Jul 2026· Biotechnology for Biofuels and Bioproducts· 0 citations
TL;DR
A PDC-responsive biosensor is developed to facilitate high-throughput screening of improved PDC-producing strains and enables real-time, specific detection of PDC produced from lignin-derived aromatic compounds, including those in black liquor extract derived from lignocellulosic biomass.
Abstract
Lignin is the most abundant renewable aromatic resource on Earth, yet its efficient conversion into value-added chemicals through biological funneling remains a key challenge.
Sphingobium lignivorans
SYK-6 exhibits broad metabolic capacity for lignin-derived aromatic compounds, ranging from monomers to dimers, making it a suitable host for biological funneling. Production systems for the polymer building block 2-pyrone-4,6-dicarboxylic acid (PDC) from lignin-derived aromatic compounds have been constructed using SYK-6. In this study, we developed a PDC-responsive biosensor to facilitate high-throughput screening of improved PDC-producing strains.
PDC-assimilating bacteria were isolated from environmental samples, and their genomes were analyzed to obtain candidate PDC-responsive transcriptional regulatory systems. Among these, the LysR-type transcriptional regulator PdcR and its target promoter P
PDC
from
Cupriavidus
sp. 8B were identified and characterized as a PDC-responsive system, in which PdcR is suggested to act as both a repressor and an activator. A reporter plasmid pCup1 carrying these components was introduced into a PDC hydrolase gene-deficient strain (Δ
ligI
) of SYK-6 to construct the PDC biosensor Δ
ligI
(pCup1). The biosensor was specific for PDC, with no response observed for its upstream metabolites. When vanillic acid or protocatechuic acid was supplied as a substrate, concentration-dependent fluorescence responses were observed over a range of 0.1–5 mM, demonstrating that PDC production from lignin-derived aromatic compounds can be monitored. Fluorescence increased synchronously with PDC accumulation, suggesting real-time monitoring of PDC production. Furthermore, the biosensor responded to PDC produced from aromatic compounds in softwood black liquor extract.
Δ
ligI
(pCup1) enables real-time, specific detection of PDC produced from lignin-derived aromatic compounds, including those in black liquor extract derived from lignocellulosic biomass, demonstrating applicability to screening for lignin valorization. Integration of this biosensor with diverse strain engineering approaches, including random mutagenesis, adaptive laboratory evolution, and metagenomics-based enzyme discovery, would provide a platform for efficient selection of SYK-6 strains with enhanced PDC production capability.
Lignin depolymerization by white-rot fungi generates diverse aromatic compounds derived from hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. Although the metabolic pathways for G- and H-unit–derived aromatics have been studied, the enzymatic step responsible for the oxidative decarboxylation of the S-unit intermediate syringic acid (SA) has remained unknown. Here, we identify PcMNX1, a group A flavoprotein monooxygenase (FPMO) from the white-rot fungus Phanerochaete chrysosporium, as the enzyme catalyzing this missing step. Recombinant PcMNX1 catalyzed the NAD(P)H-dependent oxidative decarboxylation of SA to dimethoxyhydroquinone and also converted other lignin-derived aromatics, including vanillic acid and 4-hydroxybenzoic acid, with markedly higher catalytic efficiency than the closely related enzyme GsMNX1 from Gelatoporia (Ceriporiopsis) subvermispora. The crystal structure of PcMNX1 was determined at 2.00 Å resolution, revealing a typical group A FPMO fold with FAD bound in the “out” conformation. Structure-guided mutagenesis demonstrated that His247 functions as the catalytic base required for decarboxylative hydroxylation. Comparative structural analysis with bacterial 3-hydroxybenzoate 6-hydroxylase indicated that subtle substitutions in active-site residues alter substrate positioning and reaction outcomes. Consistent with this hypothesis, introduction of PcMNX1-type residues into 3-hydroxybenzoate 6-hydroxylase conferred decarboxylation activity toward lignin-derived aromatics. Furthermore, enlargement of the PcMNX1 active-site cavity through the L264A substitution markedly enhanced SA conversion. Together, these findings demonstrate that PcMNX1 catalyzes the oxidative decarboxylation of SA and reveal how subtle active-site remodeling diversifies the catalytic repertoire of closely related group A FPMOs involved in lignin-derived aromatic metabolism.
Reini Mori, Hiromitsu Suzuki, T. Ishida et al.· Journal of Biological Chemis...· 0 citations
2-Pyrone-4,6-dicarboxylic acid (PDC) is a promising pseudo-aromatic bio-based monomer for the production of biodegradable polyesters. Here, we combined metabolic engineering with fermentation process optimization to establish efficient de novo PDC biosynthesis in Corynebacterium glutamicum. To enhance the availability of protocatechuic acid (PCA), the direct precursor of PDC, endogenous PCA degradation was blocked, and precursor supply through the shikimate pathway was strengthened. Subsequently, the ligABC cluster from Sphingomonas paucimobilis SYK-6 was heterologously introduced to enable PDC formation from glucose. Medium screening identified a corn steep liquor-containing formulation that increased the PDC titer by 1.4-fold in shake-flask cultures. Further optimization of corn steep liquor concentration, dissolved oxygen, and glucose feeding rate in a 5-L bioreactor increased the PDC titer to 71.1 g/L at 90 h, with an average productivity of 0.8 g/L/h, the highest productivity for C. glutamicum-based PDC production reported to date.
Meng Chai, Rui-Tao Yu, Yi-Tong Li et al.· Fermentation· 0 citations
1,3-Propanediol is a versatile C3 diol serving as a key building block used in various industries. Klebsiella pneumoniae represents an attractive microbial chassis for 1,3-PDO production due to its endogenous 1,3-PDO and Vitamin B₁₂ biosynthetic pathways. However, low carbon flux and by-product formation remain bottlenecks to high-efficiency 1,3-PDO production. Here, a novel co-substrate (glucose-glycerol) strategy was reported to address these limitations. First, the glycerol oxidation and 3-hydroxypropionic acid biosynthetic pathways were eliminated, driving glycerol to serve exclusively as the substrate for 1,3-PDO synthesis. Glucose was then dedicated to support cell growth and energy generation. To maximize glucose-to-energy conversion, the pentose phosphate pathway, Entner-Doudoroff pathway, Embden-Meyerhof-Parnas pathway, and tricarboxylic acid cycle were systematically engineered. The engineered strain KPS8-6 produced 18.8g/L of 1,3-PDO in shake flask cultivations, with a yield of 0.97mol/mol glycerol, and 1 mole of glucose supported the production of 4.27 mole of 1,3-PDO. 91.62g/L 1,3-PDO was produced after 60h of cultivation in fed-batch fermentations, and the yield of 1,3-PDO from glycerol was 0.99mol/mol. This work establishes a robust, co-substrate-driven strategy for high-yield 1,3-PDO production.
Shao-Qi Sun, Ai Chen, Wen-Qi Wang et al.· Journal of Biotechnology· 0 citations
Efficient valorization of lignin-derived aromatics remains a key challenge in sustainable chemistry. Biobased aromatic alcohols and esters are important value-added compounds widely used in fragrances, pharmaceutical intermediates, fine chemicals, and functional materials. In this study, a whole-cell biocatalytic system co-expressing alcohol dehydrogenase (ADH6) from Saccharomyces cerevisiae and glucose dehydrogenase (GDH) was developed for the efficient conversion of syringaldehyde to syringyl alcohol. The GDH-based cofactor regeneration module enabled in situ reduced nicotinamide adenine dinucleotide phosphate (NADPH) supply, thereby improving catalytic efficiency while reducing the dependence on external cofactors. Under optimized conditions, the system achieved a high analytical yield of 98.3% at 40 mM syringaldehyde in an aqueous system, representing, to the best of our knowledge, the highest substrate concentration reported so far for this biocatalytic conversion. The substrate spectrum was further evaluated using representative lignin-derived aromatic aldehydes, including cinnamaldehyde, p-anisaldehyde, vanillin, and 3,4-dimethoxybenzaldehyde. These substrates were converted into cinnamyl alcohol, 4-methoxybenzyl alcohol, vanillyl alcohol, and 3,4-dimethoxybenzyl alcohol, respectively. Cinnamaldehyde, p-anisaldehyde, and 3,4-dimethoxybenzaldehyde afforded the corresponding alcohols with analytical yields of 99%, whereas vanillin gave vanillyl alcohol with an analytical yield of 86% after 4 h. Molecular docking indicated that these substrates could bind within the ADH6 active pocket through similar orientations, supported by hydrogen-bonding and hydrophobic interactions, providing structural explanation for the observed substrate adaptability. In addition, coupling with immobilized lipase Novozym 435 enabled the conversion of syringyl alcohol into syringyl acetate via transesterification. This work established an efficient tandem biocatalysis with reductase and lipase for upgrading of lignin-derived aromatic compounds.
Haoyu Chai, Xin Li, Yu-Cai He et al.· Bioresource Technology· 0 citations
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.
Kathryn M. Mains, Dillon T. Hofsommer, Michael A. Gapuz et al.· bioRxiv· 1 citation
L-Theanine is a health-beneficial tea-specific amino acid. This amino acid is synthesized through the catalytic action of L-theanine synthase or its isoenzymes, utilizing ethylamine and L-glutamic acid as primary substrates. As the rate-limiting substance, traditional ethylamine synthesis relies on tea root metabolism, and exploring new synthetic pathways can boost L-theanine production and explore environmental application values. Self-developed Tongji_Software was used to predict a potential pathway for ethylamine synthesis. A cell-free protein synthesis (CFPS) system was adopted for verification, and genome engineering was performed to construct a recombinant strain. According to the software predictions, atrazine chlorohydrolase (AtzA) and hydroxyatrazine deethylaminase (AtzB) could convert the herbicide atrazine into ethylamine. CFPS based on Pseudomonas knackmussii (P. knackmussii) produced 88.5 μM L-theanine within a 24-h period, while the engineered strain P. knackmussii-AtzAB yielded 445.7 μM with the conversion rate of 44.6% over 84 h of fermentation. In summary, we revealed a novel pathway for ethylamine synthesis, which can promote L-theanine accumulation in tea trees and degrade environmental pollutants.
S. Yu, J. Feng, G. Zhou et al.· Plant biology· 0 citations
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