title: Production of (R)-3-hydroxybutyric acid by Arxula adeninivorans.
abstract: (R)-3-hydroxybutyric acid can be used in industrial and health applications. The synthesis pathway comprises two enzymes, β-ketothiolase and acetoacetyl-CoA reductase which convert cytoplasmic acetyl-CoA to (R)-3-hydroxybutyric acid [(R)-3-HB] which is released into the culture medium. In the present study we used the non-conventional yeast, Arxula adeninivorans, for the synthesis enantiopure (R)-3-HB. To establish optimal production, we investigated three different endogenous yeast thiolases (Akat1p, Akat2p, Akat4p) and three bacterial thiolases (atoBp, thlp, phaAp) in combination with an enantiospecific reductase (phaBp) from Cupriavidus necator H16 and endogenous yeast reductases (Atpk2p, Afox2p). We found that Arxula is able to release (R)-3-HB used an existing secretion system negating the need to engineer membrane transport. Overexpression of thl and phaB genes in organisms cultured in a shaking flask resulted in 4.84 g L-1(R)-3-HB, at a rate of 0.023 g L-1h-1over 214 h. Fed-batch culturing with glucose as a carbon source did not improve the yield, but a similar level was reached with a shorter incubation period [3.78 g L-1of (R)-3-HB at 89 h] and the rate of production was doubled to 0.043 g L-1h-1which is higher than any levels in yeast reported to date. The secreted (R)-3-HB was 99.9% pure. This is the first evidence of enantiopure (R)-3-HB synthesis using yeast as a production host and glucose as a carbon source.
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Correnstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Biernacki
authors.affiliation: Jäckering Mühlen- und Nährmittelwerke GmbH, Vorsterhauser Weg 46, 59007, Hamm, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Riechen
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Correnstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany.
authors.forename: Urs
authors.initials: U
authors.lastname: Hähnel
authors.affiliation: Jäckering Mühlen- und Nährmittelwerke GmbH, Vorsterhauser Weg 46, 59007, Hamm, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Roick
authors.affiliation: School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
authors.forename: Kim
authors.initials: K
authors.lastname: Baronian
authors.affiliation: Institute of Microbiology, University of Greifswald, Jahnstr. 15, 17487, Greifswald, Germany.
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Bode
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Correnstr. 3, 06466, Gatersleben, Saxony-Anhalt, Germany. kunzeg@ipk-gatersleben.de.
authors.forename: Gotthard
authors.initials: G
authors.lastname: Kunze
keywords: (R)-3-HB; Acetoacetyl-CoA reductase; Arxula adeninivorans; β-Ketothiolase