Doubled production of cordycepin analogs in cultured Cordyceps militaris by addition of Andrea droppings.

Cordyceps sinensis is a traditional Chinese medicine with various biological activities. With its limited natural supply, cultured C. militaris has become the major alternative source, and the culture conditions may affect the chemical compositions. To improve the production of chemical ingredients, C. militaris was cultured with three different media, including rice only, rice plus 3% tea leaves, and rice plus 3% droppings of Andraca theae. The fractions of dried C. militaris cultured with rice were chromatographic separated to afford ten compounds: phenylalanine, dimerumic acid, nicotinic acid, tryptophan, N6-(2-hydroxyethyl)-adenosine, uracil, uridine, cordycepin, ergosterol, and mannitol. Of these, in the cultured medium of rice plus 3% Andraca droppings, the amount of one major compound cordycepin is about two folds than the highest reported data, and dimerumic acid and N6-(2-hydroxyethyl)-adenosine were isolated for the first time from this species.[Figure: see text].

Dekkera bruxellensis, a beer yeast that specifically bioconverts mogroside extracts into the intense natural sweetener siamenoside I.

In response to growing concerns about the consumption of artificial sweeteners, the demand for natural sweeteners has recently increased. Mogroside V is a common natural sweetener extracted from the fruit of Siraitia grosvenorii, but its taste should be improved for marketability. Here, we screened various microbes for the ability to perform selective hydrolysis of glycosidic bonds in mogroside V, converting it to siamenoside I, which has a higher sweetening power and better taste than other mogrosides. Dekkera bruxellensis showed the most promising results in the screen, and the Exg1 gene (coding for a ß-glucosidase) of D. bruxellensis was cloned and purified. We then used HPLC-MS/MS to assess the ß-glucosidase activity of purified enzymes on p-nitrophenyl ß-glucoside and mogroside V. The results demonstrated that D. bruxellensis had a unique enzyme that can selectively hydrolyze mogrol glycosides and promote the conversion of the natural sweetener mogroside V to siamenoside I.

Characterization of an extracellular ß-glucosidase from Dekkera bruxellensis for resveratrol production.

Polygonum cuspidatum is a widely grown crop with a rich source of polydatin (also called piceid) for resveratrol production. Resveratrol is produced from piceid via enzymatic cleavage of the sugar moiety of piceid. In this study, Dekkera bruxellensis mutants were selected based on their high p-nitrophenyl-ß-d-glucopyranoside and piceid conversion activities. The enzyme responsible for piceid conversion was a heterodimeric protein complex that was predominantly secreted to the extracellular medium and consisted of two subunits at an equal ratio with molecular masses of 30.5 kDa and 48.3 kDa. The two subunits were identified as SCW4p and glucan-ß-glucosidase precursor in D. bruxellensis. Both proteins were individually expressed in Saccharomyces cerevisiae exg1Δ mutants, which lack extracellular ß-glucosidase activity, to confirm each protein's enzymatic activities. Only the glucan-ß-glucosidase precursor was shown to be a secretory protein with piceid deglycosylation activity. Our pilot experiments of piceid bioconversion demonstrate the possible industrial applications for this glucan-ß-glucosidase precursor in the future.

Pilot scale repeated fed-batch fermentation processes of the wine yeast Dekkera bruxellensis for mass production of resveratrol from Polygonum cuspidatum.

Resveratrol has long been used as an ingredient in functional foods. Currently, Polygonum cuspidatum extract is the greatest natural source for resveratrol because of high concentrations of glycosidic-linked resveratrol. Thus, developing a cost-effective procedure to hydrolyze glucoside could substantially enhance resveratrol production from P. cuspidatum. This study selected Dekkera bruxellensis from several microorganisms based on its bioconversion and enzyme-specific activities. We demonstrated that the cells could be reused at least nine times while maintaining an average of 180.67U/L ß-glucosidase activity. The average resveratrol bioconversion efficiency within five rounds of repeated usage was 108.77±0.88%. This process worked effectively when the volume was increased to 1200L, a volume at which approximately 35mgL-1h-1 resveratrol per round was produced. This repeated fed-batch bioconversion process for resveratrol production is comparable to enzyme or cell immobilization strategies in terms of reusing cycles, but without incurring additional costs for immobilization.

Hyperproduction of ß-Glucanase Exg1 Promotes the Bioconversion of Mogrosides in Saccharomyces cerevisiae Mutants Defective in Mannoprotein Deposition.

Bacteria and fungi can secrete extracellular enzymes to convert macromolecules into smaller units. Hyperproduction of extracellular enzymes is often associated with alterations in cell wall structure in fungi. Recently, we identified that Saccharomyces cerevisiae kre6Δ mutants can efficiently convert mogroside V into mogroside III E, which has antidiabetic properties. However, the underlying efficient bioconversion mechanism is unclear. In the present study, the mogroside (MG) bioconversion properties of several cell wall structure defective mutants were analyzed. We also compared the cell walls of these mutants by transmission electron microscopy, a zymolyase sensitivity test, and a mannoprotein release assay. We found zymolyase-sensitive mutants (including kre1Δ, las21Δ, gas1Δ, and kre6Δ), with defects in mannoprotein deposition, exhibit efficient MG conversion and excessive leakage of Exg1; such defects were not observed in wild-type cells, or mutants with abnormal levels of glucans in the cell wall. Thus, yeast mutants defective in mannoprotein deposition may be employed to convert glycosylated bioactive compounds.