Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

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Cloning and expression of a chicken α-amylase gene

We have isolated and sequenced a genomic clone for a pancreatic α-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic α-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the α-amylase produced by the yeast cells was identical to that of the native chicken enzyme.     

Cloning and expression of the α-amylase gene and oxytetracycline production in Streptomyces rimosus

An 8.4 kb Sau3AI DNA fragment containing the Streptomyces rimosus TM-55 -amylase gene (amy) was ligated to a vector pIJ702, named pCYL01, and cloned into amylase deficient mutant S. lividans M2 (amy ^–). Subcloning study showed that the amy gene was localized in 3.3 kbKpnI-PstI fragment. The molecular weight of the purified -amylases of S. lividans M2/pCYL01 and S. rimosus TM-55 were estimated to be 65.7 kDa. Different sizes of recombinant plasmids carrying the amy gene had been retransferred into the parental strain of S. rimosus TM-55. Among these S. rimosus transformants, TM-55/pCYL01, TM-55/pCYL12 and TM-55/pCYL36 showed amylase activity 1.36- to 2.05-fold at the seventh day (1.61 to 2.42 units vs 1.18 units), and oxytetracycline (OTC) production 2.00- to 2.50-fold at the ninth day (approximate 140 to 170 g ml^–1 vs 72 g ml^–1), higher than that of S. rimosus TM-55 alone, respectively. These results showed that industrial microorganisms could be improved by genetic and metabolic engineering.     

Expression and secretion of barley α-amylase and A.niger glucoamylase inSaccharomyces cerevisiae

cDNAs of barley α-amylase andA. niger glucoamylase were cloned in oneE. coli-yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed intoS. cerevisiae GRF18 by protoplast transformation. The barley α-amylase andA. niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18 (pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used for the production of ethanol.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis α-amylase gene

The gene encoding Schwanniomyces occidentalis -amylase (AMY) was introduced into the chromosomal sequences of an industrial strain of Saccharomyces cerevisiae. To obtain a strain suitable for commercial use, an -integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker. The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p). The -amylase activity of Sacc. cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch. occidentalis. The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.     

Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Transfer and expression of a Bacillus licheniformis α-amylase gene in Zymomonas mobilis

The gene from Bacillus licheniformis coding for a thermostable -amylase was subcloned into the broad-host-range plasmid pKT210 in Escherichia coli. The recombinant plasmid pGNB6 was transferred into Zymomonas mobilis ATCC 31821 by conjugation. Plasmid pGNB6 was stably maintained in E. coli and unstable in Z. mobilis. The amylase gene was expressed in Z. mobilis at a lower level (25%) than in E. coli and regulation of enzyme biosynthesis was different in the host cells. Almost all the -amylase activity was recovered in the culture medium of Z. mobilis. This enzyme localization seemed to be the result of protein secretion rather than cell lysis. Integration of the amylase gene into a cryptic plasmid of Z. mobilis was observed. The amylase gene was still expressed, although at a lower level, and the -amylase activity, associated with a protein of molecular mass 62,000 daltons, was immunologically identical in Z. mobilis, E. coli and B. licheniformis.     

A gene encoding Achlya bisexualis β-amylase and its expression in Saccharomyces cerevisiae

Part of a -amylase genomic DNA sequence from the oomycete, Achlya bisexualis was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotide primers derived from the conserved regions of other known -amylase sequences. The 5- and 3-regions of the -amylase gene were amplified by genome walking method. The Ach. bisexualis -amylase gene consisted of a 1338bp open reading frame, encoding a protein of 446 amino acids with a molecular weight of 49 381Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 67% similarity to the -amylase of Saprolegnia ferax, followed by 40% similarity to that ofArabidopsis thaliana. The -amylase gene was expressed in Saccharomyces cerevisiae placing it under the control of the alcohol dehydrogenase gene (ADC1) promoter.     

Physiological and genetic modulation of inducible expression ofEscherichia coli β-galactosidase inSaccharomyces cerevisiae

Summary Saccharomyces cerevisiae cells transformed with plasmids bearing thelacZ gene fromEscherichia coli, under the control of the inducible GAL1-10/CYC1 promoter, produce the highest amount of -galactosidase during a transient physiological condition corresponding to the early stationary phase of growth. This enhanced enzyme expression is characteristic of active cycling cells down-modulated by the nutrients. By increasing the dosage of the GAL4 gene, after transformation of yeast cells with a multicopy plasmid bearing the GAL4 gene, a positive but limited enhancement of enzyme expression is induced.     

Cloning and expression of the α–L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

 First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1 _P ) and terminator (PGK1 _T ) sequences on a multicopy episomal plasmid. The resulting construct PGK1 _P -abf B-PGK1 _T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively. Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996     

Cloning of the thermostable α-amylase gene from Pyrococcus woesei in Escherichia coli

Pyrococcus woesei (DSM 3773) α-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)α-amyl and pYTB2α-amyl vectors obtained were used for expression of thermostable α-amylase or fusion of α-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of α-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation—they exhibit only 35% of total cell activity—and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable α-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75°C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95°C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120°C. Maltose was the main end product of starch hydrolysis catalyzed by this α-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Cloning and expression in Saccharomyces cerevisiae of an endo-β-glucanase gene from a thermophilic cellulolytic anaerobe

Summary The gene encoding heat-stable -glucanase from a thermophilic cellulolytic anaerobe was recloned in Saccharomyces cerevisiae. Yeast transformant expressed the heat-stable endo--glucanase and produced a level of enzyme activity similar to the Escherichia coli transformant.This work was supported in part by the Biomass Conversion Project of the Ministry of Agriculture, Forestry and Fisheries, Japan     

Hyper expression ofBacillus stearothermophilus α-amylase gene inBacillus subtilis

Summary Utilizing plasmids pUB110, pBR322 and pRG71, we have constructed a number of vectors which could be utilized for expression of cloned genes both in gram positive and gram negative bacteria. This report discusses the hyperexpression of -amylase gene ofB. stearothermophilus BR135 using these vectors.     

Cloning of the Bacillus pumilusβ-xylosidase gene (xynB ) and its expression in Saccharomyces cerevisiae

A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the β-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique. The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2 _P ) and terminator (ADH2 _T ) sequences on a multicopy episomal plasmid (pDLG11). The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone α-factor (MFα1 _S ) before insertion between the ADH2 _P and ADH2 _T sequences on a similar multicopy episomal plasmid (pDLG12). The resulting construct ADH2 _P -MFα1 _S -xynB-ADH2 _T was designated XLO1. Both plasmids pDLG11 and pDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional β-xylosidase. The total β-xylosidase activity remained cell-associated with a maximum activity of 0.09 nkat/ml obtained when the recombinant S. cerevisiae strain was grown for 143 h in synthetic medium. The temperature and pH optima of the recombinant Xlo1 enzyme were 45–50 °C and pH 6.6 respectively. The enzyme was thermostable at 45 °C; however, at 60 °C most of the Xlo1 was inactive after 5 min. Received: 11 July 1996 / Received revision: 23 October 1996 / Accepted: 25 October 1996