FTIR spectroscopic characterization of differently cultivated food related yeasts.

The application of Fourier Transform Infrared Spectroscopy for characterization of yeasts is growing rapidly. Since it is known that the phenotypic expression of yeast cells depends sensitively on the nutrients that are available in the growth medium, one standardized growth medium is usually used for identification and characterization purposes in order to obtain reproducible FTIR signals. Since our recently developed high-throughput micro-cultivation protocol has the capacity to use more than one standardized growth medium, we wanted to investigate if the parallel use of multiple growth media can improve identification results. For this purpose, five different cultivation media (YP, YPD, YMB, SAB and SD) were used. In total 91 food spoilage yeast strains of 12 different genera were cultivated in different cultivation media and subsequently characterized by FTIR spectroscopy. For spectral identifications, Radial Basis Function-Partial Least Squares (RBF-PLS) was used in combination with cross-model validation where an inner cross-validation loop was used to optimize the model, while in an outer loop an independent test set was kept aside to test the optimized model. Sensitivity and specificity were evaluated for each studied genus class. The results show that the YMB selective medium gave the best discrimination results for 9 of the 12 genera with sensitivity above 90%. Only three genera showed better identification results on other media (Clavispora and Metschnikowia on medium SD, Debaryomyces on medium YPD). We therefore suggest to use the media SD, YPD in combination with the YMB medium for the identification of food spoilage yeasts.

Use of the Saccharomyces cerevisiae endopolygalacturonase promoter to direct expression in Escherichia coli.

In Saccharomyces cerevisiae, an endopolygalacturonase encoded by the PGL1 gene catalyzes the random hydrolysis of the α-1,4 glycosidic linkages in polygalacturonic acid. To study the regulation of the PGL1 gene, we constructed a reporter vector containing the lacZ gene under the control of PGL1 promoter. Surprisingly, when Escherichia coli DH5α was transformed by this vector, cells harboring the constructed plasmid produced blue colonies. Sequence analysis of this promoter revealed that E. coli consensus sequences required to express an in-frame lacZ alpha product were present. We next decided to investigate how the PGL1 promoter is regulated in E. coli compared to yeast. In this study, we examined the modulation of the PGL1 promoter in E. coli, and the results indicated that its activity is greatly induced by saturated digalacturonic acid and is indirectly regulated by the transcriptional regulators the 2-keto-3-deoxygluconate repressor. Moreover, PGL1 expression is enhanced under aerobic conditions. We found that ß-galactosidase activity in E. coli could reach 180 units, which is 40-fold greater than the activity produced in S. cerevisiae, and greater than recombinant protein expression previously reported by other researchers. We thus demonstrate that this vector can be considered as a dual expression plasmid for both E. coli and S. cerevisiae hosts. So far, no modulation of endoPG promoters expressed in E. coli has been reported.

Regulation of the expression of endopolygalacturonase gene PGU1 in Saccharomyces.

Previous work in our laboratory has shown that Saccharomyces bayanus strain SCPP is the only reported yeast expressing the three types of pectolytic enzymes: pectin esterases, pectin lyases and polygalacturonases. One of these enzymes, the endopolygalacturonase (endoPG), hydrolyses plant-specific polysaccharide pectin. The endoPG encoding gene (PGU1) is also present in Saccharomyces cerevisiae. It has been shown that this endoPG is required for the development of pseudohyphae. Using genomic DNA, the PGU1-1 and PGU1-2 promoters of these strains have been amplified and used to construct gene fusions with the beta-galactosidase gene. On the basis of beta-galactosidase measurements, we compared the expression of both promoters in different environmental conditions in order to identify their modulation. We have shown that the PGU1 gene is upregulated by the presence of the pectin and the product resulting from endopolygalacturonase activity. Moreover, expression of the PGU1 is also enhanced under respiratory and filament formation conditions.

Purification and characterization of acidic endo-polygalacturonase encoded by the PGL1-1 gene from Saccharomyces cerevisiae.

The PGL1 gene of the yeast Saccharomyces cerevisiae has been shown to encode polygalacturonase. Cloning of the PGL1 open reading frame behind the ADH1 promoter allowed overexpression of polygalacturonase activity in S. cerevisiae. This enzyme was purified to apparent homogeneity from cultures of recombinant S. cerevisiae on synthetic medium using one-step purification by anionic exchange chromatography. The enzyme, named Pgl1P, had an apparent M(r) of 42 kDa as shown by SDS-PAGE. Pgl1P was active from pH 3 to 5.5, with an optimum temperature at 25 degrees C. This enzyme hydrolyzed polygalacturonic acid as an endo-polygalacturonase as demonstrated by independent methods. The purified protein was N-glycosylated. However, the activity remained in the N-deglycosylated form. The N-terminal amino acid sequence was also determined as D-S-C-T-L-T-G-S-S-L.

Cloning, sequence analysis and overexpression of a Saccharomyces cerevisiae endopolygalacturonase-encoding gene (PGL1).

Only a few yeast strains produce pectin-degrading enzymes such as pectin esterases and depolymerases (hydrolases and lyases). Strain SCPP is the only known Saccharomyces strain to produce these pectinases. One of these pectolytic enzymes. PGL1-encoded endopolygalacturonase (EC 3.2.1.15), hydrolyses the alpha-1,4-glycosidic bonds within the rhamnogalacturonan chains in pectic substances. This paper presents the cloning and sequencing of the first S. cerevisiae gene involved in pectin degradation. Few differences were found between the two deduced amino acid sequences encoded by PGL1-1 from a pectolytic (PG+) strain (SCPP) and PGL1-2 from a non-pectolytic (PG-) strain (X2180-1B). Similarities were found with other polygalacturonases from plants and other microorganisms. Of the two S. cerevisiae genes, only the one isolated from strain SCPP was able, by overexpression, to confer endopolygalacturonase activity to a laboratory strain of S. cerevisiae. Overexpression of PGL1-1 gene in a non-pectolytic strain resulted in halo formation on polygalacturonic acid-containing agar plates stained with ruthenium red.