High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous.
To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae.
Verwaal
René
R
Fungal Genomics, Laboratory of Microbiology, Wageningen University, Dreijenlaan 2, Wageningen, The Netherlands.
Wang
Jing
J
Meijnen
Jean-Paul
JP
Visser
Hans
H
Sandmann
Gerhard
G
van den Berg
Johan A
JA
van Ooyen
Albert J J
AJ
D001483:Base Sequence; D001487:Basidiomycota / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004875:Ergosterol / Q000096:biosynthesis; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D024243:HMGB1 Protein / Q000235:genetics; D010957:Plasmids; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic; D019207:beta Carotene / Q000096:biosynthesis* / Q000235:genetics*
D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D024243:HMGB1 Protein; C114542:RFX1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D019207:beta Carotene; D004875:Ergosterol