Expression and activity of heterologous hydroxyisocaproate dehydrogenases in Synechocystis sp. PCC 6803 Δ hoxYH

Valentina Jurkaš; Christoph K Winkler; Silvan Poschenrieder; Paulo Oliveira; Catarina C Pacheco; Eunice A Ferreira; Florian Weissensteiner; Piera De Santis; Selin Kara; Robert Kourist; Paula Tamagnini; Wolfgang Kroutil

doi:10.1016/j.engmic.2021.100008

Expression and activity of heterologous hydroxyisocaproate dehydrogenases in Synechocystis sp. PCC 6803 Δ hoxYH

Eng Microbiol. 2021 Nov 26;2(1):100008. doi: 10.1016/j.engmic.2021.100008. eCollection 2022 Mar.

Authors

Valentina Jurkaš¹, Christoph K Winkler¹, Silvan Poschenrieder¹, Paulo Oliveira^{2

3

4}, Catarina C Pacheco^{2

3}, Eunice A Ferreira^{2

3

5}, Florian Weissensteiner¹, Piera De Santis⁶, Selin Kara⁶, Robert Kourist⁷, Paula Tamagnini^{2

3

4}, Wolfgang Kroutil^{1

8

9}

Affiliations

¹ Institute of Chemistry, University of Graz, NAWI Graz, Heinrichstrasse 28, 8010 Graz, Austria.
² i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal.
³ IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal.
⁴ Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal.
⁵ ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal.
⁶ Aarhus University, Department of Engineering, Biological and Chemical Engineering Section, Biocatalysis and Bioprocessing Group, Gustav Wieds Vej 10, DK 8000 Aarhus, Denmark.
⁷ Institute of Molecular Biotechnology, Graz University of Technology, 8010 Graz, Austria.
⁸ Field of Excellence BioHealth, University of Graz, 8010 Graz, Austria.
⁹ BioTechMed Graz, 8010 Graz, Austria.

Abstract

Exploiting light to drive redox reactions is currently a hot topic since light is considered as an environmentally friendly source of energy. Consequently, cyanobacteria, which can use light e.g., for generating NADPH, are in the focus of research. Previously, it has been shown that various heterologous redox enzymes could be expressed in these microorganisms. Here we demonstrated the successful inducer-free expression of α-keto-acid dehydrogenases (L-HicDH and D-HicDH) from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008 in Synechocystis sp. PCC 6803 ΔhoxYH mutant using replicative plasmids. While the L-HicDH showed poor activity limited by the amount of expressed enzyme, the D-HicDH was applied both in vivo and in vitro, transforming the selected α-keto acids to the corresponding optically pure (R)-α-hydroxy acids (ee >99%) in up to 53% and 90% conversion, respectively.

Keywords: Biocatalysis; Cyanobacteria; Hydroxyisocaproate dehydrogenase; Ketoacid dehydrogenase; Photobiotransformation; Synechocystis sp. PCC 6803.