Ralstonia/Burkholderia sp. strain DSM 6920, which grows with 6-methylnicotinate and regioselectively hydroxylates this substrate in the C2 position by the action of 6-methylnicotinate-2-oxidoreductase to yield 2-hydroxy-6-methylnicotinate (Tinschert et al. 1997). In the present study we show that this enzymatic activity can be used for the preparation of a series of hydroxylated heterocyclic carboxylic acid derivatives. The following products were obtained from the unhydroxylated educts by biotransformation using resting cells: 2-hydroxynicotinic acid, 2-hydroxy-6-methylnicotinic acid, 2-hydroxy-6-chloronicotinic acid, 2-hydroxy-5,6-dichloronicotinic acid, 3-hydroxypyrazine-2-carboxylic acid, 3-hydroxy-5-methylpyrazine-2-carboxylic acid and 3-hydroxy-5-chloropyrazine-2-carboxylic acid. Thus the respective educts were all regioselectively mono-hydroxylated at the carbon atom between the ring-nitrogen and the ring-carbon atom carrying the carboxyl group. In contrast to its relatively broad biotransformation abilities, the strain shows a limited heterocyclic nutritional spectrum. It could grow only with three of the seven transformed educts: 6-methylnicotinate, 2-hydroxy-6-methylnicotinate and 5-methylpyrazine-2-carboxylate. 2-Hydroxynicotinate, 2-hydroxy-6-chloronicotinate, 2-hydroxy-5,6-dichloronicotinate, 3-hydroxypyrazine-2-carboxylate and 3-hydroxy-5-chloropyrazine-2-carboxylate were not degraded by the strain. Therefore, unlike 6-methylnicotinate-2-oxidoreductase, which has a broad substrate spectrum, the second enzyme of the 6-methylnicotinate pathway seems to have a much more limited substrate range. Among 28 aromatic heterocyclic compounds tested as the sole source of carbon and energy, only pyridine-2,5-dicarboxylate was found as a further growth substrate, and this was degraded by a pathway which did not involve 6-methylnicotinate-2-oxidoreductase. To the best of our knowledge the microbial production of 2-hydroxy-6-chloronicotinic acid, 2-hydroxy-5,6-dichloronicotinic acid and 3-hydroxy-5-methylpyrazine-2-carboxylic acid have not been reported before. Strain DSM 6920 is so far the only known strain which allows the microbial production of both these compounds and 3-hydroxypyrazine-2-carboxylic acid and 3-hydroxy-5-chloroypyrazine-2-carboxylic acid.

2-Hydroxynicotinic acid is an important building block for herbicides and pharmaceuticals. Enrichment strategies to increase the chances of finding microorganisms capable of hydroxylating at the C2 position and to avoid the degradation of nicotinic acid via the usual intermediate, 6-hydroxynicotinic acid, were used. Three bacterial strains (Mena 23/3-3c, Mena 25/4-1, and Mena 25/ 4-3) were isolated from enrichment cultures with 6-methylnicotinic acid as the sole source of carbon and energy. Partial characterization of these strains indicated that they represent new bacterial species. All three strains completely degraded 6-methylnicotinic acid, and evidence is presented that the first step in the degradation pathway of strain Mena 23/3-3c is hydroxylation at the C2 position. Resting cells of this strain grown on 6-methylnicotinic acid also hydroxylated nicotinic acid at the C2 position, but did not further degrade the product. Strain Mena 23/ 3-3c showed the highest degree of 16S rRNA sequence similarity to members of the genera Ralstonia and Burkholderia.

The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions.