TUNGSTEN BIOCATALYSIS – HEAVY METAL ENZYMES FOR SUSTAINABLE INDUSTRIAL BIOCATALYSIS

Project Information

W-BioCat

Grant agreement ID: 101129798

DOI

10.3030/101129798

EC signature date 29 September 2023

Start date 1 February 2024

End date 31 January 2028

Funded under

• The European Innovation Council (EIC)

Total cost

€ 2 922 332,25

EU contribution

€ 2 922 332,25

Coordinated by

TECHNISCHE UNIVERSITEIT DELFT
Netherlands

Periodic Reporting for period 1 - W-BioCat (TUNGSTEN BIOCATALYSIS – HEAVY METAL ENZYMES FOR SUSTAINABLE INDUSTRIAL BIOCATALYSIS)

Reporting period: 2024-02-01 to 2025-01-31

Summary of the context and overall objectives of the project

Europe needs a sustainable chemical industry which will only be realized by new breakthrough technologies. Industrial biotechnology is established in chemical manufacturing, offering more efficient, more specific, safer and less energy demanding production, but is held back by the limited number of enzyme classes in industrial use. This project opens up an important new enzyme class of tungsten-containing enzymes (W-enzymes) which catalyse amazing chemical reactions involving challenging low redox potential reduction reactions, but are currently impossible to obtain economically and on scale to match industrial needs. We need to produce W-enzymes using an industrial workhorse micro-organism such as E. coli. Yet, we discovered that W-cofactor biosynthesis is the bottleneck preventing successful production of W-enzymes in E. coli. W-BioCat project can solve this challenge by using cutting-edge computational enzyme design approaches we recently developed, to create a completely new W-cofactor biosynthesis pathway for E. coli. The WBioCat strains developed in this project will enable expression of new W-enzymes from genetic databases, and facilitate production of new engineered W-enzymes. The catalytic potential of these new W-enzymes will be established and implemented in new processes. Exciting new reaction scope in biocatalytic CO2 reduction to valuable chemicals and Birch reduction of aromatic compounds will be explored, alongside the already-established and broadly applicable carboxylic acid reductions. W-BioCat will be the breakthrough to make W-enzymes accessible for industry. As a proof of concept, a hydrogen-driven process to convert plant-derived oleic acid to the emollient ester oleyl oleate will be created. Oleyl oleate is used in many cosmetic products used daily by millions of people. This process will be demonstrated in multi-gram yield in scalable, industrially-relevant hydrogenation reactors, together with market research to address a pathway to commercialisation.

Objective 1. Debottleneck W-enzyme expression
Objective 2. Explore and establish the biocatalytic scope of W-enzymes
Objective 3. Discover and produce new natural and engineered W-enzymes
Objective 4. Develop hydrogen based biocatalytic process for the reduction of fatty acids to high value aldehydes and alcohols

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

Within WP1, in silico designs of Pfu AOR, the tungsten uptake system WtpABC and the W-cofactor biosynthesis enzymes MoeA1, MoeA2 and MoaB (involved in metal insertion) have been generated. In addition, constructs for co-expression of different combinations the designs of the AOR and selected W-cofactor biosynthesis enzymes have been produced. In parallel different WT Pfu AOR and Methanococcus maripaludis GAPOR expression systems have been prepared and tested for cofactor incorporation and activity. PfAOR was successfully expressed as a soluble and thermostable enzyme, which could be isolated after a single heat step and buffer exchange and showing a low crotonaldehyde oxidation activity. The expression of WT PfAOR has been the main focus up to now because of these promising preliminary results.

WP2 activities focused on exploring the scope of W-AORs from Pyrococcus furiosus. The production and isolation of W-AORs from Pyrococcus furiosus has been successfully completed. In addition, the chemical analysis methods (based on GC) for the simultaneous quantitative detection of the aldehyde, carboxylic acid and alcohol substrate/product samples were successfully developed. Preliminary results for acid reduction and aldehyde oxidation were obtained with P. furiosus whole cells, cell extract and purified AOR.

Within WP3, a new protocol for the identification of tungsten sites in protein sequences has been developed. In addition, it has been used to analyze the tungsten protein content of 9089 bacterial, 303 archaeal and 2770 eukaryotic organisms. A set of tungsten proteomes has been obtained which constitutes the Deliverable D3.1 of the project.

WP4 activities have been delayed and the experimental work has recently started. Therefore there are no achievements to report.

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)

A PROSS designed W-cofactor biosynthesis pathway was produced, which is beyond what has been done so far using this approach as it normally focuses on a single enzyme only. The resulting designs will have to be validated experimentally.

Recombinant PfAOR with at least some functionality was produced in a particular E. coli strain, which is beyond the current state of the art as until now no functional W-AOR expression in this organism was reported. The functionality of the recombinant AOR can still be improved by a factor of 100, based on the activity of the native enzyme.

The set of predicted tungsten proteomes delivered in D3.1 is per se an advancement of knowledge in the field of bioinorganic chemistry, as it provides an unprecedented view of the biodiversity of W-AOR enzymes, and of W-proteins in general. In addition, it represents a key achievement towards the pursuit of further results of the project, as it will drive protein engineering approaches aimed at producing W-AORs with novel, optimized catalytic properties.

Structure prediction of P. furiosus GAPOR, an AOR family enzyme with a strict substrate selectivity, was not possible until recently, however using AlphaFold 3 we obtained good quality predicted structures including the cofactor and substrate. These promising results will be used to speculate on structural basis of substrate selectivity in AOR family enzymes and to improve the experimental structural characterization for this enzyme.

W-BioCat overview: making W-enzymes accessible for a sustainable chemical industry