T. Bayer, T. Wiesinger, S. Milker, M. Winkler, M.D. Mihovilovic, F. Rudroff:
"In vivo synthesis of polyhydroxylated compounds from a 'hidden reservoir' of toxic aldehyde species";
Synthetic enzyme cascades in living cells often lack efficiency due to formation of by-products by endogenous enzymes, or toxicity of cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, leading to undesired side reactions and decrease yields. Due to the metabolic background of Escherichia coli (E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase (ADH) from Pseudomonas putida (P. putida). Introduction of a carboxylic acid reductase from Norcadia iowensis (CARNi), allowed retrieving the target compound from the carboxylate sink. Further reduction of aldehydes to the alcohols by endogenous E. coli enzymes completes the equilibration between alcohols, aldehydes, and carboxylic acids.Thus, aldehyde concentrations remained below non-viable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone (DHA) dependent aldolase (Fsa1-A129S, E coli) demonstrated, that the reactive aldehyde species are freely available and resulted in the formation of aldol product (3S,4R)-1,3,4-trihydroxy-5-phenylpentan-2-one in 70% isolated yield within short reaction times.
synthetic enzyme cascade . redox funneling . carboxylic acid reductase . in vivo aldol production
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