Abstract
BackgroundUnderstanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae. Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites.ResultsUnbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively.ConclusionsThe heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.
Highlights
Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae
The oxidoreductive xyloseassimilating pathway consisting of XYL1, XYL2, and XYL3 coding for xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase, respectively, from Scheffersomyces stipitis [8] was introduced into S. cerevisiae D452-2 by multicopy integration [9]
hierarchical cluster analysis (HCA) of the intracellular metabolites of strain EJ4 grown on different carbon sources For comparing the intracellular metabolite profiles of strain EJ4 grown on the four different sugars, sampling of culture broth was performed twice for each sugar in the exponential phase of growth as follows: glucose, at 12 and 16 h; galactose, at 16 and 20 h; xylose, at 24 and 33 h; and cellobiose, at 33 and 45 h (Fig. 1a, b)
Summary
Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae. We studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renew‐ able biomass by profiling intracellular and extracellular metabolites. For efficient utilization of various sugars generated from biomass feedstocks including lignocellulose by S. cerevisiae, strain improvement, enabling the assimilation of the sugars that are naturally non-fermentable by S. cerevisiae such as xylose [4] and cellobiose [5] and the simultaneous fermentation of mixed sugars, have been attempted on many occasions by means of genetic perturbations [5, 6]. S. cerevisiae has been successfully engineered to catabolize xylose and cellobiose simultaneously (resulting in strain EJ4) using multiple genetic perturbations and rational and laboratory evolution, as follows [7]. Through serial subculturing of strain EJ1 in the presence of cellobiose, strain EJ4 with efficient and simultaneous fermenting capability for xylose and cellobiose was created [7]
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