Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds. (9th November 2017) - Wiesinger, Thomas
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. Abstract : Cascades in cells : A synthetic cascade for the transformation of primary alcohols into polyhydroxylated compounds through in situ preparation of cytotoxic aldehyde intermediates and subsequent aldolase‐mediated C−C bond formation is investigated. The optimized whole‐cell catalyst, combined with a refined solid‐phase extraction downstream purification protocol gives optically pure aldol products.