caa a22 4500 605506485 CHVBK 20210128100627.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s00253-014-6297-z doi (NATIONALLICENCE)springer-10.1007/s00253-014-6297-z Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol [Elektronische Daten] [Marion Rauter, Alexandra Prokoph, Jakub Kasprzak, Karin Becker, Keith Baronian, Rüdiger Bode, Gotthard Kunze, H.- Vorbrodt] The yeast Arxula adeninivorans was used for the overexpression of an ADH gene of Lactobacillus brevis coding for (R)-specific alcohol dehydrogenase (LbADH) to synthesise enantiomerically pure 1-(R)-phenylethanol. Glucose dehydrogenase gene from Bacillus megaterium (BmGDH) or glucose 6-phosphate dehydrogenase of Bacillus pumilus (BpG6PDH) were coexpressed in Arxula to regenerate the cofactor NADPH by oxidising glucose or glucose 6-phosphate. The yeast strain expressing LbADH and BpG6PDH produced 5200U l-1 ADH and 370U l-1 G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700U l-1 ADH and 170U l-1 GDH activity. However, the crude extract of both strains reduced 40mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99% in 60min without detectable by-products. An increase in yield was achieved using immobilised crude extracts (IEs), Triton X-100 permeabilised cells (PCs) and permeabilised immobilised cells (PICs) with PICs being most stable with GDH regeneration over 52 cycles. Even though the activity and synthesis rate of 1-(R)-phenylethanol with the BpG6PDH and LbADH coexpressing strain was higher, the BmGDH-LbADH strain was more stable over successive reaction cycles. This, combined with its higher total turnover number (TTN) of 391 mol product per mole NADP+, makes it the preferred strain for continuous reaction systems. The initial non-optimised semi-continuous reaction produced 9.74gl−1day−1 or 406gkg−1 dry cell weight (dcw) day−1 isolated 1-(R)-phenylethanol with an ee of 100% and a TTN of 206mol product per mole NADP+. In conclusion, A. adeninivorans is a promising host for LbADH and BpG6PDH or BmGDH production and offers a simple method for the production of enantiomerically pure alcohols. Springer-Verlag Berlin Heidelberg, 2014 1-(R)-Phenylethanol nationallicence Lactobacillus brevis ADH nationallicence Bacillus megaterium GDH nationallicence Bacillus pumilus BpG6PDH nationallicence Arxula adeninivorans nationallicence Cell permeabilisation nationallicence Calcium alginate immobilisation nationallicence Rauter Marion Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany aut Prokoph Alexandra Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany aut Kasprzak Jakub Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany aut Becker Karin Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany aut Baronian Keith School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand aut Bode Rüdiger Institute of Microbiology, University of Greifswald, Felix-Hausdorffstr. 4, 17487, Greifswald, Germany aut Kunze Gotthard Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany aut Vorbrodt H.-. Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/11(2015-06-01), 4723-4733 0175-7598 99:11<4723 2015 99 253 https://doi.org/10.1007/s00253-014-6297-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6297-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Rauter Marion Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany aut NATIONALLICENCE 700 1- Prokoph Alexandra Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany aut NATIONALLICENCE 700 1- Kasprzak Jakub Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany aut NATIONALLICENCE 700 1- Becker Karin Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany aut NATIONALLICENCE 700 1- Baronian Keith School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand aut NATIONALLICENCE 700 1- Bode Rüdiger Institute of Microbiology, University of Greifswald, Felix-Hausdorffstr. 4, 17487, Greifswald, Germany aut NATIONALLICENCE 700 1- Kunze Gotthard Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany aut NATIONALLICENCE 700 1- Vorbrodt H.-. Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/11(2015-06-01), 4723-4733 0175-7598 99:11<4723 2015 99 253