Engineered PQQ-Dependent Alcohol Dehydrogenase for the Oxidation of 5-(Hydroxymethyl)furoic Acid

Abstract

Furan-2,5-dicarboxylic acid (FDCA) is a bio-based platform chemical with the potential to replace terephthalic acid in the production of polymers. A critical step for enzymatic and whole-cell production of FDCA from 5-(hydroxymethyl)­furfural (HMF) is the transformation of 5-(hydroxymethyl)­furoic acid (HMFA) into 5-formylfuroic acid (FFA). Here, we establish periplasmic pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases (ADHs) as biocatalytic tools for the oxidation of HMFA, HMF, and 5-formylfurfural (FFF). Further, we identify several amino acid residues including the “lid loop” of the substrate channel as promising targets for future engineering steps toward a fully periplasmic oxidation pathway to FDCA.

Furfuryl Alcohol Production with High Selectivity by a Novel Visible-Light Driven Biocatalysis Process

“…Because of the ring-shaped structure of FAL, a steric hindrance would be formed when it combined with the activity site of the enzymes. In previous research, although the ADH was confirmed to have high activity of the CO bonds, the high selectivity of furan alcohols can be achieved by whole-cell catalysts with overexpressed ADH . Nevertheless, the binding state of FAL in ADH remained unclear.…”

“…In previous research, although the ADH was confirmed to have high activity of the CO bonds, the high selectivity of furan alcohols can be achieved by whole-cell catalysts with overexpressed ADH. 53 Nevertheless, the binding state of FAL in ADH remained unclear. In this section, to better understand the mechanism of the visible light-driven biocatalysis process, molecular docking simulation was carried out to confirm the binding between FAL and ADH.…”

“…To date, the investigation of the enzymatic catalysis process for FOL from FAL remains limited because of the difficulties in cofactor regeneration. In previous reports, whole-cell catalysis is selected to realize furan reduction; thus, the NADH can be regenerated by the glycolysis of sugars in the living cells. , However, this process is limited by the high toxicity and low tolerance of microorganisms to both of the furan substrates and furan alcohol products. ,, Besides, because sugars were metabolized by cells continuously, the residual carbon source and byproducts would contaminate the reactant, resulting in many difficulties in the separation of the products.…”

Pseudomonas putida KT2440 endures temporary oxygen limitations

“…Recently, proteins of the ped cluster have been shown to be involved in the oxidation of alcohols by P. putida KT2440 (Bator, Karmainski, et al, 2020; Li et al, 2020; Thompson et al, 2020). Subsequently, using cytochrome c , electrons are transferred to the cytochrome c oxidase (PP_2675, PedF), which was also less abundant here, in turn reducing O 2 as part of the respiratory electron transfer chain (García‐Horsman et al, 1994; Wehrmann et al, 2020). The downregulation of the ped can thus be reasoned with the temporary low abundance of molecular O 2 , impeding the functionality of PedF and potentially the PQQ biosynthesis pathway, which requires molecular O 2 itself.…”

(Chemo)biocatalytic Upgrading of Biobased Furanic Platforms to Chemicals, Fuels, and Materials: A Comprehensive Review

“…GO M 3–5 was reported to rapidly oxidize 50 mM HMFCA to FFCA within 1 h in the presence of HRP and CAT, although Dd GO displayed low catalytic activity toward HMFCA . A periplasmic PQQ-dependent ADH from P. putida KT2440 (PedH) was engineered to oxidize HMFCA to FFCA . The wild-type (WT) enzyme showed no activity toward HMFCA, while the variant F412 V/W561A had the specific activity of about 1 U/mg.…”