Isolation of a new gene (SW A2) encoding an α‐amylase from Schwanniomyces occidentalis and its expression in Saccharomyces cerevisiae

Abarca, Dolores; Fernàndez-Lubato, M.; Pozo, Lourdes Del; Jiménez, Antonio Moreno

doi:10.1016/0014-5793(91)80245-x

Cited by 19 publications

(24 citation statements)

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“…The nonconventional yeast Schwanniomyces occidentalis (also Debaryomyces occidentalis) has been regarded for years as a biotechnologically interesting organism because of its ability to grow in a broad range of inexpensive carbon sources, such as starch and inulin, using a number of activities (12)(13)(14)(15), as well as for its efficient extracellular secretion of high molecular mass proteins (16,17) and its low attached glycosylation (18). An extracellular invertase/fructofuranosidase activity has also been reported in this yeast when lactose was used as the carbon source (14).…”

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confidence: 99%

Structural and Kinetic Analysis of Schwanniomyces occidentalis Invertase Reveals a New Oligomerization Pattern and the Role of Its Supplementary Domain in Substrate Binding

Álvaro-Benito

Polo

González

et al. 2010

Journal of Biological Chemistry

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Schwanniomyces occidentalis invertase is an extracellular enzyme that hydrolizes sucrose and releases ␤-fructose from various oligosaccharides and essential storage fructan polymers such as inulin. We report here the three-dimensional structure of Sw. occidentalis invertase at 2.9 Å resolution and its complex with fructose at 1.9 Å resolution. The monomer presents a bimodular arrangement common to other GH32 enzymes, with an N-terminal 5-fold ␤-propeller catalytic domain and a C-terminal ␤-sandwich domain for which the function has been unknown until now. However, the dimeric nature of Sw. occidentalis invertase reveals a unique active site cleft shaped by both subunits that may be representative of other yeast enzymes reported to be multimeric. Binding of the tetrasaccharide nystose and the polymer inulin was explored by docking analysis, which suggested that medium size and long substrates are recognized by residues from both subunits. The identified residues were mutated, and the enzymatic activity of the mutants against sucrose, nystose, and inulin were investigated by kinetic analysis. The replacements that showed the largest effect on catalytic efficiency were Q228V, a residue putatively involved in nystose and inulin binding, and S281I, involved in a polar link at the dimer interface. Moreover, a significant decrease in catalytic efficiency against inulin was observed in the mutants Q435A and Y462A, both located in the ␤-sandwich domain of the second monomer. This highlights the essential function that oligomerization plays in substrate specificity and assigns, for the first time, a direct catalytic role to the supplementary domain of a GH32 enzyme.Fructans, the fructose-rich polymers derived biosynthetically from sucrose, are important storage oligosaccharides and polysaccharides in many bacteria and fungi and numerous plant species. Furthermore, sucrose is one of the most widespread disaccharides in nature and is especially ubiquitous in higher plants as the first free sugar resulting from photosynthesis. It is the major transport compound to bring energy and carbon skeletons from source to sink tissues. Carbohydrate partitioning and sugar sensing are intimately connected to sucrose metabolism; these processes are vital throughout plant development. Therefore, the enzymes involved in fructans and sucrose processing are essential to plant cell metabolism.The enzymes that hydrolyze sucrose are referred to collectively as invertases or ␤-fructofuranosidases (EC 3.2.1.26) and catalyze the release of ␤-fructose from the nonreducing end of various ␤-D-fructofuranoside substrates (Fig. 1). The cleavage of the ␤-glycosidic bond is carried out by a double displacement catalytic mechanism that retains the configuration of the fructose anomeric carbon, two conserved residues, an aspartic and a glutamic acid, being the nucleophile and the general acid-base catalyst, respectively. On the basis of the amino acid sequences (1) they are classified into family 32 of the glycosylhydrolases (GH32), 3 which are included in...

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Structural and Kinetic Analysis of Schwanniomyces occidentalis Invertase Reveals a New Oligomerization Pattern and the Role of Its Supplementary Domain in Substrate Binding

Álvaro-Benito

Polo

González

et al. 2010

Journal of Biological Chemistry

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“…Several of its amylolytic enzymes have been characterized, including amylases and glucoamylase (1,9), as well as an invertase (17). In addition, we also characterized an extracellular ␤-fructofuranosidase (Ffase) from this yeast that hydrolyzes sucrose, 1-kestose, and nystose (5).…”

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New Insights into the Fructosyltransferase Activity ofSchwanniomyces occidentalisβ-Fructofuranosidase, Emerging from Nonconventional Codon Usage and Directed Mutation

Álvaro-Benito

Abreu

Portillo

et al. 2010

Appl Environ Microbiol

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Schwanniomyces occidentalis ␤-fructofuranosidase (Ffase) releases ␤-fructose from the nonreducing ends of ␤-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k cat /K m ) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.

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“…A majority of S. occidentalis promoters of analysed genes worked in S. cerevisiae cells, e.g. ADE2 (Klein and Favreau, 1988), SWA1 and SWA2 (genes encoding amylases; Abarca et al, 1988Abarca et al, , 1991. However, the S. occidentalis gene encoding glucoamylase GAM1 was not expressed in S. cerevisiae from its own promoter, which had to be replaced by a S. cerevisiae promoter (Dohmen et al, , 1990.…”

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Development of a reporter system for the yeast Schwanniomyces occidentalis: influence of DNA composition and codon usage

Janatová

Costaglioli

Wesche

et al. 2003

Yeast

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In this paper we report on searching for suitable reporters to monitor gene expression and protein secretion in the amylolytic yeast Schwanniomyces occidentalis. Several potential reporter and marker genes, formerly shown to be functional in other yeasts, were cloned downstream from the homologous invertase gene (INV ) promoter and their activity was followed in conditions of repression and derepression of the INV promoter. However, neither β-glucuronidase nor β-lactamase nor phleomycin resistance-conferring gene, all originating from E. coli, were expressed in S. occidentalis cells to such a level to allow for monitoring of their activity. All the reporter genes tested have a higher percentage of GC (47-62%) in their DNA compared to the DNA composition of S. occidentalis genes that are more ATrich (36% GC). The codon usage of all the reporter genes also varies from that of 16 so far sequenced S. occidentalis genes. This suggests that an appropriate composition of DNA and a codon usage similar to S. occidentalis genes might be very important parameters for an efficient expression of a heterologous gene in Schwanniomyces occidentalis. Indeed, two genes originating from Staphylococcus aureus, with an AT-content in their DNA similar to that of S. occidentalis, were functionally expressed in S. occidentalis cells. Both a phleomycin resistance-conferring gene and a chloramphenicol acetyltransferase-encoding gene thus represent suitable reporters of gene expression and protein secretion in S. occidentalis. Additionally, we show in this work that the transcription-regulating region and the signal peptide sequence of the S. occidentalis invertase gene were efficient to direct gene expression and subsequent protein secretion in Saccharomyces cerevisiae.

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Isolation of a new gene (SW A2) encoding an α‐amylase from Schwanniomyces occidentalis and its expression in Saccharomyces cerevisiae

Cited by 19 publications

References 20 publications

Structural and Kinetic Analysis of Schwanniomyces occidentalis Invertase Reveals a New Oligomerization Pattern and the Role of Its Supplementary Domain in Substrate Binding

Structural and Kinetic Analysis of Schwanniomyces occidentalis Invertase Reveals a New Oligomerization Pattern and the Role of Its Supplementary Domain in Substrate Binding

New Insights into the Fructosyltransferase Activity ofSchwanniomyces occidentalisβ-Fructofuranosidase, Emerging from Nonconventional Codon Usage and Directed Mutation

Development of a reporter system for the yeast Schwanniomyces occidentalis: influence of DNA composition and codon usage

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