|Greenberg, Jay - UNIV OF ROCHESTER|
|Oliver, Richard - MURDOCH UNIV - AUSTRALIA|
|Sherman, Fred - UNIV OF ROCHESTER|
|Rustchenko, Elena - UNIV OF ROCHESTER|
Submitted to: Yeast
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 16, 2005
Publication Date: July 19, 2005
Citation: Greenberg, J.R., Price, N.P., Oliver, R.P., Sherman, F., Rustchenko, E. 2005. Candida albicans SOU1 encodes a sorbose reductase required for L-sorbose utilization. Yeast 2005. 22:957-969. Interpretive Summary: Mannitol is a naturally-occurring sweetener with potential as a low-calorie dietary sugar substitute. Mannitol is presently made from fructose by a non-selective chemical route or by a biologically-based approach recently developed at USDA-NCAUR. We now report the discovery of an enzyme from the common yeast C. albicans that converts fructose to mannitol in high yield. We have purified this enzyme by over-expressing its gene (called SOU1) and have used it to convert fructose (corn sugar) to mannitol in high yield. As well as converting fructose to mannitol, SOU1 is also able to interconvert several other sugars with potential value to the food industry. This represents a clean, enzyme-based preparation of high-value mannitol from a relatively low-cost agricultural product, fructose.
Technical Abstract: Previous work in our laboratory showed that L-sorbose utilization in Candida albicans is subject to a novel form of regulation which involves a reversible increase or decrease in the copy number of chromosome 5. Furthermore, the structural gene SOU1 is required for L-sorbose utilization and encodes a member of the short chain dehydrogenase family. However, the precise function of SOU1 was not known, nor was the pathway for L-sorbose utilization. We have now expressed SOU1 at a high level from a replicative plasmid having a constitutive ADH1 promotor and purified a version of SOU1p tagged with the FLAG epitope at the N-terminus. Sou1FLAGNp has a sorbose reductase activity which utilizes NADPH as a cofactor and converts L-sorbose to D-sorbitol. It can also less efficiently utilize fructose as a substrate with NADPH as a cofactor, converting fructose to mannitol. In agreement with prediction, the purified enzyme has a subunit molecular weight of 31 kDa and a pI of about 4.8. It probably consists of four identical subunits and has a pH optimum of 6.2. The L-sorbose utilization pathway in C. albicans probably converts L-sorbose to fructose-6-phophate via D-sorbitol as an intermediate. The first step is catalyzed by Sou1p. We also found the C. albicans extracts have a D-sorbitol-6-phosphate dehydrogenase activity not encoded by SOU1 which utilizes NADP as a cofactor. This activity has not been previously described in yeasts and may be involved in the conversation of phosphorylated D-sorbitol to fructose-6-phosphate or glucose-6-phosphate.