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Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

Hans Marx1, Diethard Mattanovich12* and Michael Sauer12

Author Affiliations

1 Institute of Applied Microbiology, Department of Biotechnology, BOKU – University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190, Wien, Austria

2 School of Bioengineering, FH Campus Wien – University of Applied Sciences, Muthgasse 18, 1190, Wien, Austria

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Microbial Cell Factories 2008, 7:23  doi:10.1186/1475-2859-7-23

Published: 29 July 2008



High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes.


Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1) is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) increases the riboflavin accumulation significantly.


The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established.