Mario De Simone
@itqb.unl.pt
Instituto de Tecnologia Química e Biológica António Xavier
RESEARCH, TEACHING, or OTHER INTERESTS
Biotechnology, Biochemistry
Scopus Publications
Scholar Citations
Scholar h-index
Scholar i10-index
Scopus Publications
Mario De Simone, Lur Alonso-Cotchico, Maria Fátima Lucas, Vânia Brissos, and Lígia O. Martins
Elsevier BV
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, and Marco W. Fraaije
Wiley
AbstractVanillin 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.
Mario De Simone, Laura Alvigini, Lur Alonso-Cotchico, Vânia Brissos, Jonatan Caroli, Maria Fátima Lucas, Emanuele Monza, Eduardo Pinho Melo, Andrea Mattevi, and Lígia O. Martins
American Chemical Society (ACS)
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.
Luca Zuliani, Annabel Serpico, Mario De Simone, Nicola Frison, and Salvatore Fusco
MDPI AG
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.