| 摘要 |
An isolated nucleic acid sequence coding for a polypeptide having acetohydroxy acid synthase (AHAS) activity is new. The isolated nucleic acid sequence cited above further comprises: (a) a fully defined sequence of 519 bp (SEQ ID NO: 1 or 3); (b) a sequence comprising in position 21 and 22 a base triplet coding for Asp and Phe, respectively; (c) a sequence hybridizing under stringent conditions with (a) or (b); (d) a sequence having a homology of at least 70% with those of (a) or (b); (e) a sequence coding for a polypeptide having at least 80% homology on amino aid level with the polypeptide coded by (a) or (b); (f) a nucleic acid coding for a polypeptide with improved activity and/or selectivity and/or stability as compared with the polypeptide coded by (a) or (b), prepared by mutagenesis of a nucleic acid of (a) or (b), ligating the nucleic acid sequence obtainable from a suitable vector followed by transformation into a suitable expression system and expression and detection of the critical polypeptide with improved activity and/or selectivity and/or stability; or (g) polynucleotide containing at least 15 successive bases of the polynucleotide sequences of (a)-(f). Independent claims are also included for: (1) a polypeptide coded by the nucleic acid sequence cited above, comprising a fully defined sequence of 173 amino acids (SEQ ID NO: 2 or 4), or a polypeptide which is at least 84% homologous to SEQ ID NO: 2 or 4, without the activity and/or selectivity and/or stability of the polypeptide being substantially reduced as compared with the polypeptide with SEQ ID NO: 2 or 4; (2) plasmids, vectors, micro-organisms comprising one or more nucleic acid sequences cited above; (3) primers for preparing, by means of PCR, or hybridization probes for detecting the nucleic acid sequences cited above; (4) preparing improved rec-polypeptides with AHAS activity starting from nucleic acid sequences cited above, comprising subjecting the nucleic acid sequences to mutagenesis, obtained nucleic acid sequences are cloned in a suitable vector and transferred into a suitable expression system, and detecting and isolating formed polypeptides with improved activity and/or selectivity and/or stability; (5) rec-polypeptides or encoding nucleic acid sequences, obtained by the process of (4); (6) production of a branched-chain amino acid using the polypeptide of (1); (7) a vector pECKA or pECKA/ilvBNC; and (8) microorganisms such as DSM15652, DAM15651 and DSM15650. |
