Abstract
(Englisch)
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A novel, molecular-level mathematical model for glucose and maltose uptake in Saccharomyces cerevisiac was extended and applied to suggest new genetic strategies to enhance mixed-sugar utilization. Using this model as a foundation, we formulated two strategies for the alleviation of glucose control in a polyploid. The first strategy, expression of a glucose insensitive maltose permease, was predicted, both in the absence and presence of mig1 disruption, to enhance maltose utilization. In this case, an increased level of maltose enters the cell, binds to malR, which in turn up-regulates the expression of the permeaselmaltase at higher glucose levels. The success of this strategy can be directly traced to the duality of character of malR. A second strategy involves the reengineering of rnigl binding sites on the malR, malT (permease) and malS (maltase) genes. It is predicted that targeted disruption of mig1 binding affinity for the control sites of malT/malS, while leaving malR untouched, substantially enhances maltose utilization. In this instance, the sensitivity of permease/maltase expression to migl is removed, while malR expression remains repressed.
In parallel with this EC Project, other research in our laboratory, in collaboration with Prof. K. Wüthrich at the ETH Zurich, showed potential for convenient, information rich-assays of metabolic flux distributions in cells. Such information can be extremely valuable in aiding genetic design, and also for evaluating and improving mathematical models of cell factories. Opportunities for important applications of this new technique to reveal carbon flux patterns in yeasts important in this project were identified in collaboration of the laboratory of Prof. B. Hahn-Hagerdahl. Recognizing the importance of pentose utilization from hemicellulosic substrates, the Hahn-Ha~gerdahl lab tried to confer this phenotype from the efficient pentose utilizer P. stipitis to a recombinant S. cerevisiae. In this recombinant strain, however, xylose utilization was sub-optimal, although enzymes catalyzing the conversion of xylose into an intermediate of the pentose phosphate pathway (PPP) were successfully expressed. Using recently metabolic flux ratio (METAFoR) analysis , which is based on two-dimensional [13C,1H] NMR, we found an unusually low upper bound for the fraction of PEP molecules derived from pentose in S. cerevisiae (3% vs. 50% in Pichia), illustrating a more active PPP in P. stipitis, compared to wild-type S. cerevisiae. Additionally, P. stipitis exhibited a higher relative flux through the tricarboxylic acid (TCA) cycle compares to S. cerevisiae, indicating a higher capacity for oxidative catabolism in the former. These data indicate a potential limitation of xylose catabolism in the PPP of the recombinant S. cerevisiae strain and suggest overexpression of transaldolase and/or transketolase as a necessary next step for engineering efficient xylose-utilizing S. cerevisiae.
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