Project description:In Scheffersomyces stipitis and related fungal species the genes for L-rhamnose catabolism RHA1, LRA2, LRA3 and LRA4 but not LADH are clustered. We find that located next to the cluster is a transcription factor, TRC1, which is conserved among related species.Our transcriptome analysis shows that all the catabolic genes and all genes of the cluster are up-regulated on L-rhamnose. Among the genes that were also up-regulated on L-rhamnose were two transcription factors including the TRC1. In addition, in 16 out of the 32 analysed fungal species only RHA1, LRA2 and LRA3 are in a cluster. The clustering of RHA1, LRA3 and TRC1 is also conserved in species not closely related to S. stipitis. Since the LRA4 is often not part of the cluster and it has several paralogs in L-rhamnose utilising yeasts we analysed the function of one of the paralogs, RHA41 by heterologous expression and biochemical characterization. Rha41p has similar catalytic properties but the transcript was not up-regulated on L-rhamnose. The RHA1, LRA2, LRA4 and LADH genes were previously characterized in Sheffersomyces (Pichia) stipitis. We expressed the L-rhamnonate dehydratase, Rha3p, in S. cerevisiae, estimated the kinetic constants of the protein and showed that it indeed has activity with L-rhamnonate.
Project description:In Scheffersomyces stipitis and related fungal species the genes for L-rhamnose catabolism RHA1, LRA2, LRA3 and LRA4 but not LADH are clustered. We find that located next to the cluster is a transcription factor, TRC1, which is conserved among related species.Our transcriptome analysis shows that all the catabolic genes and all genes of the cluster are up-regulated on L-rhamnose. Among the genes that were also up-regulated on L-rhamnose were two transcription factors including the TRC1. In addition, in 16 out of the 32 analysed fungal species only RHA1, LRA2 and LRA3 are in a cluster. The clustering of RHA1, LRA3 and TRC1 is also conserved in species not closely related to S. stipitis. Since the LRA4 is often not part of the cluster and it has several paralogs in L-rhamnose utilising yeasts we analysed the function of one of the paralogs, RHA41 by heterologous expression and biochemical characterization. Rha41p has similar catalytic properties but the transcript was not up-regulated on L-rhamnose. The RHA1, LRA2, LRA4 and LADH genes were previously characterized in Sheffersomyces (Pichia) stipitis. We expressed the L-rhamnonate dehydratase, Rha3p, in S. cerevisiae, estimated the kinetic constants of the protein and showed that it indeed has activity with L-rhamnonate. A six chip study using total RNA recovered from three separate cultures of S. stipitis CBS 6054 grown glucose and respectively three separate cultures grown on rhamnose
Project description:Liu2012 - Genome-scale metabolic network of Scheffersomyces stipitis (iTL885)
This model is described in the article:
A constraint-based model of
Scheffersomyces stipitis for improved ethanol production.
Liu T, Zou W, Liu L, Chen J.
Biotechnol Biofuels 2012; 5(1):
72
Abstract:
UNLABELLED: BACKGROUND: As one of the best xylose
utilization microorganisms, Scheffersomyces stipitis exhibits
great potential for the efficient lignocellulosic biomass
fermentation. Therefore, a comprehensive understanding of its
unique physiological and metabolic characteristics is required
to further improve its performance on cellulosic ethanol
production. RESULTS: A constraint-based genome-scale metabolic
model for S. stipitis CBS 6054 was developed on the basis of
its genomic, transcriptomic and literature information. The
model iTL885 consists of 885 genes, 870 metabolites, and 1240
reactions. During the reconstruction process, 36 putative sugar
transporters were reannotated and the metabolisms of 7 sugars
were illuminated. Essentiality study was conducted to predict
essential genes on different growth media. Key factors
affecting cell growth and ethanol formation were investigated
by the use of constraint-based analysis. Furthermore, the
uptake systems and metabolic routes of xylose were elucidated,
and the optimization strategies for the overproduction of
ethanol were proposed from both genetic and environmental
perspectives. CONCLUSIONS: Systems biology modelling has proven
to be a powerful tool for targeting metabolic changes. Thus,
this systematic investigation of the metabolism of S. stipitis
could be used as a starting point for future experiment designs
aimed at identifying the metabolic bottlenecks of this
important yeast.
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