Periodic Reporting for period 1 - ROBUSTOO (Robust industrial biocatalysts with peroxygenase, phenol-oxidase or furfuryl-oxidase activities from bacterial and fungal hosts)
Reporting period: 2024-01-01 to 2025-06-30
High-level production, catalytic efficiency and robustness are main requirements for the implementation of enzyme biocatalysts in the industry. In ROBUSTOO we aim to demonstrate how such limitations can be overcome for three enzyme types with peroxygenase (unspecific peroxygenases, UPOs), phenol oxidase (laccases) or furfuryl oxidase (hydroxymethyl furfural oxidases, HMFOs) activities. The choice of oxidative enzymes is relevant from an industrial point of view, as there are few examples of oxidative enzymes widely available on the market compared to the numerous hydrolytic enzymes currently used in industrial processes.
Laccases are multicopper-oxidases well known for their ligninolytic activity. UPOs are heme-thiolate peroxygenases considered as dream biocatalysts for a plethora of oxygenation reactions difficult to obtain by chemical synthesis. HMFOs convert 5-hydroxymethylfurfural into 2,5-furandicarboxylic acid, a versatile building block for bioplastics, through a multi-step oxidation route.
The three enzymes are at different stages of research and development: numerous studies have reported the fundamentals and applications of laccases, while thousand members of the more recently-described UPOs and HMFOs await investigation. Nevertheless, all of them show great biotechnological potential as biocatalysts for the topical bio-based applications addressed in ROBUSTOO: the synthesis of oxygenated lipophilic ingredients and fine chemicals (by UPOs); the valorisation of industrial lignins into phenolic building blocks and polymers (by laccases); and the production of sugar-derived plastic building blocks (by HMFOs).
To solve the aforesaid hurdles for industrial deployment of these biocatalysts and demonstrate the technical and environmental feasibility of such biotransformations, we use computational, synthetic biology and protein engineering tools to:
1) Improve the applicability of laccases, UPOs and HMFOs by conferring robustness and superior activity/selectivity on the target substrates under industrial operating conditions.
2) Produce at pilot scale the best candidates as recombinant enzymes in customized microbial hosts.
3) Develop more efficient and sustainable processes to produce bio-based products of commercial interest.
Laccases are multicopper-oxidases well known for their ligninolytic activity. UPOs are heme-thiolate peroxygenases considered as dream biocatalysts for a plethora of oxygenation reactions difficult to obtain by chemical synthesis. HMFOs convert 5-hydroxymethylfurfural into 2,5-furandicarboxylic acid, a versatile building block for bioplastics, through a multi-step oxidation route.
The three enzymes are at different stages of research and development: numerous studies have reported the fundamentals and applications of laccases, while thousand members of the more recently-described UPOs and HMFOs await investigation. Nevertheless, all of them show great biotechnological potential as biocatalysts for the topical bio-based applications addressed in ROBUSTOO: the synthesis of oxygenated lipophilic ingredients and fine chemicals (by UPOs); the valorisation of industrial lignins into phenolic building blocks and polymers (by laccases); and the production of sugar-derived plastic building blocks (by HMFOs).
To solve the aforesaid hurdles for industrial deployment of these biocatalysts and demonstrate the technical and environmental feasibility of such biotransformations, we use computational, synthetic biology and protein engineering tools to:
1) Improve the applicability of laccases, UPOs and HMFOs by conferring robustness and superior activity/selectivity on the target substrates under industrial operating conditions.
2) Produce at pilot scale the best candidates as recombinant enzymes in customized microbial hosts.
3) Develop more efficient and sustainable processes to produce bio-based products of commercial interest.
The high-performance computational pipeline for enzyme bioprospecting and design has been specifically adapted to each type of enzyme using cutting-edge machine learning tools, structural modelling and molecular simulations, and feedback from experimental data. All this has enabled massive database searches to identify new enzymes with anticipated properties and predict beneficial mutations.
The development of microbial strains specifically tailored to produce each type of enzyme, combined with the optimization of culture conditions, the design of genetic constructs to enhance protein solubility, and the use of high-throughput techniques have provided a broad panel of novel recombinant oxidative enzymes, some of which are produced at high yields.
The biochemical characterization of the recombinant enzymes obtained so far, and their evaluation in target reactions served to recognize the best enzyme candidates and identify bottlenecks and engineering goals to be addressed in the next period.
For instance, laccase engineering has resulted in notable enhancements of enzyme robustness and activity or in new functionalities. Preliminary lignin oxidation tests have confirmed the stronger modification of the polymer by the new engineered laccases at the required process conditions. In addition, remarkable progress was made in the formulation of lignin-based phenolic resins as adhesives in wood panels.
To sum up, we have identified, expressed, characterized and/or engineered an exclusive assortment of new UPOs, HMFOs and laccases, several of which show strong potential as biocatalysts for the reactions aimed in the project.
The development of microbial strains specifically tailored to produce each type of enzyme, combined with the optimization of culture conditions, the design of genetic constructs to enhance protein solubility, and the use of high-throughput techniques have provided a broad panel of novel recombinant oxidative enzymes, some of which are produced at high yields.
The biochemical characterization of the recombinant enzymes obtained so far, and their evaluation in target reactions served to recognize the best enzyme candidates and identify bottlenecks and engineering goals to be addressed in the next period.
For instance, laccase engineering has resulted in notable enhancements of enzyme robustness and activity or in new functionalities. Preliminary lignin oxidation tests have confirmed the stronger modification of the polymer by the new engineered laccases at the required process conditions. In addition, remarkable progress was made in the formulation of lignin-based phenolic resins as adhesives in wood panels.
To sum up, we have identified, expressed, characterized and/or engineered an exclusive assortment of new UPOs, HMFOs and laccases, several of which show strong potential as biocatalysts for the reactions aimed in the project.
The outstanding computing facilities and front-line computational strategies used in ROBUSTOO have opened unique possibilities for the identification and design of unexplored enzymes with expected properties. As the collections of enzyme sequences and computational tools will be publicly available after the project, a broaden impact in computational biotechnology to develop other enzyme types or in drug discovery, can be expected.
The computational strategy has been reinforced with experimental enzyme engineering, obtaining promising candidates compared to reference enzymes. In addition, data from protein engineering (and laboratory validation of the newly discovered enzymes) is key for refining the computational pipeline.
Moreover, the different approaches adopted to improve enzyme expression not only pave the way for scaling up the production of a next-generation of recombinant oxidative enzymes, but the knowledge gained will also be available for other biotechnological goals in the fields of production strains and industrial enzymes in the long term.
In addition, the analytical techniques developed or fine-tuned during this period have allowed to test a wide range of oxidation and oxyfunctionalisation reactions and identify promising enzyme candidates. The progress made on the characterization of a wide range of UPOs and HMFOs, and their evaluation in target reactions, will have a significant impact on the understanding of the fundamentals and applicability prospects of these still largely unknown enzyme families.
Furthermore, we expect to prove the applicability of the new enzymes in the chemical and materials industry, which could be extended to other sectors in the long term due to their versatile oxidative capabilities.
The computational strategy has been reinforced with experimental enzyme engineering, obtaining promising candidates compared to reference enzymes. In addition, data from protein engineering (and laboratory validation of the newly discovered enzymes) is key for refining the computational pipeline.
Moreover, the different approaches adopted to improve enzyme expression not only pave the way for scaling up the production of a next-generation of recombinant oxidative enzymes, but the knowledge gained will also be available for other biotechnological goals in the fields of production strains and industrial enzymes in the long term.
In addition, the analytical techniques developed or fine-tuned during this period have allowed to test a wide range of oxidation and oxyfunctionalisation reactions and identify promising enzyme candidates. The progress made on the characterization of a wide range of UPOs and HMFOs, and their evaluation in target reactions, will have a significant impact on the understanding of the fundamentals and applicability prospects of these still largely unknown enzyme families.
Furthermore, we expect to prove the applicability of the new enzymes in the chemical and materials industry, which could be extended to other sectors in the long term due to their versatile oxidative capabilities.