title: Biased mutagenesis in the N-terminal region by degenerate oligonucleotide gene shuffling enhances secretory expression of barley alpha-amylase 2 in yeast.
abstract: Recombinant barley alpha-amylase 1 (rAMY1) and 2 (rAMY2), despite 80% sequence identity, are produced in very different amounts of 1.1 and <0.05 mg/l, respectively, by Saccharomyces cerevisiae strain S150-2B. The low yield of AMY2 practically excludes mutational analysis of structure-function relationships and protein engineering. Since different secretion levels of AMY1/AMY2 chimeras were previously ascribed to the N-terminal sequence, AMY1 residues were combinatorially introduced at the 10 non-conserved positions in His14-Gln49 of AMY2 using degenerate oligonucleotide gene shuffling (DOGS) coupled with homologous recombination in S.cerevisiae strain INVSc1. Activity screening of a partial library of 843 clones selected six having a large halo size on starch plates. Three mutants, F21M/Q44H, A42P/A47S and A42P rAMY2, also gave higher activity than wild-type in liquid culture. Only A42P showed wild-type stability and enzymatic properties. The replacement is located to a beta-->alpha loop 2 that interacts with domain B (beta-->alpha loop 3) protruding from the catalytic (beta/alpha)(8)-barrel. Most remarkably Pichia pastoris strain GS115 secreted 60 mg/l A42P compared with 3 mg/l of wild-type rAMY2. The crystal structure of A42P rAMY2 was solved and found to differ marginally from the AMY2 structure, suggesting that the high A42P yield stems from stabilization of the mature and/or intermediate form owing to the introduced proline residue. Moreover, the G to C substitution for the A42P mutation might have a positive impact on protein translation.
authors.affiliation: Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark.
authors.forename: Kenji
authors.initials: K
authors.lastname: Fukuda
authors.forename: Malene H
authors.initials: MH
authors.lastname: Jensen
authors.forename: Richard
authors.initials: R
authors.lastname: Haser
authors.forename: Nushin
authors.initials: N
authors.lastname: Aghajari
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D004795:Enzyme Stability / Q000235:genetics; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D013213:Starch / Q000737:chemistry / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013213:Starch; D000516:alpha-Amylases