Periodic Reporting for period 1 - Monopoly (Baeyer Villiger Monooxygenases as Biocatalytic Parts for Monomers of New Lactone-based Polymeric Materials)
Berichtszeitraum: 2019-07-15 bis 2021-07-14
Baeyer-Villiger monooxygenases (BVMOs) are flavoenzymes and belong to the class of oxidoreductases. They catalyse the oxidation of linear, cyclic and aromatic ketones to the corresponding esters or lactones, highly similar to the chemical Baeyer-Villiger oxidation. During the enzymatic oxidation, one atom of molecular oxygen is incorporated into a carbon-carbon bond of a non-activated ketone. Lactones can be produced biocatalytically using Baeyer–Villiger monooxygenases (BVMOs), which are members of a diverse class of flavoprotein monooxygenases. The use of protecting groups and formation of by-products can be avoided in enzymatic processes, simplifying synthesis. BVMOs can show excellent stereoselectivity, and react under relatively mild conditions. Oxidation of ketones by BVMOs, however, is often regio-divergent, generating two lactone products with some substrates. The isomers are produced via migration of the nucleophilic carbon centre, to form the ‘normal’ and ‘abnormal’ isomer, respectively. In Monopoly, we aimed to gain a deeper understanding from a structural/engineering perspective of enzyme catalysis in relation to lactone production by new members of the BVMO family. The overall aim of this Marie Curie CAR fellowship programme is therefore to exploit enzyme kinetics, biocatalysis, structure determination, biophysical analysis and modelling as enabling disciplines to build a tool kit of novel Bayer Villiger monooxygenases as part of a self-consistent Design-Build-Test cycle.
Biomaterials composed of lactone monomers have been used to produce polyurethanes. For example, the synthesis of poly--caprolactones takes place via ring-opening polymerisation (ROP) of a monomeric -caprolactones. Additionally, ketal lactones (oxo-carboxylic ketals) are useful in the production of surfactants, plasticisers, solvents and polymers. Monopoly will lead to the development of new lactone monomers from which new polymers could be generated with improved properties and novel applications, especially in the healthcare arena. Biodegradable poly-lactone materials derived from petrochemical routes are already used widely in industry (e.g. speciality polyurethanes, additives, plasticisers), dentistry (as Resilon composites in splints and fillings) and healthcare (e.g. scaffolds for tissue engineering, drug delivery, bio-adhesives and orthopaedic castings). The sustainable production of monomers for these and related polymers is likely to impact in these areas, especially in the healthcare and dentistry markets where high quality/low volume products are sought.
Biomaterials composed of lactone monomers have been used to produce polyurethanes. For example, the synthesis of poly--caprolactones takes place via ring-opening polymerisation (ROP) of a monomeric -caprolactones. Additionally, ketal lactones (oxo-carboxylic ketals) are useful in the production of surfactants, plasticisers, solvents and polymers. Monopoly will lead to the development of new lactone monomers from which new polymers could be generated with improved properties and novel applications, especially in the healthcare arena. Biodegradable poly-lactone materials derived from petrochemical routes are already used widely in industry (e.g. speciality polyurethanes, additives, plasticisers), dentistry (as Resilon composites in splints and fillings) and healthcare (e.g. scaffolds for tissue engineering, drug delivery, bio-adhesives and orthopaedic castings). The sustainable production of monomers for these and related polymers is likely to impact in these areas, especially in the healthcare and dentistry markets where high quality/low volume products are sought.
1. Biocatalyst identification:
BVMO were chosen based on their product formation, substrate range, and expression and purification details availability. The following constructs gave very good expression and soluble protein production: Brevibacterium sp.HCU
(CHMO B1), Rhodococcus jostii (strain RHA1), (BVMO RJ), Rhodococcus pyridinivorans (BVMO RP), Comamonas sp. (strain NCIMB 9872) (CPMO C), Pseudomonas sp. HI-70 (CPDMO), Rhodococcus sp. Phi1 (CHMO R), Dietzia sp. (strain D5)
(BVMO D4).
2. BVMO expression and purification:
His trap NINTA AKTA chromatography and gel filtration was used to purify BVMOs. The protein was very pure after gel filtration. After purification, crystal screens were set up and analytical biotransformations were done.
3. Photocatalysis experiments: Photocatalysis has recently found its way into biocatalysis as a mild, cheap, and environmentally benign method for the production and/or recycling of cofactors in situ. Recent studies have revealed that enzymes harboring natural cofactors with photosensitizing properties trigger unprecedented reactivities when irradiated with light (mostly oxidoreductases). Photochemistry of flavins: Participation of these compounds in photochemistry and photobiology processes is of particular importance in the fields of biology, chemistry, and medicine. The idea was to subject different BVMOs to photocatalysis and check the product formation by GC/MS. Different BVMOS were used for photocatalysis experiments under different conditions like aerobic, anaerobic, different substrates, range of time period for the reaction to take place, normal day light, and blue light. Outcome of the photocatalysis experiment above:
- We observed that very little amount of caprolactone was formed in photocatalysis as compared to BVMO reference reaction.
- There was no unique product formation under the effect of light, further conditions need to be explored for new lactone formation.
Product formation was assayed by GC-MS.
4. Wild type and mutant Cyclohexanone monooxygenase (CHMO) enzyme kinetics:
Previous work from our lab (Biochemistry 2018, 57, 1997−2008) has shown that wild type CHMO from Rhodococcus yields abnormal lactone and its triple mutant yields normal lactone in the presence of Dihydrocarvone (DHC) as substrate. We attempted to study the kinetics of wild type and mutant enzymes to study the details of differences in product formation. For CHMO wild type kinetics the enzyme concentration was 20 nm and Dihydrocarvone concentration was from 1 µm to 2 mm. Km was observed to be 0.263 mM which was comparable to other BVMOs as published on BRENDA. For CHMO mutant kinetics the enzyme concentration was 0.5 µM and substrate dihydrocarvone concentration was from 50 µm to 10 mm. Km was calculated to be 1.860 mM. Further stopped-flow experiments need to be done to fully understand the mechanism of different product formations.
BVMO were chosen based on their product formation, substrate range, and expression and purification details availability. The following constructs gave very good expression and soluble protein production: Brevibacterium sp.HCU
(CHMO B1), Rhodococcus jostii (strain RHA1), (BVMO RJ), Rhodococcus pyridinivorans (BVMO RP), Comamonas sp. (strain NCIMB 9872) (CPMO C), Pseudomonas sp. HI-70 (CPDMO), Rhodococcus sp. Phi1 (CHMO R), Dietzia sp. (strain D5)
(BVMO D4).
2. BVMO expression and purification:
His trap NINTA AKTA chromatography and gel filtration was used to purify BVMOs. The protein was very pure after gel filtration. After purification, crystal screens were set up and analytical biotransformations were done.
3. Photocatalysis experiments: Photocatalysis has recently found its way into biocatalysis as a mild, cheap, and environmentally benign method for the production and/or recycling of cofactors in situ. Recent studies have revealed that enzymes harboring natural cofactors with photosensitizing properties trigger unprecedented reactivities when irradiated with light (mostly oxidoreductases). Photochemistry of flavins: Participation of these compounds in photochemistry and photobiology processes is of particular importance in the fields of biology, chemistry, and medicine. The idea was to subject different BVMOs to photocatalysis and check the product formation by GC/MS. Different BVMOS were used for photocatalysis experiments under different conditions like aerobic, anaerobic, different substrates, range of time period for the reaction to take place, normal day light, and blue light. Outcome of the photocatalysis experiment above:
- We observed that very little amount of caprolactone was formed in photocatalysis as compared to BVMO reference reaction.
- There was no unique product formation under the effect of light, further conditions need to be explored for new lactone formation.
Product formation was assayed by GC-MS.
4. Wild type and mutant Cyclohexanone monooxygenase (CHMO) enzyme kinetics:
Previous work from our lab (Biochemistry 2018, 57, 1997−2008) has shown that wild type CHMO from Rhodococcus yields abnormal lactone and its triple mutant yields normal lactone in the presence of Dihydrocarvone (DHC) as substrate. We attempted to study the kinetics of wild type and mutant enzymes to study the details of differences in product formation. For CHMO wild type kinetics the enzyme concentration was 20 nm and Dihydrocarvone concentration was from 1 µm to 2 mm. Km was observed to be 0.263 mM which was comparable to other BVMOs as published on BRENDA. For CHMO mutant kinetics the enzyme concentration was 0.5 µM and substrate dihydrocarvone concentration was from 50 µm to 10 mm. Km was calculated to be 1.860 mM. Further stopped-flow experiments need to be done to fully understand the mechanism of different product formations.
Lactones can be produced biocatalytically using Baeyer–Villiger monooxygenases (BVMOs), which are members of a diverse class of flavoprotein monooxygenases. The use of protecting groups and formation of by-products can be avoided in enzymatic processes, simplifying synthesis. BVMOs can show excellent stereoselectivity, and react under relatively mild conditions. Oxidation of ketones by BVMOs, however, is often regio-divergent, generating two lactone products with some substrates. The isomers are produced via migration of the nucleophilic carbon centre, to form the ‘normal’ and ‘abnormal’ isomer, respectively. There is interest in using polymenthide and polycarvomenthide as renewable thermoplastic elastomers and components of pressure sensitive adhesives. However there is currently lack of a comprehensive repository of BVMO enzymes suitable for building a number of production pathways to valuable polymers based on lactones.
By the end of this project we identified six BVMOs, expressed and purified them. Kinetic studies and photocatalysis experiments were done to get insights into normal, abnormal, and other product formation. Future reserach plan on these biocatalysts will be to integrate these into existing terpene production strains available in the host laboratory to engineer improved microbial strains that produce lactone based monomers sustainably (fermentation approach), or to use them to drive whole cell biotransformation reactions (expanding chemical diversity by substrate feeding).
By the end of this project we identified six BVMOs, expressed and purified them. Kinetic studies and photocatalysis experiments were done to get insights into normal, abnormal, and other product formation. Future reserach plan on these biocatalysts will be to integrate these into existing terpene production strains available in the host laboratory to engineer improved microbial strains that produce lactone based monomers sustainably (fermentation approach), or to use them to drive whole cell biotransformation reactions (expanding chemical diversity by substrate feeding).