Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli

Abstract

Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90–99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2–C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production.

Microbial Engineering for Aldehyde Synthesis

“…Although the problem of alcohol byproduct formation has been described extensively, very few studies of alkane biosynthesis have used strains engineered with deletions of aldehyde reductases. Rodriguez and Atsumi discussed the relevance of their strain for alkane synthesis but did not demonstrate alkane production in their study (56). Production of propane was recently reported by Kallio and colleagues using engineered E. coli that displayed decreased endogenous conversion of butyraldehyde to butanol due to deletions of ahr and yqhD (63).…”

“…Soon after aromatic aldehyde accumulation was reported, Ro- (56). Given that the consequential gene deletions in the RARE strain form a subset of the genes deleted by Rodriguez and Atsumi, both strains are likely capable of accumulating most aromatic and aliphatic aldehydes of interest under aerobic conditions at the shake flask scale.…”

Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids

“…Further complicating matters in vivo is the presence of endogenous aldehyde reductases that have the potential to reduce any alcohol groups oxidized to aldehydes back to the corresponding alcohols (Rodriguez and Atsumi, 2014).…”

Microbial production of scent and flavor compounds

“…The production of 10 g/L isobutanol in these strains suggested that further gene deletion may be necessary to eliminate isobutanol production. Upon identification and deletion of five additional uncharacterized aldehyde reductase encoding genes in E. coli (13 total), a variety of aldehydes could be produced from 2-keto acids without significant flux to alcohol byproducts [26].…”