Improved furfural tolerance in Escherichia coli mediated by heterologous NADH-dependent benzyl alcohol dehydrogenases

Abstract

While lignocellulose is a promising source of renewable sugars for microbial fermentations, the presence of inhibitory compounds in typical lignocellulosic feedstocks, such as furfural, has hindered their utilisation. In Escherichia coli, a major route of furfural toxicity is the depletion of NADPH pools due to its use as a substrate by the YqhD enzyme that reduces furfural to its less toxic alcohol form. Here, we examine the potential of exploiting benzyl alcohol dehydrogenases as an alternative means to provide this same catalytic function but using the more abundant reductant NADH, as a strategy to increase the capacity for furfural removal. We determine the biochemical properties of three of these enzymes, from Pseudomonas putida, Acinetobacter calcoaceticus, and Burkholderia ambifaria, which all demonstrate furfural reductase activity. Furthermore, we show that the P. putida and B. ambifaria enzymes are able to provide substantial increases in furfural tolerance in vivo, by allowing more rapid conversion to furfuryl alcohol and resumption of growth. The study demonstrates that methods to seek alternative co-factor dependent enzymes can improve the intrinsic robustness of microbial chassis to feedstock inhibitors.

Bioconversion of Furanic Compounds by Chlorella vulgaris—Unveiling Biotechnological Potentials

“…Comparing conversion products from Chlorella vulgaris to furan metabolites from known metabolic pathways in other organisms [ 1 , 4 , 14 , 15 , 16 , 19 , 21 ] using LC-QTOF-IM-MS, the products were identified as 5-Hydroxymethyl-2-Furoic Acid (HMFA) and furoic acid (FA) ( Figure 5 ). Oxidation reactions to detoxify HMF and furfural are common in other microorganisms ( Table 1 ), but the responsible enzymes vary.…”

“…Therefore, bioconversions would be a feasible tool for the detoxification of furanic compounds. Increasing research activities are currently focused on the implementation of microbial processes, including native pathways [ 1 , 14 , 15 ] of furan detoxification or generating recombinant strains [ 16 , 17 ], expressing identified enzymes isolated from strains that do possess these abilities.…”

Unique alcohol dehydrogenases involved in algal sugar utilization by marine bacteria

“…Both ADHs possessed predominantly activity for aromatic substances, resulting in a substrate specificity resembling partially those of cinnamyl alcohol and/or benzyl alcohol dehydrogenases (Larroy et al 2002 ; Willson et al 2022 ). However, the highest activity was observed for pyridine-3-carbaldehyde and furan derivatives.…”

Metabolic engineering in lignocellulose biorefining for high-value chemicals: recent advances, challenges, and outlook for enabling a bioeconomy

“…In addition to this, several other studies have been conducted to identify and use oxidoreductase for tolerance improvement but were not evaluated using LCB hydrolysate (Kim et al, 2022b;Ramzi et al, 2022;Willson et al, 2022;Chang et al, 2023;Wang et al, 2023). Zou et al, 2021 generated inhibitor-tolerant P. putida by disrupting the glucose metabolism (phosphorylation and oxidation route) by deleting gtsABCD (ATP-dependent sugar transporter) and gcd (glucose dehydrogenase).…”