Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by resting cells ofE. coliRARE strains

Abstract

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 ofEscherichia colistrains 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 toE. coliRARE cells grown under aerobic conditions, they remain stabilized on the timescale of days, whereas when these same aldehydes are supplemented to resting cell preparations ofE. coliRARE that had been grown under the same conditions, we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in theE. coligenome 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 theE. coliROAR strain. Seeking to apply this new strain to resting cell biocatalysis, we compared the capability to synthesize 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.

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

“…As a first step towards testing whether inactivation of aldehyde reductases could eliminate TPAL reduction, we evaluated TPAL stability in the previously engineered RARE.Δ6 strain under aerobic growth. Although we recently verified that the RARE.Δ6 strain reliably stabilizes a broad range of aromatic aldehydes under these conditions, 43 we were surprised to observe TPAL reduction at our first time point of 4 h with greater reduction seen at 12 and 24 h (Figure 1C). However, we observed that cultures of the RARE.Δ6 strain at all timepoints were able to stabilize the mono‐aldehyde 4HMB, eliminating the complete reduction of TPAL to BDM.…”

Reductive amination cascades in cell-free and resting whole cell formats for valorization of lignin deconstruction products

“…For expression, all CAR plasmids were transformed into E. coli ROAR (Butler et al, 2023) for whole cell biocatalysis purposes and BL21 (DE3) for purification. 250 mL of LB-Lennox medium (LBL: 10 g/L bacto tryptone, 5 g/L sodium chloride, 5 g/L yeast extract), in 1 L baffled flasks were supplemented with 5 mM glucose, 2.5 mM MgSO 4 , and 30 μg/mL kanamycin (Fisher Scientific) was inoculated with 1% of saturated overnight culture and induced at mid-exponential phase (OD600 0.5-0.8) with 0.1 µg/mL anhydrotetracycline (ATC, Cayman Chemical).…”

“…For all samples analyzed over HPLC, any concentrations above the linear range were appropriately diluted prior to analysis to ensure the measurements fell within the linear portion of the standard curve (Supporting Information: Figure S6a-d). In parallel, we observed that the duration of culturing prior to harvesting resting cell biocatalysts could affect aldehyde stability (Butler et al, 2023). As such, we chose to harvest cells after shorter durations of culturing, and we tested WC5 cells prepared in this manner for their reusability across multiple batch reactions.…”

“…We report the biosynthesis of 2c, 4c, and 5c from their carboxylic acid precursors for the first time. Our whole cell biocatalysis efforts utilize a unique and recently engineered strain of Escherichia coli that exhibits reduced oxidation and reduction of aryl aldehydes (ROAR) (Butler et al, 2023), which built upon prior efforts that focused solely on mitigating aldehyde reduction in E. coli (Kunjapur et al, 2014). Overall, our work fills a void in the existing arsenal of biomass upcycling approaches, thereby enhancing the prospects for efficient valorization of lignin deconstruction streams.…”

Reductive amination cascades in cell‐free and resting whole cell formats for valorization of lignin deconstruction products

“…For all samples analyzed over HPLC, any concentrations above the linear range were appropriately diluted before analysis to ensure the measurements fell within the linear portion of the standard curve (Supporting Information S1: Figure 6a−d). In parallel, we observed that the duration of culturing before harvesting resting cell biocatalysts could affect aldehyde stability (Butler et al, 2023). As such, we chose to harvest cells after shorter durations of culturing, and we tested WC5 cells prepared in this manner for their reusability across multiple batch reactions.…”