Vanillin from lignin-chemicals platform: An overview on current chemo- and biocatalytic processes Mario De Simone, Lígia O. Martins Sustainable Chemistry for the Environment, 2025 Lignin is a major aromatic biopolymer in plant cell walls and represents a promising renewable carbon source. Upon depolymerization, lignin can yield a variety of aromatic compounds, forming a versatile chemical platform for producing high-value chemicals, among them vanillin, a widely used flavouring agent across multiple industries. However, due to its structural complexity and inherent recalcitrance, efficient lignin depolymerization and conversion remain significant challenges. This review outlines both chemical and enzymatic lignin depolymerization strategies, focusing on recent advances in valorisation into vanillin. Enzymatic approaches are highlighted for their sustainability and potential to achieve high yields. Moreover, techniques such as protein engineering, whole-cell bioconversions, and enzyme immobilization are discussed to overcome current limitations in enzymatic processes. The review concludes with perspectives on future developments in enzyme design, process optimization, and integrated lignin biorefinery strategies, emphasizing the central role of protein engineering in driving the circular bioeconomy forward.
Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis Mario De Simone, Lur Alonso-Cotchico, Maria Fátima Lucas, Vânia Brissos, Lígia O. Martins Journal of Biotechnology, 2024 Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Å from the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.
A One-Pot, Whole-Cell Biocatalysis Approach for Vanillin Production using Lignin Oil Ivana Marić, Yiming Guo, Maximilian J. L. J. Fürst, Korneel Van Aelst, Sander Van den Bosch, Mario De Simone, Lígia O. Martins, Bert F. Sels, Marco W. Fraaije Advanced Synthesis and Catalysis, 2023 Vanillin is a popular and versatile flavor compound, almost entirely produced from petroleum‐derived phenol by a multi‐step chemical synthesis. The process is hazardous to the environment and unsustainable for its fossil oil usage. Therefore, developing environmentally friendly, efficient, and sustainable routes to biobased vanillin is essential. Here, we report on vanillin production from 4‐n‐propylguaiacol (4PG), one of the main components in lignin oil obtained through reductive catalytic fractionation (RCF) of soft wood, by employing recombinant Escherichia coli cells. Conversion is based on the expression of two engineered oxidative enzymes: a 4‐n‐propylguaiacol oxidase and an isoeugenol dioxygenase. A high yield of vanillin, 66% from 4PG in RCF lignin oil was achieved through rounds of optimisation of the whole‐cell conversion process. This high‐performance strategy was readily scaled up to produce vanillin at an unprecedented 18% and 3% yield based on lignin oil and spruce wood respectively. The whole‐cell bioconversion process shows good tolerance even at high loadings of starting material, showcasing the robustness and applicability of the employed biocatalysts. This work paves the way for further development towards the efficient production of high‐titer biobased vanillin using depolymerised lignin as the feedstock.
Rationally Guided Improvement of NOV1 Dioxygenase for the Conversion of Lignin-Derived Isoeugenol to Vanillin Mario De Simone, Laura Alvigini, Lur Alonso-Cotchico, Vânia Brissos, Jonatan Caroli, Maria Fátima Lucas, Emanuele Monza, Eduardo Pinho Melo, Andrea Mattevi, Lígia O. Martins Biochemistry, 2023 Biocatalysis is a key tool in both green chemistry and biorefinery fields. NOV1 is a dioxygenase that catalyzes the one-step, coenzyme-free oxidation of isoeugenol into vanillin and holds enormous biotechnological potential for the complete valorization of lignin as a sustainable starting material for biobased chemicals, polymers, and materials. This study integrates computational, kinetic, structural, and biophysical approaches to characterize a new NOV1 variant featuring improved activity and stability compared to those of the wild type. The S283F replacement results in a 2-fold increased turnover rate (kcat) for isoeugenol and a 4-fold higher catalytic efficiency (kcat/Km) for molecular oxygen compared to those of the wild type. Furthermore, the variant exhibits a half-life that is 20-fold higher than that of the wild type, which most likely relates to the enhanced stabilization of the iron cofactor in the active site. Molecular dynamics supports this view, revealing that the S283F replacement decreases the optimal pKa and favors conformations of the iron-coordinating histidines compatible with an increased level of binding to iron. Importantly, whole cells containing the S283F variant catalyze the conversion of ≤100 mM isoeugenol to vanillin, yielding >99% molar conversion yields within 24 h. This integrative strategy provided a new enzyme for biotechnological applications and mechanistic insights that will facilitate the future design of robust and efficient biocatalysts.
Biorefinery gets hot: Thermophilic enzymes and microorganisms for second-generation bioethanol production Luca Zuliani, Annabel Serpico, Mario De Simone, Nicola Frison, Salvatore Fusco Processes, 2021 To mitigate the current global energy and the environmental crisis, biofuels such as bioethanol have progressively gained attention from both scientific and industrial perspectives. However, at present, commercialized bioethanol is mainly derived from edible crops, thus raising serious concerns given its competition with feed production. For this reason, lignocellulosic biomasses (LCBs) have been recognized as important alternatives for bioethanol production. Because LCBs supply is sustainable, abundant, widespread, and cheap, LCBs-derived bioethanol currently represents one of the most viable solutions to meet the global demand for liquid fuel. However, the cost-effective conversion of LCBs into ethanol remains a challenge and its implementation has been hampered by several bottlenecks that must still be tackled. Among other factors related to the challenging and variable nature of LCBs, we highlight: (i) energy-demanding pretreatments, (ii) expensive hydrolytic enzyme blends, and (iii) the need for microorganisms that can ferment mixed sugars. In this regard, thermophiles represent valuable tools to overcome some of these limitations. Thus, the aim of this review is to provide an overview of the state-of-the-art technologies involved, such as the use of thermophilic enzymes and microorganisms in industrial-relevant conditions, and to propose possible means to implement thermophiles into second-generation ethanol biorefineries that are already in operation.
RECENT SCHOLAR PUBLICATIONS
Vanillin from Lignin-Chemicals Platform: An Overview on Current Chemo-and Biocatalytic Processes M De Simone, LO Martins Sustainable Chemistry for the Environment, 100297 , 2025 2025.0 Citations: 1
Enhancing enzymes with a state-of-the-art computational pipeline: targeting distal mutations for improved stability L Tiessler-Sala, L Alonso-Cotchico, M Canyelles-Niño, F Casilli, ... Book of abstracts, 16 , 2025 2025.0
Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis M De Simone, L Alonso-Cotchico, MF Lucas, V Brissos, LO Martins Journal of Biotechnology 391, 92-98 , 2024 2024.0 Citations: 12
Improvement of Isoeugenol Dioxygenases for the Sustainable Production Of Lignin-Derived Vanillin M De Simone PQDT-Global , 2024 2024.0
A one‐pot, whole‐cell biocatalysis approach for vanillin production using lignin oil I Marić, Y Guo, MJLJ Fürst, K Van Aelst, S Van den Bosch, M De Simone, ... Advanced Synthesis & Catalysis 365 (22), 3987-3995 , 2023 2023.0 Citations: 23
Rationally Guided improvement of NOV1 dioxygenase for the conversion of lignin-derived isoeugenol to vanillin M De Simone, L Alvigini, L Alonso-Cotchico, V Brissos, J Caroli, MF Lucas, ... Biochemistry 62 (2), 419-428 , 2022 2022.0 Citations: 16
Biorefinery gets hot: Thermophilic enzymes and microorganisms for second-generation bioethanol production L Zuliani, A Serpico, M De Simone, N Frison, S Fusco Processes 9 (9), 1583 , 2021 2021.0 Citations: 40
Sustainable Chemistry for the Environment M De Simone, LO Martins
MOST CITED SCHOLAR PUBLICATIONS
Biorefinery gets hot: Thermophilic enzymes and microorganisms for second-generation bioethanol production L Zuliani, A Serpico, M De Simone, N Frison, S Fusco Processes 9 (9), 1583 , 2021 2021.0 Citations: 40
A one‐pot, whole‐cell biocatalysis approach for vanillin production using lignin oil I Marić, Y Guo, MJLJ Fürst, K Van Aelst, S Van den Bosch, M De Simone, ... Advanced Synthesis & Catalysis 365 (22), 3987-3995 , 2023 2023.0 Citations: 23
Rationally Guided improvement of NOV1 dioxygenase for the conversion of lignin-derived isoeugenol to vanillin M De Simone, L Alvigini, L Alonso-Cotchico, V Brissos, J Caroli, MF Lucas, ... Biochemistry 62 (2), 419-428 , 2022 2022.0 Citations: 16
Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis M De Simone, L Alonso-Cotchico, MF Lucas, V Brissos, LO Martins Journal of Biotechnology 391, 92-98 , 2024 2024.0 Citations: 12
Vanillin from Lignin-Chemicals Platform: An Overview on Current Chemo-and Biocatalytic Processes M De Simone, LO Martins Sustainable Chemistry for the Environment, 100297 , 2025 2025.0 Citations: 1
Enhancing enzymes with a state-of-the-art computational pipeline: targeting distal mutations for improved stability L Tiessler-Sala, L Alonso-Cotchico, M Canyelles-Niño, F Casilli, ... Book of abstracts, 16 , 2025 2025.0
Improvement of Isoeugenol Dioxygenases for the Sustainable Production Of Lignin-Derived Vanillin M De Simone PQDT-Global , 2024 2024.0
Sustainable Chemistry for the Environment M De Simone, LO Martins
