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Selective Modifications of ARomatics through Biocatalytic Oxidations

Periodic Reporting for period 2 - SMARTBOX (Selective Modifications of ARomatics through Biocatalytic Oxidations)

Reporting period: 2020-11-01 to 2022-04-30

SMARTBOX will develop 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.
O1. Accelerate, simplify, and reduce the costs of computational enzyme engineering: Framework for Rapid Enzyme Stabilization by Computational libraries” (FRESCO) was successfully applied as a computational method in order to improve thermostability and activity and has been continuously applied to further refine the mutant enzymes in terms of efficiency with excellent results. Relying on the advanced engineering platform, SMARTBOX developed the one-enzyme conversion of HMF into FDCA. Following initial trials with chemically synthesized FDCA, the quality of the polymer achieved with the enzymatically produced FDCA was confirmed to be the same.
O2: Improve the production of FDCA from HMF: FDCA had been obtained from HMF via the HMF oxidase enzyme at pilot scale with comparative levels of purity to the standards and could be used by AVA in the synthesis of PEF. The unique feature of SMARTBOX is that reductive catalytic fractionation (RCF) will be used to selectively produce specific lignin monomers from biomass. Due to the smart combination between oxidative biocatalysis and RCF, the production of added-value bio-aromatics will proceed with higher yields than the state of the art.
The SMARTBOX project being within the second half of its runtime, is following its objectives and has achieved some interesting results. 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, while 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.
There are 2 publications accepted within the enzyme development work package written by UniPavia, RUG, (Discovery, Biocatalytic Exploration and Structural Analysis of a 4-Ethylphenol Oxidase from Gulosibacter chungangensis) and the 2nd as joint article by ITQB, Zymvol, UniPavia (Rationally Guided Improvement of NOV1 Dioxygenase for the Conversion of Lignin-Derived Isoeugenol to Vanillin) (open access expected July 2023).
The intermediates WorkPackage with KUL as its lead partner has also published on their scientific work with a paper called “Identification and quantification of lignin monomers and oligomers from reductive catalytic fractionation of pine wood with GC × GC – FID/MS – Published 04 Dec. 2021”.
The in silico FRESCO computational method (Wiljma et al 2014) was applied successfully by partners (RUG/Zymvol) to several oxidative enzymes to improve the stability of these enzymes and increase the resistance to co-solvents. The in silico computational method (FRESCO) used to create more thermostable variants of oxidative enzymes is now part of the Zymvol bioinformatic toolbox. Within the first reporting period the first application turning HMF into FDCA the best candidates resulting from an HMFO mutant library generated by computational design (ZYMVOL) was screened by RUG. The industrial production up to 150L scale of enzymatic FDCA via bioconversion and its following purification steps was done successfully by BBEPP, making the bio-based building block available at high polymer grade purity and in bigger quantities. In the last 18 month further work has progressed on the enzymatic synthesis of FDCA and DFF from HMF. A refined HMFO has been delivered for the production of FDCA by partner RUG.
The selective production of specific lignin monomers through RCF is successfully done at lab-scale and first intermediates have been send to partners for enzymatic conversion experiments. The basic design of the prototype for the mini-pilot reactor to produce intermediates on kg scale has been finished.
The framework of the sustainability and risk assessment with a common basis for the evaluation of the investigated SMARTBOX technologies has been 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 preliminary analysis of 2 value chains within SMARTBOX could be delivered, the finalisation of this data is ongoing with latest inputs from partner.
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 different enzymes. These results confirm the potential of using this computational platform for the engineering of new enzymes in the future. The final goal is to have a special-purpose engineering platform for oxidative enzymes. At this stage of the project we can confirm that the computational tools are valuable to focus engineering of enzymes. On the other hand, these new enzymes are currently being studied for exploitation and their introduction as new products.
We 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 (needed to reach 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 will provide a TEA, LCA and Natural and Social Capital Valuation assessment of the four value chains investigated, from raw material extraction to related end-use products. The advantages in terms of sustainability, technology and economy will be compared with the current available state-of-the-art technology, providing a complete picture of the benefits of SMARTBOX.
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