Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol

Abstract

The commercial application of genetically modified industrial microorganisms has been problematic due to public concerns. We constructed a "self-cloning" sake yeast strain that overexpresses the ATF1 gene encoding alcohol acetyltransferase, to improve the flavor profile of Japanese sake. A constitutive yeast overexpression promoter, TDH3p, derived from the glyceraldehyde-3-phosphate dehydrogenase gene from sake yeast was fused to ATF1; and the 5' upstream non-coding sequence of ATF1 was further fused to TDH3p-ATF1. The fragment was placed on a binary vector, pGG119, containing a drug-resistance marker for transformation and a counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the ATF1 locus of a sake yeast strain. This integration constructed tandem repeats of ATF1 and TDH3p-ATF1 sequences, between which the plasmid was inserted. Loss of the plasmid, which occurs through homologous recombination between either the TDH3p downstream ATF1 repeats or the TDH3p upstream repeat sequences, was selected by growing transformants on counter-selective medium. Recombination between the downstream repeats led to reversion to a wild type strain, but that between the upstream repeats resulted in a strain that possessed TDH3p-ATF1 without the extraneous DNA sequences. The self-cloning TDH3p-ATF1 yeast strain produced a higher amount of isoamyl acetate. This is the first expression-controlled self-cloning industrial yeast.

Deletion or Overexpression of Mitochondrial NAD + Carriers in Saccharomyces cerevisiae Alters Cellular NAD and ATP Contents and Affects Mitochondrial Metabolism and the Rate of Glycolysis

“…A strain overexpressing NDT1 (ndt1-over strain) was obtained by the one-step substitution of the chromosomal NDT1 promoter for the TDH3 promoter (P TDH3 ). Its sequence has been demonstrated to drive the constitutive and strong overexpression of downstream genes (16). To this end, we constructed a promoter-substitution cassette, with NatMX4 as a selectable marker for nourseothricin resistance, by cloning P TDH3 amplified from S. cerevisiae genomic DNA into the NdeI and HindIII sites of the pAG25 vector (13).…”

Progress in Metabolic Engineering of Saccharomyces cerevisiae

“…In general, it has been assumed that self-cloned yeasts have a greater potential for commercialization because of better consumer acceptance compared to transgenic organisms. In fact, yeast genetic engineers have expended significant effort on reinforcing self-cloning strategies (4,14,123,339,385). It is also obvious that the focus on self-cloning in food-related yeast metabolic engineering will, in the future, direct more attention to inverse metabolic engineering approaches, i.e., exploiting the natural diversity of yeast.…”

Large-Scale Selection and Breeding To Generate Industrial Yeasts with Superior Aroma Production

“…Genetic engineering of genes coding for key enzymes, such as ATF1 and ATF2, has been demonstrated to yield remarkable changes in the production of acetate esters (5,7,8).…”