Engineering of biotin-prototrophy in Pichia pastoris for robust production processes.
Biotin plays an essential role as cofactor for biotin-dependent carboxylases involved in essential metabolic pathways. The cultivation of Pichia pastoris, a methylotrophic yeast that is successfully used as host for the production of recombinant proteins, requires addition of high dosage of biotin. As biotin is the only non-salt media component used during P. pastoris fermentation (apart from the carbon source), nonconformities during protein production processes are usually attributed to poor quality of the added biotin. In order to avoid dismissed production runs due to biotin quality issues, we engineered the biotin-requiring yeast P. pastoris to become a biotin-prototrophic yeast. Integration of four genes involved in the biotin biosynthesis from brewing yeast into the P. pastoris genome rendered P. pastoris biotin-prototrophic. The engineered strain has successfully been used as production host for both intracellular and secreted heterologous proteins in fed-batch processes, employing mineral media without vitamins. Another field of application for these truly prototrophic hosts is the production of biochemicals and small metabolites, where defined mineral media leads to easier purification procedures.
Gasser
Brigitte
B
Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190 Vienna, Austria. brigitte.gasser@boku.ac.at
Dragosits
Martin
M
Mattanovich
Diethard
D
D057005:Bioengineering; D001710:Biotin / Q000096:biosynthesis* / Q000235:genetics*; D003470:Culture Media; D005285:Fermentation; D005822:Genetic Vectors; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D003470:Culture Media; D011994:Recombinant Proteins; D001710:Biotin