Cloning, sequence analysis and expression in yeasts of a cDNA containing a Lipomyces kononenkoae alpha-amylase-encoding gene.
The yeast Lipomyces kononenkoae (Lk) secretes a highly active raw starch-degrading alpha-amylase (alpha Amy) that liberates reducing groups from glucose polymers containing both alpha-1,4 and alpha-1,6 bonds. The LKA1 gene encoding this industrially important alpha Amy was cloned as a 2261-bp cDNA fragment from a glucose-derepressed mutant (IGC4052B) of Lk and characterized. The nucleotide (nt) sequence of the cDNA fragment was determined, revealing an open reading frame of 1872 bp, encoding a 596 amino-acid (aa) mature protein (LKA1) with a calculated M(r) of 65,706. The similarity between the aa sequence of LKA1 and those of other alpha Amy showed four common conserved regions characteristic of the alpha Amy protein family: (A) 264DIVVNH269, (B) 349GLRIDTVKH357, (B') 376GEVFD380 and (C) 439FLENQD444. The deduced aa sequence revealed significant homology to the aa sequences of the Aspergillus oryzae, Schwanniomyces occidentalis and Saccharomycopsis fibuligera alpha Amy, various bacterial cyclomaltodextrin glucanotransferases, a beta-amylase and the 5'-region of a glucoamylase. LKA1 was expressed in Saccharomyces cerevisiae (Sc) under the control of the phosphoglycerate kinase (PGK1) promoter and Northern blot analysis showed the presence of a single 2.3-kb transcript. The 28-aa signal peptide of the LKA1 protein efficiently directed its secretion into the medium when expressed in Sc.
Department of Microbiology, University of Stellenbosch, South Africa.
A J
AJ
Steyn
J
J
Marmur
I S
IS
Pretorius
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013045:Species Specificity; D015003:Yeasts / Q000235:genetics*; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000516:alpha-Amylases