Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds

Chembiochem. 2018 Feb 16;19(4):361-368. doi: 10.1002/cbic.201700464. Epub 2017 Nov 9.


A synthetic cascade for the transformation of primary alcohols into polyhydroxylated compounds in Escherichia coli, through the in situ preparation of cytotoxic aldehyde intermediates and subsequent aldolase-mediated C-C bond formation, has been investigated. An enzymatic toolbox consisting of alcohol dehydrogenase AlkJ from Pseudomonas putida and the dihydroxyacetone-/hydroxyacetone-accepting aldolase variant Fsa1-A129S was applied. Pathway optimization was performed at the genetic and process levels. Three different arrangements of the alkJ and fsa1-A129S genes in operon, monocistronic, and pseudo-operon configuration were tested. The last of these proved to be most beneficial with regard to bacterial growth and protein expression levels. The optimized whole-cell catalyst, combined with a refined solid-phase extraction downstream purification protocol, provides diastereomerically pure carbohydrate derivatives that can be isolated in up to 91 % yield over two reaction steps.

Keywords: alcohols; biocatalysis; domino reactions; enzymes; solid-phase extraction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Dehydrogenase / genetics
  • Alcohol Dehydrogenase / metabolism*
  • Biocatalysis
  • Carbohydrates / biosynthesis*
  • Carbohydrates / chemistry
  • Molecular Structure
  • Pseudomonas putida / enzymology*
  • Pseudomonas putida / growth & development
  • Stereoisomerism


  • Carbohydrates
  • Alcohol Dehydrogenase