Cloning and characterization of a second α-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae

Date
2003
Authors
Eksteen J.M.
Steyn A.J.C.
van Rensburg P.
Cordero Otero R.R.
Pretorius I.S.
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Abstract
Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. α-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 α-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second α-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55 307 Da. The LKA2-encoded α-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the α-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1p) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active α-amylase. The LKA2-encoded α-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing α-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60°C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient α-amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S. cerevisiae for the efficient bioconversion of starch into commercially important commodities. The nucleotide sequence of the LKA2 gene has been assigned GenBank Accession No. AF443872. Copyright © 2002 John Wiley & Sons, Ltd.
Description
Keywords
amino acid, amylase, bacterial enzyme, complementary DNA, cyclomaltodextrin glucanotransferase, fungal protein, glucan 1,4 alpha glucosidase, nucleotide, phosphoglycerate kinase, starch, Aspergillus nidulans, biotransformation, controlled study, Debaryomyces, Endomycetales, enzyme specificity, fungal gene, fungal strain, gene expression, genetic analysis, genetic code, genome analysis, intron, medical research, molecular cloning, molecular weight, nonhuman, nucleotide sequence, open reading frame, pH measurement, priority journal, promoter region, review, Saccharomyces cerevisiae, Schizosaccharomyces pombe, sequence analysis, sequence homology, temperature measurement, yeast, alpha-Amylase, Amino Acid Sequence, Base Sequence, Cloning, Molecular, DNA, Complementary, Hydrogen-Ion Concentration, Molecular Sequence Data, Saccharomyces cerevisiae, Saccharomycetales, Substrate Specificity, Aspergillus, Bacteria (microorganisms), Debaryomyces, Debaryomyces occidentalis, Emericella nidulans, Lipomyces kononenkoae, Saccharomyces, Saccharomyces cerevisiae, Saccharomycopsis, Saccharomycopsis fibuligera, Schizosaccharomyces, Schizosaccharomyces pombe
Citation
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