title: Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine.
abstract: Until now, pyridoxine (PN), the most commonly supplemented B6 vitamer for animals and humans, is chemically synthesized for commercial purposes. Thus, the development of a microbial fermentation process is of great interest for the biotech industry. Recently, we constructed a Bacillus subtilis strain that formed significant amounts of PN via a non-native deoxyxylulose 5'-phosphate-(DXP)-dependent vitamin B6 pathway. Here we report the optimization of the condensing reaction of this pathway that consists of the 4-hydroxy-l-threonine-phosphate dehydrogenase PdxA, the pyridoxine 5'-phosphate synthase PdxJ and the native DXP synthase, Dxs. To allow feeding of high amounts of 4-hydroxy-threonine (4-HO-Thr) that can be converted to PN by B. subtilis overexpressing PdxA and PdxJ, we first adapted the bacteria to tolerate the antimetabolite 4-HO-Thr. The adapted bacteria produced 28-34mg/l PN from 4-HO-Thr while the wild-type parent produced only 12mg/l PN. Moreover, by expressing different pdxA and pdxJ alleles in the adapted strain we identified a better combination of PdxA and PdxJ enzymes than reported previously, and the resulting strain produced 65mg/l PN. To further enhance productivity mutants were isolated that efficiently take up and convert deoxyxylulose (DX) to DXP, which is incorporated into PN. Although these mutants were very efficient to convert low amount of exogenous DX, at higher DX levels they performed only slightly better. The present study uncovered several enzymes with promiscuous activity and it revealed that host metabolic pathways compete with the heterologous pathway for 4-HO-Thr. Moreover, the study revealed that the B. subtilis genome is quite flexible with respect to adaptive mutations, a property, which is very important for strain engineering.
authors.affiliation: DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland; Department of General Microbiology, Georg-August-University of Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany. Electronic address: fcommic1@gwdg.de.
authors.forename: Fabian M
authors.initials: FM
authors.lastname: Commichau
authors.affiliation: DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland.
authors.forename: Ariane
authors.initials: A
authors.lastname: Alzinger
authors.affiliation: DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland.
authors.forename: Rafael
authors.initials: R
authors.lastname: Sande
authors.affiliation: DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland.
authors.forename: Werner
authors.initials: W
authors.lastname: Bretzel
authors.affiliation: DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Reuß
authors.affiliation: Department of General Microbiology, Georg-August-University of Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Dormeyer
authors.affiliation: DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland.
authors.forename: Bastien
authors.initials: B
authors.lastname: Chevreux
authors.affiliation: Department of Genomic and Applied Microbiology, Georg-August-University of Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Schuldes
authors.affiliation: Department of Genomic and Applied Microbiology, Georg-August-University of Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Daniel
authors.affiliation: DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands.
authors.forename: Michiel
authors.initials: M
authors.lastname: Akeroyd
authors.affiliation: DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Wyss
authors.affiliation: DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Hohmann
authors.affiliation: DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland. Electronic address: zoltan.pragai@dsm.com.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Prágai
mesh_terms: D000963:Antimetabolites / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D060847:Metabolic Engineering; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D011736:Pyridoxine / Q000096:biosynthesis*; D013912:Threonine / Q000031:analogs & derivatives* / Q000096:biosynthesis
chemical_list: D000963:Antimetabolites; D001426:Bacterial Proteins; C011285:hydroxythreonine; D013912:Threonine; D010088:Oxidoreductases; D002237:Carbohydrate Dehydrogenases; D011736:Pyridoxine
keywords: 4-Hydroxy-l-threonine; Antimetabolite; Bacillus subtilis; Promiscuous enzyme; Pyridoxine; Vitamin B6