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Selective Modifications of ARomatics through Biocatalytic Oxidations

Periodic Reporting for period 1 - SMARTBOX (Selective Modifications of ARomatics through Biocatalytic Oxidations)

Reporting period: 2019-05-01 to 2020-10-31

Although they have the potential to improve the economic and environmental sustainability of biorefineries, oxidative enzymes have not experienced a complete breakthrough yet in the biobased industries. This is mainly caused by the high cost and long time associated with traditional enzyme engineering methods such as directed evolution. 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. As this significantly improves computational predictability, the time and costs associated with oxidative enzyme engineering will be reduced 10-fold compared to state-of-the-art (SOTA) directed evolution methods.

Relying on the advanced engineering platform, SMARTBOX will develop the one-enzyme conversion of HMF into FDCA and intermediates, and the one-enzyme conversion of lignin monomers into a potential biobased building block for polycarbonates and vanillin. By adopting a one-enzyme FDCA production process, the associated production costs and carbon footprint are expected to decrease significantly compared to SOTA chemical oxidation methods. The unique feature of SMARTBOX is that reductive catalytic fractionation (RCF) will be used to selectively produce specific lignin monomers from biomass in near theoretical yields. The structural similarity of the resulting monomers with the SMARTBOX building blocks allows the development of high-yielding processes with only one enzyme. 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 first 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 for the intermediates development through reductive catalytic fractionation (RCF) by KUL ((i) on developed solvent extraction protocols that have generated ‘simplified oil fractions’ DOI: 10.1039/D0SC04182C) in the high impact Journal of Chemical Sciences, and (ii) on pending challenges of biorefinery reactor/process technology and scalability summarized in an opinion article ( and some more are in the pipeline for the enzyme engineering through the ‘Framework for Rapid Enzyme Stabilization by Computational libraries’ (FRESCO) and Zymvolver platform.

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. For 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.

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.
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 for ZYMVOL‘s portfolio is expected to have an impact on the company’s revenue.

At this point in the project, 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), as well as discussions with TRANSfarm, the host location of the pilot reactor, has led to new insights about the pending challenges of biorefinery reactor/process technology and scalability. This knowledge is used by KUL to steer its own research activities, but has also been summarized in an opinion article in the Journal Industrial & Engineering Chemistry Research, to steer the activities of the broader 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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