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Enhanced Diastereoselective Synthesis of L‐ threo ‐3,4‐dihydroxyphenylserine by Low‐specific L‐threonine aldolase mutants
Author(s) -
Baik SangHo,
Gwon HuiJeong,
Lim SangYong,
Kim DongHo,
Harayama Shigeaki
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1219.5
Subject(s) - mutant , wild type , chemistry , biochemistry , microbiology and biotechnology , stereochemistry , biology , gene
Four mutants that displayed more enhanced diastereoselectivities (de) than the wild‐type L‐TA from Streptomyces avermitilis MA4680 were successfully obtained by an error‐prone PCR followed by a high‐throughput screening. One candidate mutant, T3‐3 which showed almost the same reverse aldol condensation activity as E. coli transformed pLTA SA‐2 (wild‐type) but lower L‐ erythro ‐DOPS synthesis activity, was selected for further improvement by second error‐prone PCR. About 20,000 clones were screened as the same way, and a mutant named T3‐3m5 was finally obtained. Wild‐type L‐TA showed a very poor de of 14%, while the de of the isolated mutants was approximately 20.5%, 26.2%, 21%, 40%, respectively. Most improved diastreoselectivity was achieved from mutant T3‐3m7 showing a 2.7‐fold more increase than that of the wild‐type L‐TA after a second screening. The complete sequences of the obtained mutants were determined. Sequence analysis showed that each of these mutants had a single or several amino acid substitutions: V86I; R241C; Y306C for T2‐2, Y34C for T2‐4, R241C; A287V for T3‐2 and Y39C, Y306C for T3‐3.