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

被引:4
|
作者
Wiesinger, Thomas [1 ]
Bayer, Thomas [1 ]
Milker, Sofia [1 ]
Mihovilovic, Marko D. [1 ]
Rudroff, Florian [1 ]
机构
[1] TU Wien, Inst Appl Synthet Chem, OC 163,Getreidemarkt 9, A-1060 Vienna, Austria
基金
奥地利科学基金会;
关键词
alcohols; biocatalysis; domino reactions; enzymes; solid-phase extraction; D-FRUCTOSE-6-PHOSPHATE ALDOLASE; ORGANIC-SYNTHESIS; ESCHERICHIA-COLI; CASCADE REACTIONS; 2-STEP SYNTHESIS; D-FAGOMINE; ADDITIONS; GLYCOLALDEHYDE; DIHYDROXYACETONE; IMINOCYCLITOLS;
D O I
10.1002/cbic.201700464
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:361 / 368
页数:8
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