bioRxiv2023
Aldehydes are attractive chemical targets given applications as end products in the flavors and fragrances industry and as intermediates due to their propensity for C-C bond formation. While biosynthetic routes to diverse aldehydes have been designed, a common challenge is the stability of these aldehydes in the presence of microbial hosts of engineered pathways. Here, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation, in the presence of Escherichia coli strains that were engineered to minimize aldehyde reduction. Of heightened interest to us were resting cell conditions as they offer numerous advantages for the bioconversion of toxic metabolites. Surprisingly, when diverse aldehydes are supplemented to E. coli RARE cells grown under aerobic conditions, they remain stabilized on the timescale of days, whereas when these same aldehydes are supplemented to resting cell preparations of E. coli RARE that had been grown under the same conditions, we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 hours after their addition. Given that our newly engineered strain exhibits Reduced Oxidation And Reduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. Seeking to apply this new strain to resting cell biocatalysis, we compared the capability to synthesis the aldehyde furfural from 2-furoic acid via the carboxylic acid reductase enzyme from Nocardia iowensis. Here, we found that use of ROAR resting cells achieved 2-fold enhancement in furfural titer after 4 h and nearly 9-fold enhancement after 20 h as compared to resting cells of the RARE strain. Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity. HighlightsO_LIWhen genes that encode aldehyde reductases are knocked out in Escherichia coli strains, supplemented aldehydes can experience oxidation instead of reduction, which is catalyzed by a different set of endogenous enzymes. C_LIO_LIInterestingly, we show for a collection of aromatic aldehydes that this oxidation is far more substantial when using resting cell preparations than during aerobic fermentation. C_LIO_LIWe investigate the identity of the responsible genes by performing combinatorial gene inactivation using multiplex automatable genome engineering. C_LIO_LIThe strain that we engineer exhibits Reduced Oxidation And Reduction (the E. coli ROAR strain) and thereby enables design of more efficient aldehyde bioconversion processes under diverse formats. C_LI