title: Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol.
abstract: 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 5200 U l(-1) ADH and 370 U l(-1) G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700 U l(-1) ADH and 170 U l(-1) GDH activity. However, the crude extract of both strains reduced 40 mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99 % in 60 min 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 9.74 g l(-1) day(-1) or 406 g kg(-1) dry cell weight (dcw) day(-1) isolated 1-(R)-phenylethanol with an ee of 100 % and a TTN of 206 mol 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.
authors.affiliation: Orgentis Chemicals GmbH, Bahnhofstr. 3-5, 06466, Gatersleben, Germany.
authors.forename: Marion
authors.initials: M
authors.lastname: Rauter
authors.forename: Alexandra
authors.initials: A
authors.lastname: Prokoph
authors.forename: Jakub
authors.initials: J
authors.lastname: Kasprzak
authors.forename: Karin
authors.initials: K
authors.lastname: Becker
authors.forename: Keith
authors.initials: K
authors.lastname: Baronian
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Bode
authors.forename: Gotthard
authors.initials: G
authors.lastname: Kunze
authors.forename: H- Matthias
authors.initials: H
authors.lastname: Vorbrodt
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001592:Benzyl Alcohols / Q000378:metabolism*; D015870:Gene Expression; D042843:Glucose 1-Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D052196:Levilactobacillus brevis / Q000201:enzymology* / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D001592:Benzyl Alcohols; D011994:Recombinant Proteins; C002017:methylphenyl carbinol; D000426:Alcohol Dehydrogenase; D042843:Glucose 1-Dehydrogenase; D005954:Glucosephosphate Dehydrogenase