Selective Modifications of ARomatics through Biocatalytic Oxidations

Project Information

SMARTBOX

Grant agreement ID: 837890

DOI

10.3030/837890

Project closed

EC signature date 26 April 2019

Start date 1 May 2019

End date 31 October 2023

Funded under

• INDUSTRIAL LEADERSHIP - Leadership in enabling and industrial technologies – Biotechnology

Total cost

€ 4 520 895,83

EU contribution

€ 3 924 163,00

Coordinated by

BIO BASE EUROPE PILOT PLANT VZW
Belgium

Periodic Reporting for period 3 - SMARTBOX (Selective Modifications of ARomatics through Biocatalytic Oxidations)

Reporting period: 2022-05-01 to 2023-10-31

Summary of the context and overall objectives of the project

One of the objectives was to accelerate, simplify, and reduce the costs of computational enzyme engineering by using actively in the project the Framework for Rapid Enzyme Stabilization by Computational libraries” (FRESCO) by the University of Groningen and the ZYMVOLVER tool - In Silico Engineering of Oxidative Enzymes Applied to Lignin Revalorization Processes by Zymvol. Both tools were successfully applied as a computational method to improve thermostability and activity and has been continuously applied to further refine the mutant enzymes in terms of efficiency with excellent results. SMARTBOX has developed an advanced computational engineering platform specifically for oxidative enzymes, which can automatically screen for improved enzyme variants with minimal human intervention. This is achieved by implementing several innovations into current computational screening methods, most importantly machine learning, which allows to train the algorithms with experimental results. SMARTBOX has developed the one-enzyme conversion of HMF into FDCA. The bioconversion of HMF to FDCA using the HMF oxidase enzyme was successfully scaled up to TRL5 was achieved within Bio Base Europe Pilot Plant. The improvement of the production of FDCA from HMF was another key objective in the project. Following initial trials with chemically synthesized FDCA, the quality of the polymer achieved with the enzymatically produced FDCA was confirmed to be with comparative levels of purity to the standards and could potentially be used by AVA in the synthesis of PEF.
With the support of SMARTBOX a pilot scale 200L batch biorefinery reactor for reductive catalytic fractionation (RCF) for kg-scale lignin oil & pulp production could be commissioned having surmount lab-scale RCF process development, the design & construction of the basic RCF pilot reactor. KU Leuven with the Sels-Group can offer on pilot scale the reductive catalytic fractionation technology to selectively produce specific lignin monomers from lignocellulosic biomass.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

After a period of dedicated efforts, collaboration, and significant achievements, the Smartbox project has officially come to its end. SMARTBOX partners developed a strong joint collaboration and created an open atmosphere for fruitful discussion so, that progress and innovation on the enzyme development as biocatalysis and the upscaling process can take place. SMARTBOX is following its dissemination and exploitation strategy, by reaching out to the scientific and industrial community with its news section on the project website, posts in social media and attendance to many conferences and events. The in-silico FRESCO computational method was applied successfully by partners to several oxidative enzymes to improve the stability of these enzymes and increase the resistance to co-solvents. In -silico computational method (FRESCO) created more thermostable variants of oxidative enzymes and is now part of the Zymvol bioinformatic toolbox. Within the first reporting period the first application turning HMF into FDCA, the best candidates resulted from an HMFO mutant library generated by computational design (ZYMVOL) and was screened by RUG. The industrial production up to 150L scale of enzymatic FDCA via bioconversion and its following purification steps was performed successfully by BBEPP, making the bio-based building block available at high polymer grade purity and in significant quantities. In the meantime, further work has progressed on the enzymatic synthesis of FDCA and DFF from HMF.
The selective production of specific lignin monomers through RCF was successfully performed at lab-scale and the first intermediates were send to partners for enzymatic conversion experiments. At the end of the project the Biocon pilot facility was opened in December 2023 by KU Leuven and the SELS Group. In the last period 3 more publications were accepted within the enzyme development working group authored by UniPavia, RUG, ITQB, Zymvol, UniPavia, and 1 joint publication with the intermediates WorkPackage with KUL as its lead partner. All 9 publications can be found open access on the project website. The framework of the sustainability and risk assessment with a common basis for the evaluation of the investigated SMARTBOX technologies were defined, including (i) the goal and scope definition, value chain definition with a short description of the processes, identification of boundaries for the different assessments and (ii) the description of the methodologies used for the LCA/ TEA. 2 reports on analysis of 2 value chains within SMARTBOX could be delivered within the project lifetime. These are of confidential nature. In order to highlight some faces behind the SMARTBOX Work Packages and its results the idea was to set-up short videos in which the project partner explain their role in the project and mention their outcomes and SMARTBOX's efforts. The videos has then been released one by one on the SMARTBOX LinkedIn account.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The software FRESCO has been incorporated into the pipeline used at ZYMVOL and efforts are being made to improve the automation of the computational pipeline. The results show that the computational strategy is transversal to activity and stability as well as for different enzymes. These results confirm the potential of using this computational platform for the engineering of new enzymes in the future and especially to have a special-purpose engineering platform for oxidative enzymes. At the partner site new enzymes are being studied for exploitation and their introduction as new products in other ongoing collaborations or for future projects. Smartbox can state that the enzymatic production of FDCA via bioconversion at 150L scale and purification towards a purity higher than the benchmark chemical FDCA is the most significant technological achievement. This achievement can be labelled as a breakthrough step as before the start of the project, biocatalytic processes that converted HMF into FDCA had only been performed at lab-scale. The objective of producing PEF with FDCA obtained by enzymatic bioconversion is still relevant as it shows that bioconversion can be used to produce polymer grade FDCA which could provide scientific and technological breakthrough potential. The developed solvent extraction protocols have generated ‘simplified oil fractions’ which led to a beyond-the-state-of-the-art understanding of the structure of complex lignin molecules in the RCF oil. The results have been published in the high impact Journal of Chemical Sciences and will provide valuable information for both the scientific community as well as interested industry, for instance to link structural motives to specific product properties. Extensive collaboration and discussions with the engineering firm during basic design of the planned pilot-scale RCF reactor (reached TRL5) has led to new insights about the pending challenges of biorefinery reactor/process technology and scalability. This knowledge has been summarized in an opinion article in the Journal Industrial & Engineering Chemistry Research, to steer the activities of the scientific community around lignin and biorefinery research and development. SMARTBOX has provided a TEA, LCA and Natural and Social Capital Valuation (public available) assessment of 2 value chains investigated, from raw material extraction to related end-use products.