title: Overexpression of a non-native deoxyxylulose-dependent vitamin B6 pathway in Bacillus subtilis for the production of pyridoxine.
abstract: Vitamin B6 is a designation for the vitamers pyridoxine, pyridoxal, pyridoxamine, and their respective 5'-phosphates. Pyridoxal 5'-phosphate, the biologically most-important vitamer, serves as a cofactor for many enzymes, mainly active in amino acid metabolism. While microorganisms and plants are capable of synthesizing vitamin B6, other organisms have to ingest it. The vitamer pyridoxine, which is used as a dietary supplement for animals and humans is commercially produced by chemical processes. The development of potentially more cost-effective and more sustainable fermentation processes for pyridoxine production is of interest for the biotech industry. We describe the generation and characterization of a Bacillus subtilis pyridoxine production strain overexpressing five genes of a non-native deoxyxylulose 5'-phosphate-dependent vitamin B6 pathway. The genes, derived from Escherichia coli and Sinorhizobium meliloti, were assembled to two expression cassettes and introduced into the B. subtilis chromosome. in vivo complementation assays revealed that the enzymes of this pathway were functionally expressed and active. The resulting strain produced 14mg/l pyridoxine in a small-scale production assay. By optimizing the growth conditions and co-feeding of 4-hydroxy-threonine and deoxyxylulose the productivity was increased to 54mg/l. Although relative protein quantification revealed bottlenecks in the heterologous pathway that remain to be eliminated, the final strain provides a promising basis to further enhance the production of pyridoxine using B. subtilis.
authors: DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland; Department of General Microbiology, Georg-August-University of Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany. Electronic address: fcommic1@gwdg.de.
authors: Fabian M
authors: FM
authors: Commichau
authors: DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland.
authors: Ariane
authors: A
authors: Alzinger
authors: DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland.
authors: Rafael
authors: R
authors: Sande
authors: DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland.
authors: Werner
authors: W
authors: Bretzel
authors: DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland.
authors: Frederik M
authors: FM
authors: Meyer
authors: DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland.
authors: Bastien
authors: B
authors: Chevreux
authors: DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland.
authors: Markus
authors: M
authors: Wyss
authors: DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland.
authors: Hans-Peter
authors: HP
authors: Hohmann
authors: DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland. Electronic address: zoltan.pragai@dsm.com.
authors: Zoltán
authors: Z
authors: Prágai
mesh_terms: D001412:Bacillus subtilis / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D011736:Pyridoxine / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics; D025101:Vitamin B 6 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014996:Xylulose / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D014996:Xylulose; C029724:1-deoxy-2-pentulose; D025101:Vitamin B 6; D011736:Pyridoxine
keywords: Antimetabolite; Bacillus subtilis; Heterologous gene expression; Pyridoxine; Vitamin B6