On the role of GAPDH isoenzymes during pentose fermentation in engineered Saccharomyces cerevisiae.

In the metabolic network of the cell, many intermediary products are shared between different pathways. d-Glyceraldehyde-3-phosphate, a glycolytic intermediate, is a substrate of GAPDH but is also utilized by transaldolase and transketolase in the scrambling reactions of the nonoxidative pentose phosphate pathway. Recent efforts to engineer baker's yeast strains capable of utilizing pentose sugars present in plant biomass rely on increasing the carbon flux through this pathway. However, the competition between transaldolase and GAPDH for d-glyceraldehyde-3-phosphate produced in the first transketolase reaction compromises the carbon balance of the pathway, thereby limiting the product yield. Guided by the hypothesis that reduction in GAPDH activity would increase the availability of d-glyceraldehyde-3-phosphate for transaldolase and thereby improve ethanol production during fermentation of pentoses, we performed a comprehensive characterization of the three GAPDH isoenzymes in baker's yeast, Tdh1, Tdh2, and Tdh3 and analyzed the effect of their deletion on xylose utilization by engineered strains. Our data suggest that overexpression of transaldolase is a more promising strategy than reduction in GAPDH activity to increase the flux through the nonoxidative pentose phosphate pathway.

Biosynthesis of cis,cis-muconic acid and its aromatic precursors, catechol and protocatechuic acid, from renewable feedstocks by Saccharomyces cerevisiae.

Adipic acid is a high-value compound used primarily as a precursor for the synthesis of nylon, coatings, and plastics. Today it is produced mainly in chemical processes from petrochemicals like benzene. Because of the strong environmental impact of the production processes and the dependence on fossil resources, biotechnological production processes would provide an interesting alternative. Here we describe the first engineered Saccharomyces cerevisiae strain expressing a heterologous biosynthetic pathway converting the intermediate 3-dehydroshikimate of the aromatic amino acid biosynthesis pathway via protocatechuic acid and catechol into cis,cis-muconic acid, which can be chemically dehydrogenated to adipic acid. The pathway consists of three heterologous microbial enzymes, 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase composed of three different subunits, and catechol 1,2-dioxygenase. For each heterologous reaction step, we analyzed several potential candidates for their expression and activity in yeast to compose a functional cis,cis-muconic acid synthesis pathway. Carbon flow into the heterologous pathway was optimized by increasing the flux through selected steps of the common aromatic amino acid biosynthesis pathway and by blocking the conversion of 3-dehydroshikimate into shikimate. The recombinant yeast cells finally produced about 1.56 mg/liter cis,cis-muconic acid.

Tracking adult neovascularization during ischemia and inflammation using Vegfr2-LacZ reporter mice.

The vascular endothelial growth factor/vascular endothelial growth factor receptor 2 (VEGF/VEGFR-2) signal transduction system plays a key role during embryonic vascular development and adult neovascularization. In contrast to many endothelial genes, VEGFR-2 is expressed at low levels in most adult vessels but is strongly upregulated during neovascularization, leading to a pro-angiogenic response. Here, we analyzed the activity of regulatory sequences of the murine Vegfr2 gene during neovessel formation in vivo under ischemic and inflammatory conditions. Hindlimb ischemia was induced in transgenic mice, expressing the LacZ reporter gene under the control of Vegfr2 promoter/enhancer elements. Most vessels in the ischemic muscle tissue showed strong endothelium-specific reporter gene expression, whereas nearly no LacZ-expressing capillaries were observed in untreated control tissue. Cutaneous punch wounds were created to induce angiogenesis under inflammatory conditions, leading to robust LacZ expression in the majority of the blood vessels in the wound tissue. Since the cornea is physiologically avascular, the functionality of these promoter/enhancer elements exclusively in newly formed vessels was confirmed using the cornea micropocket assay. Taken together, our results show that these Vegfr2 regulatory elements are active during adult neovessel formation in general. Therefore, these sequences may prove to be valuable targets for novel endothelium-specific anti-angiogenic as well as pro-angiogenic treatment strategies. They may especially allow directing therapeutic gene expression to sites of adult neovascularization. Moreover, the Vegfr2/LacZ reporter mice represent a powerful model to generally analyze the transcriptional control mechanisms involved in the induction of Vegfr2 expression during adult neovascularization.

Establishing a yeast-based screening system for discovery of human GLUT5 inhibitors and activators.

Human GLUT5 is a fructose-specific transporter in the glucose transporter family (GLUT, SLC2 gene family). Its substrate-specificity and tissue-specific expression make it a promising target for treatment of diabetes, metabolic syndrome and cancer, but few GLUT5 inhibitors are known. To identify and characterize potential GLUT5 ligands, we developed a whole-cell system based on a yeast strain deficient in fructose uptake, in which GLUT5 transport activity is associated with cell growth in fructose-based media or assayed by fructose uptake in whole cells. The former method is convenient for high-throughput screening of potential GLUT5 inhibitors and activators, while the latter enables detailed kinetic characterization of identified GLUT5 ligands. We show that functional expression of GLUT5 in yeast requires mutations at specific positions of the transporter sequence. The mutated proteins exhibit kinetic properties similar to the wild-type transporter and are inhibited by established GLUT5 inhibitors N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) and (-)-epicatechin-gallate (ECG). Thus, this system has the potential to greatly accelerate the discovery of compounds that modulate the fructose transport activity of GLUT5.