Development of a screening method for the identification of a novel Saccharomyces cerevisiae mutant over-expressing Trichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) from Trichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme from Saccharomycopsis fibuligera significantly hindered its expression and secretion in Saccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes in S. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) in S. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenised S. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain of S. cerevisiae, WM91-CBGL1, which exhibited up to 200 U L-1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with the T. reesei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.8 page(s

Effect of the cellulose-binding domain on the catalytic activity of a β-glucosidase from Saccharomycopsis fibuligera

Enzyme engineering was performed to link the β-glucosidase enzyme (BGL1) from Saccharomycopsis fibuligera to the cellulose-binding domain (CBD2) of Trichoderma reesei cellobiohydrolase (CBHII) to investigate the effect of a fungal CBD on the enzymatic characteristics of this non-cellulolytic yeast enzyme. Recombinant enzymes were constructed with single and double copies of CBD2 fused at the N-terminus of BGL1 to mimic the two-domain organization displayed by cellulolytic enzymes in nature. The engineered S. fibuligera β-glucosidases were expressed in Saccharomyces cerevisiae under the control of phosphoglycerate-kinase-1 promoter (PGK1 P) and terminator (PGK1 T) and yeast mating pheromone α-factor secretion signal (MFα1 S ). The secreted enzymes were purified and characterized using a range of cellulosic and non-cellulosic substrates to illustrate the effect of the CBD on their enzymatic activity. The results indicated that the recombinant enzymes of BGL1 displayed a 2-4-fold increase in their hydrolytic activity toward cellulosic substrates like avicel, amorphous cellulose, bacterial microcrystalline cellulose, and carboxy methyl cellulose in comparison with the native enzyme. The organization of the CBD in these recombinant enzymes also resulted in enhanced substrate affinity, molecular flexibility and synergistic activity, thereby improving the ability of the enzymes to act on and hydrolyze cellulosic substrates, as characterized by adsorption, kinetics, thermal stability, and scanning electron microscopic analyses.9 page(s

Different genetic backgrounds influence the secretory expression of the LKA1-encoded Lipomyces kononenkoae α-amylase in industrial strains of Saccharomyces cerevisiae

A haploid laboratory strain and four industrial (baking, brewing, wine, ATCC) strains of Saccharomyces cerevisiae were transformed with the Lipomyces kononenkoae α-amylase-encoding gene (LKA1). These transformants displayed significant differences in terms of the level of secretory expression of LKA1 under control of the PGK1 promoter and terminator, as well as their ability to produce and secrete the LKA1-encoded rawstarch-degrading ?-amylase and to ferment starch. These results demonstrate the importance of the selection of appropriate host strains for yeast development pursuant to starch conversion into commercially important commodities via consolidated bioprocessing.6 page(s

Distinct genetic code expansion strategies for selenocysteine and pyrrolysine are reflected in different aminoacyl-tRNA formation systems

Selenocysteine and pyrrolysine, known as the 21st and 22nd amino acids, are directly inserted into growing polypeptides during translation. Selenocysteine is synthesized via a tRNA-dependent pathway and decodes UGA (opal) codons. The incorporation of selenocysteine requires the concerted action of specific RNA and protein elements. In contrast, pyrrolysine is ligated directly to tRNA(Pyl) and inserted into proteins in response to UAG (amber) codons without the need for complex re-coding machinery. Here we review the latest updates on the structure and mechanisms of molecules involved in Sec-tRNA(Sec) and Pyl-tRNA(Pyl) formation as well as the distribution of the Pyl-decoding trait. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved

Different genetic backgrounds influence the secretory expression of the LKA1-encoded Lipomyces kononenkoae alpha-amylase in industrial strains of Saccharomyces cerevisiae

The original publication is available at www.springerlink.com.Includes bibliography.A haploid laboratory strain and four industrial (baking, brewing, wine, ATCC) strains of Saccharomyces cerevisiae were transformed with the Lipomyces kononenkoae alpha-amylase-encoding gene (LKA1). These transformants displayed significant differences in terms of the level of secretory expression of LKA1 under control of the PGK1 promoter and terminator, as well as their ability to produce and secrete the LKA1-encoded rawstarch-degrading alpha-amylase and to ferment starch. These results demonstrate the importance of the selection of appropriate host strains for yeast development pursuant to starch conversion into commercially important commodities via consolidated bioprocessing

Branchiostoma floridae has separate healing and sealing enzymes for 5′-phosphate RNA ligation

Animal cells have two tRNA splicing pathways: (i) a 5′-P ligation mechanism, where the 5′-phosphate of the 3′ tRNA half becomes the junction phosphate of the new phosphodiester linkage, and (ii) a 3′-P ligation process, in which the 3′-phosphate of the 5′ tRNA half turns into the junction phosphate. Although both activities are known to exist in animals, in almost three decades of investigation, neither of the two RNA ligases has been identified. Here we describe a gene from the chordate Branchiostoma floridae that encodes an RNA ligase (Bf RNL) with a strict requirement for RNA substrates with a 2′-phosphate terminus for the ligation of RNAs with 5′-phosphate and 3′-hydroxyl ends. Unlike the yeast and plant tRNA ligases involved in tRNA splicing, Bf RNL lacks healing activities and requires the action of a polynucleotide kinase (PNK) and a cyclic phosphodiesterase (CDPase) in trans. The activities of these two enzymes were identified in a single B. floridae protein (Bf PNK/CPDase). The combined activities of Bf RNL and Bf PNK/CPDase are sufficient for the joining of tRNA splicing intermediates in vitro, and for the functional complementation of a tRNA ligase-deficient Saccharomyces cerevisiae strain in vivo. Hence, these two proteins constitute the 5′-P RNA ligation pathway in an animal organism

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA(Pyl) by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements required for recognition and aminoacylation of tRNA(Pyl) in vivo by using the Pyl analog N-ε-cyclopentyloxycarbonyl-l-lysine. Forty-two Methanosarcina barkeri tRNA(Pyl) variants were tested in Escherichia coli for suppression of the lac amber A24 mutation; then relevant tRNA(Pyl) mutants were selected to determine in vivo binding to M. barkeri PylRS in a yeast three-hybrid system and to measure in vitro tRNA(Pyl) aminoacylation. tRNA(Pyl) identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51:C63, and the anticodon flanking nucleotides U33 and A37. Transplantation of the tRNA(Pyl) identity elements into the mitochondrial bovine tRNA(Ser) scaffold yielded chimeric tRNAs active both in vitro and in vivo. Because the anticodon is not important for PylRS recognition, a tRNA(Pyl) variant could be constructed that efficiently suppressed the lac opal U4 mutation in E. coli. These data suggest that tRNA(Pyl) variants may decode numerous codons and that tRNA(Pyl):PylRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code

Domain engineering of Saccharomyces cerevisiae exoglucanases

To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg⁻¹ respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.8 page(s