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Content archived on 2024-05-14

Engineering of yeast glycerol metabolism towards optimised yield of fermentation end products and improved tolerance to osmotic stress

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By deletion of the two isogenes GDP1 and GDP2, encoding glycerol 3 phosphate dehydrogenase, we produced a mutant having a block in the pathway for the glycerol biosynthesis. This mutant was shown to accumulate NADH and arrest the ethanol production and growth when subjected to anaerobiosis, unless provided with an artificial acceptor of reducing equivalents, such as acetoine or acetaldehyde thus, glycerol production appears to be the only available alternative for oxidation of excess NADH under anaerobic growth conditions. This opens for the possibility of using the excess reducing power accumulated under anaerobic conditions to drive other desired reductive reactions by introducing suitable heterologous reductases. A practical advantage of our mutant strain is that it can be cultivated easily under aerobic standard conditions and the shifted to anaerobic conditions to carry out the appropriate reductive step under controlled conditions. We have already observed that the heterlologous reduction of xylose to the artificial sweetener xylitol can be improved by a factor of three in a gpd1 gpd2 strain.
We have previously fitted the Fsp1 gene from the Saccharpmyces cerevisiae, which encodes a glycerol channel protein. The main physiological role of Fsp1p on yeast is to regulate the export of glycerol. This export function is gated by means of an N-terminal extension. Deletion of Fsp1 gene increases glycerol retention whereas truncation of the N-terminal extension of Fps1p causes a constitutive high glycerol export activity. Yeast cells expressing the constitutive active form of Fsps1p cannot efficiently retain glycerol and compensate for the loss of the osmolyte by glycerol over production. This feature can be used to enhance glycerol production. This has been tested, e.g. in a model wine yeast strain and has led to an about 2.5 fold enhanced total glycerol production. A number of polyols beyond glycerol are of actual or potential industrial importance, for instances as precursors for the production of plastics or as sweeteners. Fps1p can also transport other polyols. In addition, homologues of Fp21 from the MIP family of channel proteins have also been shown to transport a variety of polyols and other uncharged substances, but not amino acids or sugars. This suggests that members of the MIP family, such as Fps1p, could be used in metabolic engineering of microbial metabolism in general, to steer the production of polyols by providing an export path from the cell and thereby driving the reaction equilibrium towards the desired point. We plan to develop this idea further. In a presently ongoing project funded by the Commission we are searching for novel MIP channels with interesting transport specificity and we test them in production of xylitol by genetically engineered yeast. We also plan to test such channels in genetically engineered yeast's that produce e.g. mannitol. Clearly, however, the concept will have implications beyond yeast and for metabolic engineering and biotransformation.

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