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Periodic Report Summary 2 - BIOOX (Developing a validated technology platform for the application of oxygen dependent enzymes in synthesis and transformation of alcohols)

Project Context and Objectives:
BIOOX brings together a consortium of academic and industrial excellence to address the challenges of developing new technologies to fundamentally change chemical manufacturing in Europe and beyond. This collaborative project draws 11 partners in 6 European countries from academia, innovative SMEs, and major industrial partners supplying chemical markets worldwide. BIOOX will explore technology development from enzyme discovery and optimisation, through process development and scale-up, to product evaluation. BIOOX aims to develop and validate a technology platform providing industrial biocatalysts for transformation of industrially relevant compounds in viable process applications.

Oxidations are amongst the most important reactions in industrial chemistry, used in the production of fine and bulk chemicals, consumer products, and treatment of waste streams. However, chemical oxidations are both hazardous and have high environmental impacts. Biocatalytic oxidations are well studied in academic laboratories but few have been applied on an industrial scale. Biooxidations are characterised by relatively benign reaction conditions and exquisite selectivity, often achieving transformations of cheap and accessible precursors which are difficult or impossible via traditional chemistry. Due to inherent difficulties associated with large scale chemical oxidation methods, robust ODEs have great potential for application in industrial production of both bulk and high-value chemicals.

BIOOX incorporates a fully integrated project structure in order to ensure that the process requirements are considered from the earliest stage of the project. This integration enables efficient translation of research expertise into scalable process development, with outputs that can be transferred directly to industrial partners for evaluation. The project is split into 7 research Work Packages (WPs) in order to provide the necessary focus to address the challenges: WPs 1-4 focus on development of suitable biocatalysts; WPs 5-7 on the associated process technologies and metrics.

WP1 is focused on determining target reactions; identifying new enzymes through analysis of bioinformatics databases; screening and characterizing ODEs for application in oxidation reactions; creating engineered strains to analyse the application of model enzymes for in vivo production.

WP2 covers enzyme engineering for improvement and optimization of activity towards target substrates: 1) development of bioinformatics tools to identify mutation hotspots; 2) selection of target substrates for oxidations; 3) creation and screening of ODE variant libraries; 4) creation of engineered strains for substrate and enzyme production.

WP3 is for development and delivery of whole cell biocatalysts for syntheses of hydroxylated fatty acids, alkenes and oxyfunctionalised terpenes: 1) development of robust and selective oxidising enzymes in whole cells; 2) fermentation strains suitable for application; 3) optimization of fermentation expression and production conditions.

WP4 will develop immobilized enzymes and enzyme formulations with improved stability, applicability and recyclability for ODEs from WP1-3. To develop economical and environmentally-friendly scale up, WP4 will input to WP5 and 6.

WP5 is focused on evaluation of reactor configurations and oxygen feeding strategies, and how these influence stability and kinetics of ODEs. Evaluation will be based on isolated enzymes, immobilized enzymes, and whole-cell biocatalysts.

WP6 is for scalable demonstration of technologies developing in other WPs, including production of demonstration scale batches of formulated enzymes, and testing of BIOOX synthesis and transformation technologies at relevant scales.

WP7 is focussed on economic and environmental process evaluation, developing economic and environmental models for evaluation of oxidase bioprocesses.

Project Results:
WP1: A key early output was a Vision for Success, which defined BIOOX enzyme targets, productivity, yields, and reactor types for the selected oxidations. A tool for automated mining of the patent literature has been developed, which will allow rapid identification of patented technologies with relevance to BIOOX. New enzyme discovery by bioinformatics approaches, as well as studies of in-house enzymes, have so far allowed screening of hundreds of ODEs for activity towards substrates identified by the industrial partners, as well as the auxiliary enzymes for cofactor recycling. New metagenomic technologies have allowed the identification and production of large panels of completely novel enzymes, including new classes of activity, with freedom to operate.

WP2: Bioinformatics databases were created for ODEs in several superfamilies, a range of substrates were chosen as oxidation targets, and libraries of enzyme variants have been created and screened. As a result of these activities, a highly productive ODE system for production of an industrially relevant target has been identified, and will be developed for scaled demonstration in other WPs. Other industrial ODE systems are being developed by a combination of enzyme and strain engineering. A modular fungal expression vector has been developed to allow FTO high-yield, industrial protein production in Aspergillus.

WP3: Protocols were established for stable, reliable, and reproducible expression of ODEs in E. coli whole cells. The whole cell biocatalysts were further optimised by strain engineering, and tested towards relevant oxidation reactions. A reliable and optimized “fermenter in a flask system” for high-density bacterial liquid cultivation was developed for production of ODEs in BIOOX. A prototype engineered strain was developed for production of valuable flavours and fragrances from cheap starting materials. Other industrially relevant biotransformations using ODEs were established.

WP4: Enzyme immobilization technologies were designed and developed for use in innovative reactor configurations and down-stream processing to economically recover products from scaled processes. Two technologies have been developed which can help to overcome process limitations when applying the ODEs developed in WP1-3. Immobilized biocatalysts will be developed for use in novel flow reactors, enabling comparison studies with other reactor technologies.

WP5: Suitable reactor configuration options were identified for experimental evaluation in batch and continuous flow reactors, and three oxygen supply strategies were considered: bubble aeration; membrane assisted aeration; oxygen supply by H2O2 decomposition. These systems were used to study effects of reactor configurations and oxygen supply on biocatalyst operational stability and reaction rates, allowing design of appropriate systems for the application of BIOOX ODEs. These data form inputs to WP6 and 7. A flowsheet has been developed for the ODE system identified for demonstration in WP2. Cofactor regeneration alternatives for oxidation of alcohols have been further investigated. Flowsheets based on the reactor configurations evaluated in WP5, result in process models that allow comparison in WP7 of costs and environmental impact of process alternatives, and enable identification of process bottlenecks that can be addressed in WP6.

WP8 focusses on management of IP, publications, dissemination and engagement to wider audiences including the chemical industry, scientific community, and general public. BIOOX aims to increase uptake of biotechnology, and also to enhance public understanding of biotechnology, and particularly biocatalysis for production of chemical products. The two editions of the BIOOX Newsletter have been distributed to a range of stakeholders, and a webinar on the discovery and production of ODEs was made available via the project website. The BIOOX project has been presented at conferences and public outreach events.

Potential Impact:
The Consortium brings together a wealth of experience in pan-European collaborative research, and many were coordinators or partners in other major EU projects. Their further work in BIOOX will build upon that expertise, and represents and ongoing commitment to maintain the EU IB Community as world leaders in innovation and research.

The technologies developed in BIOOX will have applications in diverse markets, including chemicals and intermediates, biopolymers, consumer products, and flavours and fragrances. With an emphasis on quality by design, these applications must exceed the environmental, economic, and product performance of traditional methodologies, in order to increase technology uptake by European industry. By improving the efficiency, costs, and safety profiles of manufacturing processes and expanding the product portfolio produced by European industries, BIOOX will help to secure jobs in the sector and boost the contribution of chemical manufacturing to the economy of the region.

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