Metabolic engineering of Rhodococcus aromaticivorans RHA1 for muconic acid production from lignin.

Metabolic engineering of Pseudomonas sp. NGC7 for glucose-free 4-hydroxybenzoic acid production.

4-hydroxybenzoate (HBA) is an aromatic component that forms the basis of great number of specialty plastics and fine chemicals. In pursuit of a greener and sustainable production, this article described Pseudomonas sp. NGC7, which has been metabolically engineered for the production of HBA from a mixture of aromatic compounds from sugarcane bagasse fractionated by alkaline treatment. While biomass-derived saccharides will continue to be in use, alternative carbon feedstocks for bioproduction are also desirable. For this reason, our work focused on producing HBA from a complex mixture of aromatics derived from lignocellulosic biomass. Following a mild alkaline treatment of sugarcane bagasse, the extracts principally comprised an aromatic mixture of p-coumarate, ferulate, 4-hydroxybenzaldehyde, HBA, syringaldehyde, syringate, vanillin, and vanillate. An HBA hydroxylase gene (pobA) knockout of NGC7 could produce HBA while utilizing the alkaline extract as a carbon source. The regulator gene (ferR) associated with the degradation pathway of hydroxycinnamate derivatives in NGC7 was identified, and its disruption enhanced the production rate of HBA from the extract. The engineered ⊿pobA⊿ferR strain proliferated and produced >6 g/L HBA with a yield of exceeding 90 mol% efficiently from the alkaline extract in the absence of saccharides. This study proved that "glucose-free HBA production" is attainable through metabolic flux manipulation in NGC7.

Biotechnological route for biobased vanillin production from alkaline lignin using Rhodotorula glutinis Y1

Expanding the catabolic capacity of Pseudomonas putida to acetovanillone, 5-carboxyvanillate, and vanillyl glyoxylate for muconate production from kraft lignin-derived aromatics

The pulp and paper industry produces large volumes of condensed kraft lignin, which is challenging to convert to single chemical products. For this purpose, tandem chemical depolymerization and bioconversion to a single atom-efficient product is a potentially promising strategy. In this study, we conducted copper-catalyzed oxidative depolymerization using pine-derived kraft lignin to generate multiple bioavailable aromatic monomers at a yield of 4.5 weight% (wt%; g monomers per g lignin) from both C– O and C–C bond cleavage, followed by counter-current extraction with a 52 wt% monomer recovery. This resulted in an oxidized lignin product containing vanillin, vanillate, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, 5-formylvanillin, 5-carboxyvanillin, 5-carboxyvanillate, acetovanillone, and vanillyl glyoxylate. Based on this stream composition, we engineered the industrially relevant soil bacterium Pseudomonas putida KT2440 to catabolize the latter five compounds via overexpression of ten heterologous genes (acvABCDEFSYK-6, vceABSYK-6, ligW2SYK-6, and mdlCPP). We combined these engineered pathways with previously reported strategies for muconate production from G- and H-type monomers to generate P. putida KMM428, which utilized 93.6 ± 0.2 mol% of the quantified aromatic monomers in a depolymerized kraft lignin mixture, and produced muconate at a yield of 99 ± 3 mol%, on a quantified monomer basis. Together, this work increases the theoretical carbon conversion efficiency of this process by 37.6 ± 0.1 mol% through incorporation of three β-5 cleavage products, in addition to traditional G-type monomers.

Biocatalytic Production of Oxidized Furan Derivatives from Lignocellulosic Sugar-Rich Streams Using a Novel Fungal Glyoxal Oxidase

Lignocellulosic biomass is a renewable and abundant resource for producing high-value chemicals as sustainable alternatives to petroleum-derived products. Among these, 5-hydroxymethylfurfural (HMF) and furfural (FA), generated through catalytic dehydration of biomass-derived sugars, serve as pivotal intermediates for enzymatic synthesis of valuable platform chemicals like 2,5-furandicarboxylic acid (FDCA). In recent studies, glyoxal oxidases (GlyOx, EC 1.2.3.15) have emerged as promising candidates for the biotransformation of furan derivatives. This study focused on the recombinant production and characterization of a GlyOx from the medicinal basidiomycete Ganoderma lucidum capable of oxidizing HMF and FA with 51.5% and 23.0% conversion, respectively, after 48 h. Furthermore, the enzyme oxidized HMF derived from hexose-rich wheat straw hydrolysates, leading to 32.4% conversion after 72 h, as well as FA derived from beechwood hemicellulosic hydrolysates, leading to furoic acid with 17.0% conversion after 48 h, marking the first time such enzymatic activity has been demonstrated on furans originating from genuine lignocellulosic biomass sugar streams.

Biocatalytic and synthetic biology strategies for S-type lignin valorization.

Lignin, as one of the most abundant natural aromatic polymers, holds significant potential for valorization into high-value products. However, its complex and heterogeneous structure presents a major challenge for bioconversion. This review focuses on the bioconversion of lignin-derived syringyl monomers, which are characterized by 3,5-dimethoxy-substituted aromatic rings that create metabolic bottlenecks during microbial O-demethylation and ring cleavage. Recent advances are summarized in lignin depolymerization, biological funneling, and synthetic biology-driven pathway design for converting syringyl monomers into value-added products. Special attention is given to critical enzymes, including Rieske non-heme iron monooxygenases, tetrahydrofolate-dependent O-demethylase systems, and cytochrome P450 peroxygenases, which are pivotal for demethylation. Furthermore, metabolic engineering strategies are discussed to enhance the efficiency of lignin conversion and address challenges such as cofactor imbalance and intermediate toxicity. Finally, this review discusses future research priorities, including broad-substrate-range O-demethylase engineering, host tolerance improvement, validation using realistic lignin-derived streams, and industrial translation. Advancing the bioconversion of lignin syringyl monomers could enable sustainable production of renewable products and supports a circular bioeconomy.

Construction of Engineered Escherichia coli for Vanillin Production From Glucose: Pathway Module Optimization and Methyl-Donor Regulation.

Vanillin is an important flavor compound widely used in the food, fragrance, and pharmaceutical industries. Current biotransformation processes from ferulic acid or eugenol are limited by high substrate cost and low carbon efficiency, motivating de novo biosynthesis from glucose. This study employed Escherichia coli as the chassis organism to establish a modular vanillin biosynthesis system based on the phenylpropanoid metabolic pathway. Heterologous expression of sam8, sam5, and comt established a biosynthetic module for the sequential conversion of l-tyrosine to p-coumaric acid, then to caffeic acid, and finally to ferulic acid. This module enabled the production of 15.86 mg/L ferulic acid from glucose. Two ferulic acid-to-vanillin modules were compared: a CoA-dependent deacetylation pathway (fcs/ech) and an oxidative decarboxylation pathway (fdc/cso2). With ferulic acid feeding, the deacetylation route produced 445.78 mg/L vanillin, far exceeding the 3.49 mg/L obtained via oxidative decarboxylation. When integrated with the upstream module, the deacetylation pathway enabled de novo vanillin production from glucose at 4.46 mg/L, whereas the oxidative decarboxylation route yielded only 0.46 mg/L, indicating better performance of the former under the tested conditions. Metabolite profiling indicated accumulation of caffeic acid and limited ferulic acid levels, identifying O-methylation and S-adenosyl-L-methionine (SAM) supply as major bottlenecks. Implementation of SAM regeneration modules revealed that mtn overexpression enhanced the vanillin titer by about 3-fold, to 12.36 mg/L, while luxS overexpression had a negligible effect. In summary, this study establishes a functional de novo phenylpropanoid pathway for vanillin in E. coli, underscores the critical role of terminal‑pathway selection, and demonstrates that SAM regeneration effectively improves vanillin production from glucose.