H2-driven enzymatic cascades for the biosynthesis of N-heterocycles
Metabolism, Biochemistry and Genetics of Microorganisms
Final Report Abstract
Heterocycles containing nitrogen are fundamental building blocks for various agrochemicals, biologically active substances, and pharmaceuticals. The best-known simple N-heterocyclic compounds are pyrrolidines, piperidines, and piperazines, which are abundant in many natural products and pharmaceutically active compounds. In the course of this project, the application of putrescine oxidase from Rhodococcus erythropolis in an enzyme cascade and its development towards more strongly substituted diamond substrates was investigated. The first experiments showed that a high oxidation activity occurred with the natural substrate putrescine, while the activity of the enzyme was strongly reduced towards cadaverine and its methyl-substituted derivatives. Based on this initial assessment, the putrescine oxidase was optimized by enzyme engineering, and variants with increased activity for longer and substituted diamine substrates were identified. The combination of these variants with an imine reductase from Streptosporangium roseum in an enzyme cascade provided access to enantiomerically enriched, substituted N-heterocycles. The conversion of the methyl-substituted diamine substrate into the N-heterocyclic product was successfully upscaled from the shaking method to a 20 L bioreactor with increased substrate concentrations. The compatibility of putrescine oxidase and imine reductase allowed the development of a whole-cell system on the bioreactor scale under optimized reaction conditions. Our results show that this enzymatic cascade, which has been established as whole-cell biotransformation with living cells, is promising and can be applied on a large scale, opening the perspective for the production of high-quality piperidine compounds that might be difficult to obtain in organic chemistry. Next, we addressed the question of in-situ cofactor regeneration and evaluated the incorporation of an oxygen tolerant NAD+-reducing hydrogenase. Substituted pyrrolidines and piperidines were obtained with up to 97% product formation in an H2-driven one-pot reaction. Furthermore, a scalable bioelectrochemical flow system was developed to run this enzymatic cascade in vitro. H2 and O2 were generated by electrolysis and served as electron mediators for redox enzymes and ensured safe H2 conditions. Methylated N-heterocycles were generated from diamines with up to 99% product formation and label regioselectivity with deuterium of up to 99%. In addition, we evaluated the ability of the hydrogenase with respect to H2-driven flavin reduction and demonstrated efficient NAD(P)H-free regeneration for a number of flavindependent oxidoreductases. The possibility of using H2 as an energy carrier for flavin recycling opens up opportunities for highly efficient biocatalysis with high atomic efficiency and provides a versatile platform for future biotransformations and functionalization of N-heterocycles with flavin- and H2O2-dependent enzymes. Moreover, this project designed an alternative approach to C- and N-substituted piperazines using R-selective imine reductase from Myxococcus stipitatus. Biotransformations showed that a variety of novel piperazine products (75 C- and N-substituted piperazines) were produced from easily accessible 1,2-dicarbonyl and 1,2-diamine substrates. By engineering, the cofactor specificity of imine reductase from Myxococcus stipitatus could be modified. Two variants with a preference for the NADH cofactor and recovered catalytic activity were generated. This approach can be used to change the cofactor specificity from NADPH to NADH of all IREDs described above.
Publications
- „Recent advances in imine reductase-catalyzed reactions”, World J. Microbiol. Biotechnol. 2017, 33, 199
M. Lenz, N. Borlinghaus, L. Weinmann, B. M. Nestl
(See online at https://doi.org/10.1007/s11274-017-2365-8) - „Biocatalytic Access to Piperazines from Diamines and Dicarbonyls“ ACS Catal. 2018, 8; 3727-3732
N. Borlinghaus, S. Gergel, B. M. Nestl
(See online at https://doi.org/10.1021/acscatal.8b00291) - „Switching the Cofactor Specificity of an Imine Reductase“, ChemCatChem 2018, 10, 183-187
N. Borlinghaus, B. M. Nestl
(See online at https://doi.org/10.1002/cctc.201701194) - „Cascade Biotransformation to Access 3-Methylpiperidine in Whole Cells“, ChemCatChem 2019, 11, 5738-5742
N. Borlinghaus, L. Weinmann, F. Krimpzer, P. N. Scheller, A. Al-Shameri, L. Lauterbach, A. S. Coquel, C. Lattemann, B. Hauer, B. M. Nestl
(See online at https://doi.org/10.1002/cctc.201900702) - „Synthesis of N-heterocycles from diamines via H2-driven NADPH recycling in the presence of O2”, Green Chem. 2019, 21, 1396-1400
A. Al-Shameri, N. Borlinghaus, L. Weinmann, P. N. Scheller, L. Lauterbach, B. M. Nestl
(See online at https://doi.org/10.1039/c8gc03798a) - „Powering Artificial Enzymatic Cascades with Electrical Energy”, Angew. Chem. Int. Ed. 2020
A. Al-Shameri, M.-C. Petrich, K.-j. Puring, U.-P. Apfel, B. M. Nestl, L. Lauterbach
(See online at https://doi.org/10.1002/anie.202001302) - „Preparation of Imine Reductases at 15 L Scale and Their Application in Asymmetric Piperazine Synthesis” in Practical Methods for Biocatalysis and Biotransformations 4. Editors John Whittall and Peter W. Sutton 2020, John Wiley & Sons, Ltd
N. Borlinghaus, S. Gergel, B. M. Nestl, F. Thol, H. Penders
(See online at https://doi.org/10.1002/9781119487043.ch3)
