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The design and development of efficient biocatalytic cascades and biosynthetic pathways for the sustainable production of amines

Periodic Reporting for period 4 - BIOSUSAMIN (The design and development of efficient biocatalytic cascades and biosynthetic pathways for the sustainable production of amines)

Reporting period: 2018-09-01 to 2020-10-31

The main aim of this project is to design and develop efficient biocatalytic cascades using purified enzymes in vitro, and develop artificial biosynthetic pathways in whole cells microbial organisms. The targets of the biocatalytic pathways are amine products (chiral and non-chiral) that are of particular interest for the chemical industry. We introduce the amine functionality in the pivotal catalytic step of the artificial biocatalytic pathways using a new class of enzymes, namely the amine dehydrogenases (AmDHs). Notably, natural “true” AmDHs were not known in nature at the time of this proposal and therefore scientists had to engineer them in laboratory. Only two AmDHs were generated before the start of this project and they displayed a limited and similar substrate scope. Therefore, the BioSusAmin project aims at extending the tool-box of amine dehydrogenases as biocatalysts for the asymmetric reductive amination of ketones. In particular, we focus on the protein engineering of AmDHs that are active on the specific chemical targets of the designed biocatalytic cascades. Additionally, screening for more diverse substrates is being carried out. We investigate alternative scaffolds and alternative strategies to obtain diverse AmDHs. Finally, we will apply our new AmDHs in combination with other enzymes such as alcohol dehydrogenases, oxidases, alkane monooxygenases, etc., to deliver variously functionalised amines and derivatives as final products with elevated yields, perfect chemo- regio- and stereoselectivity, enhanced atom efficiency and minimum environmental impact. Such an approach will be realised through the design of new pathways that will convert inexpensive starting materials from renewable resources, encompassing the internal recycling of redox equivalents, the use of inorganic ammonia as nitrogen source and, if necessary, only molecular oxygen as the innocuous additional oxidant. Water will be the sole by-product. In summary, this project contributes to develop sustainable synthesis of high-value amines that are important for the bulk chemicals, pharmaceutical and fine chemicals industries.
We developed a hydrogen-borrowing biocatalytic cascade for the direct conversion of alcohols into enantiopure amines. The biocatalytic method possesses the highest possible atom efficiency because it requires ammonia as the nitrogen source and generates water as the sole by-product. The dual-enzyme hydrogen-borrowing amination can give direct access to highly valuable enantiopure amines that constitute the active core of a large number of pharmaceuticals and fine chemical. The results were published in the prestigious journal Science, (DOI: 10.1126/science.aac9283) and a world patent has been granted (WO 2016001362). A second-generation hydrogen-borrowing amination process was also developed (published in ChemCatChem; DOI: 10.1002/cctc.201701366). Furthermore, we have developed a method for the amination of racemic alcohols through a five-enzyme biocatalytic network operating in two concurrent and orthogonal cycles (published in Organic & Biomolecular Chemistry DOI: 10.1039/C7OB01927K). On the same line, we have developed a method for the amination of alcohols using alcohol oxidases (AOxs) and AmDHs. Interestingly, the same AOx could also be used for the development of a method for the direct conversion of alcohols into nitriles. This latter work was published in Angew. Chem Int. Ed. (DOI: 10.1002/anie.201809411) and a world patent was granted (WO 2020020844). Finally, we have implemented and extended the bioamination methodologies to the modular biocatalytic synthesis of polyamide monomers both in vitro and in vivo. As an example, different types of ADHs and AmDHs were co-expressed in a balanced way in E. coli and the in engineered strains were applied for the asymmetric amination of alcohols (work published in Green Chem. DOI:10.1039/C9GC01059A).
One of the major limitations was the relatively narrow substrate scope of the known amine dehydrogenases. Thus, the BioSusAmin team has studied the properties of the available amine dehydrogenases and their substrate scope for the reductive amination of a panel of structurally diverse prochiral ketones was elucidated. This initial work was published in Green Chemistry (IF 9.125; DOI: 10.1039/C6GC01987K). Later, the BioSusAmin project generated a tool-box of amine dehydrogenase that complements the enzymes previously available. The team applied the so called rational-guide enzyme evolution or semi-rational enzyme engineering. For example, the wild-type ε-lysine 6-dehydrogenase from Geobacillus s (Lys-EDH) was chosen as scaffold for the engineering of S-selective and R-selective AmDHs with expanded substrate scope. Our work delivered new AmDH that are active towards bulky-bulky and industrially relevant prochiral ketones. This work was published in Nat. Commun. DOI: 10.1038/s41467-019-11509-x and a world patent was granted. We also generate a second and further generation libraries of AmDHs to obtain new variants possessing dual ADH-AmDH activity. This led to the first example of “alcohol aminase” enzymes. These variants were applied in the one-enzyme hydrogen-borrowing amination of alcohols and the work was published in Chem. Eur J. DOI: 10.1002/chem.202003140. Finally, during our studies on amine dehydrogenases (AmDHs) for the synthesis of secondary and tertiary amines, we gained new insights into the catalytic mechanism of these enzymes (work published in ChemBioChem, 2019, 20, 800).
- The creation of new amine dehydrogenases and their development for organic synthesis has allowed for shortening conventional routes for the synthesis amines, and in particular α-chiral amines. One particularly important feature is that this method gives direct access to highly valuable enantiopure amines, which constitute the active core of a large number of pharmaceuticals and fine chemical.
- The develop of the first hydrogen-borrowing bioamination of alcohols (and other related methodologies) for the one-pot and concurrent enzymatic amination of alcohols.
- The introduction of these unnatural pathways into living system for performing the asymmetric bioamination of alcohols in vivo.
- A methodology for the redox-neutral conversion of inexpensive and available feedstock into polyamide monomers. The approach is flexible as it can be implemented in vitro using isolated enzymes as well as in vivo using engineered E. coli strains that harbour the genes for the required engineered enzymes.
- We have shown that alcohol dehydrogenase activity can be created from an amine dehydrogenase activity and vice versa. This novel approach will open up many new possibilities for engineering of amine dehydrogenases starting from structurally diverse alcohol dehydrogenases.
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