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Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds
Author(s) -
Wiesinger Thomas,
Bayer Thomas,
Milker Sofia,
Mihovilovic Marko D.,
Rudroff Florian
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700464
Subject(s) - aldolase a , pseudomonas putida , operon , chemistry , escherichia coli , directed evolution , aldehyde , alcohol dehydrogenase , enantioselective synthesis , yield (engineering) , combinatorial chemistry , biochemistry , enzyme , catalysis , gene , materials science , mutant , metallurgy
Abstract 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.