Construction and co-expression of a polycistronic plasmid encoding carbonyl reductase and glucose dehydrogenase for production of ethyl (S)-4-chloro-3-hydroxybutanoate.


Journal Article

Biocatalysis of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] was carried out using Escherichia coli co-expressing a carbonyl reductase gene from Pichia stipitis and a glucose dehydrogenase gene from Bacillus megaterium. An efficient polycistronic plasmid with a high-level of enzyme co-expression was constructed by changing the order of the genes, altering the Shine-Dalgarno (SD) regions, and aligned spacing (AS) between the SD sequence and the translation initiation codon. The optimal SD sequence was 5-TAAGGAGG-3, and the optimal AS distance was eight nucleotides. Asymmetric reduction of COBE to (S)-CHBE with more than 99% enantiomeric excess was demonstrated by transformants, using a water/ethyl caprylate system. The recombinant cells produced 1260 mM product in the organic phase, and the total turnover number, defined as moles (S)-CHBE formed per mole NADP(+), was 12,600, which was more than 10-fold higher than in aqueous systems.

Full Text

Cited Authors

  • Ye, Q; Cao, H; Yan, M; Cao, F; Zhang, Y; Li, X; Xu, L; Chen, Y; Xiong, J; Ouyang, P; Ying, H

Published Date

  • September 2010

Published In

Volume / Issue

  • 101 / 17

Start / End Page

  • 6761 - 6767

PubMed ID

  • 20382525

Pubmed Central ID

  • 20382525

Electronic International Standard Serial Number (EISSN)

  • 1873-2976

International Standard Serial Number (ISSN)

  • 0960-8524

Digital Object Identifier (DOI)

  • 10.1016/j.biortech.2010.03.099


  • eng