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ROBOX Report Summary

Project ID: 635734
Funded under: H2020-EU.2.1.4.

Periodic Reporting for period 1 - ROBOX (Expanding the industrial use of Robust Oxidative Biocatalysts for the conversion and production of alcohols (ROBOX))

Reporting period: 2015-04-01 to 2016-09-30

Summary of the context and overall objectives of the project

Oxygen functionalities are key functional groups in many chemicals and materials. The efficient introduction of oxygen-functionalities into raw materials are key chemical transformations in bulk and fine chemicals. Innovative biocatalytic oxidation routes using molecular oxygen (from air) under mild conditions such as ambient temperature, neutral pH and pressure can greatly improve the sustainability and economics of processes, but were so far mainly applied in the pharma segments. In this high-value segment, the high price of the end-product (> €1000/kg) justifies less than optimal enzyme production and limitations in its catalytic efficiency. In order to achieve the widening of industrial application of enzymatic biooxidation processes to also larger volume but lower price chemical markets, ROBOX will demonstrate the techno-economic viability of four types of robust oxidative enzymes catalysed biotransformations: P450 monooxygenases (P450s), Baeyer-Villiger MonoOxygenase (BVMOs), Alcohol DeHydrogenase (ADH) and Alcohol OXidase (AOX) for applications in pharma, nutrition, fine & specialty chemicals and materials applications. ROBOX will demonstrate 11 target reactions on large scale for these markets in order to prepare them for scale up to commercial-scale plants.

Societal Importance:
The potential economic impact of the ROBOX platform can be considerable as the European chemical industry accounts for €558 billion turnover, of which 25 % is generated in specialty chemicals, 20% in polymers and 28% in petrochemicals (incl. fine chemicals). However, currently only 10% of the processes in the chemical industry are estimated to be biotechnological processes, very important barriers being the robustness of the enzymes and low required cost price of the products. By addressing oxidation reactions for molecules which are relevant throughout a range of industries, the technology platform is relevant for each of the market segments addressed in ROBOX.
Since the introduction of oxidative enzymes in new market segments brings along both environmental and cost benefits, the importance of applying these enzymes is paramount. The demonstrations will take place in a broad range of processes in pharma (APIs, API metabolites), nutrition (vitamins), fine and specialty chemicals (flavour & fragrance) and materials (monomers, polymers, dispersions, pigments). The technology platform to be developed and validated in this broad range of industries therefore has ample relevance.

Overall Objectives:
The ROBOX consortium aims to make biocatalytic alternatives, based on probably the three most important types of enzymatic oxidations (oxygen introduction into C-H and C-C bonds and alcohol oxidation) with reduced environmental footprint. This will be done by the development and demonstration of industrially relevant reactions catalyzed by representatives of four different types of oxidative enzymes, while concomitantly consolidating the applied experimental and computational methods into generic platform technologies for enzyme optimization, production and application in a biocatalytic process. This approach will enable the design of highly efficient biocatalysts with an adapted range of substrates of immediate relevance for industrially important conversions.
1: To identify and engineer new robust biooxidation enzymes which feature a higher robustness in terms of enzyme stability, activity and efficiency
2: To establish competitive fermentative (platform) enzyme production systems which feature robust strains, high enzyme yields and efficient enzyme extraction methods
3: To develop efficient biocatalytic processes and technology which optimize the performance of the enzyme during the reactions
4: To bridge the gap between lab and market introduction by means of pilot scale demonstrations of the biocatalytic processes based on the new robust enzymes
5: To perform a full process evaluation of the demonstrated biocatalytic processes and the technol

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

Objective 1:
For all four enzyme classes investigated within ROBOX suitable and highly promising candidates for the application in the targeted demonstration cases have been identified either in literature and databases or in pre-existing enzymes or enzyme variant collections of the consortium members.
Objective 2:
The three platform enzyme production organisms for the four oxidative enzyme classes selected are in general Myceliophthora thermophila C1 (hyphal fungus) for oxidases, Pichia pastoris (yeast) for human and other eukaryotic P450s, and Escherichia coli (bacterium) for BVMOs, ADHs, some oxidases and most P450s.
Objective 3:
Significant progress for the ROBOX specific target reactions could be achieved. Furthermore the immobilisation of several AOXs, ADHs, BVMOs and even for the notoriously challenging P450s already succeeded applying various immobilization strategies. Investigations into reactor design for efficient oxidation reactions using molecular oxygen were initiated.
Objective 4:
The work on this objective has start only recently, but the hydroxylation of the API diclofenac was scaled up in two consecutive 100 L reactions in the pilot plant of DSM. Using oxygen from pressurized air as oxidant approximately 600 g of 4’-OH- and 5-OH-diclofenac (7:3 ratio), as they are also produced by human P450s in the human body, were produced with a P450-BM3 variant, which had been produced in an up-scaled batch fermentation.
Objective 5:
A methodology for characterizing and evaluating a biocatalytic process has been developed, describing six key process performance metrics as well as experimental lab work that is needed to determine these metrics. Additionally, the process performance required for economic viability was identified by the industry partners and formulated as targets for key process performance metrics.

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 project has managed to attain significant initial results and has progressed beyond the state-of-the-art on all Work Package levels:
An initial set of new and partially already robust enzymes have been added to already existing collection for all target enzyme classes. For the first time robust BVMOs with activity on (macro-)cyclic were identified.
The large scale production of these enzymes has so far been confirmed towards a full-scale production systems. Especially efficient production systems for P450s, BVMOs and Oxidases are now available, while they were already available for ADHs.
Initial process design and validation already resulted in efficient hydroxylation for API modification and alcohol oxidation with ADHs.
The kg scale hydroxylation of diclofenac was demonstrated on 100 l scale is unprecedented to the best of our knowledge. This offers the market introduction of the process which could result in cheaper diclofenac metabolites and spin-offs to other API metabolites.
The potential economic and ecological benefits are evaluated and compared with present conventional chemical oxidation using new benchmark tools.
Based on the current, preliminary results, the market volume addressed within ROBOX (a joint volume of over 6.000 ktons/year) is still a relevant number. This also applies to substantial reductions in cost (up to -50%), energy use (-60%), chemicals (-16%) and GHG-emissions (-50%) which the biooxidations are expected to bring.

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