Transforming Flask Reaction into Cell-Based Synthesis: Production of Polyhydroxylated Molecules via Engineered Escherichia coli

Abstract

Dihydroxyacetone phosphate (DHAP)-dependent aldolases have been intensively studied and widely used in the synthesis of carbohydrates and complex polyhydroxylated molecules. However, strict specificity toward donor substrate DHAP greatly hampers their synthetic utility. Here, we transformed DHAP-dependent aldolases-mediated by in vitro reactions into bioengineered Escherichia coli (E. coli). Such flaskto-cell transformation addressed several key issues plaguing in vitro enzymatic synthesis: (1) it solves the problem of DHAP availability by in vivo-hijacking DHAP from the glycolysis pathway of the bacterial system, (2) it circumvents purification of recombinant aldolases and phosphatase, and (3) it dephosphorylates the resultant aldol adducts in vivo, thus eliminating the additional step for phosphate removal and achieving in vivo phosphate recycling. The engineered E. coli strains tolerate a wide variety of aldehydes as acceptor and provide a set of biologically relevant polyhydroxylated molecules in gram scale.

Recent Developments in the Preparation of Carbohydrate Derivatives from Achiral Building Blocks by using Aldolases

“…Very recently, Wang and co‐workers reported an “in vivo ” methodology for the gram‐scale production of carbohydrate derivatives employing whole E. coli cells overexpressing an aldolase and a phosphatase gene simultaneously (Scheme ) . The present strategy is able to circumvent most of the limitations associated with “in vitro ” approaches by avoiding the synthesis of unstable DHAP, purification of the recombinant enzymes, and dephosphorylation steps.…”

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

“…Herein, we combined the concepts of artificial enzyme cascades in living cells, the in situ aldehyde production for subsequent stereoselective aldol reaction, and a straightforward isolation and purification protocol . Two non‐natively related enzymes, the membrane‐associated ADH AlkJ and the DHA/HA‐dependent aldolase variant Fsa1‐A129S were coexpressed in E. coli .…”

Two-step enzymatic synthesis of 6-deoxy-l-psicose

“…Therefore, numerous chemical and enzymatic methods have been explored to synthesize rare sugars from common sugars. 1c,9 Chemical strategies for rare sugars tend to require multiple protection/deprotection and complicated purification steps, suffering from low yield as a result. 10 As an alternative, enzymatic synthesis with mild conditions and high efficiency has distinct advantages in both regio- and stereo-selectivity.…”