Abstract
BackgroundHigh 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.ResultsOverexpression 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.ConclusionThe 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.
Highlights
High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins
While P. pastoris does not produce riboflavin when grown on glucose, high cell density cultures grown on methanol tend to develop yellow colored supernatants [9,10], attributed to the release of free flavins [11], which indicates that a deregulation of the flavin synthesis pathway seems feasible
A single gene overexpression renders P. pastoris flavinogenic Previously published research on the formation of riboflavin in different riboflavin overproducing organisms indicates that the enzymatic activity of guanosine triphosphate (GTP)-cyclohydrolase II (RIB1, Figure 1) plays a key regulatory role in the riboflavin biosynthetic pathway
Summary
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 given, but not pronounced when the yeast is grown on glucose. Ascomycetes like Ashbya gossypii (anamorph: Eremothecium ashbyii) [1], Candida famata [2] or Pichia guilliermondii [3] are natural overproducers of riboflavin. These, as well as other riboflavin producers have been termed flavinogenic yeasts, a phenotype which is usually induced by iron starvation [4], leading to the secretion of riboflavin in the mg/L range. Published approaches to improve riboflavin production strains employ mainly random mutagenesis and screening or selection with metabolite analoga, mainly targeting at the precursers upstream of riboflavin synthesis. For the flavinogenic fungus A. gossypii (page number not for citation purposes)
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