Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity.
An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.
Fujii
T
T
Central Laboratories of Key Technology, Kirin Brewery Co., Ltd., Gunma, Japan.
Kondo
K
K
Shimizu
F
F
Sone
H
H
Tanaka
J
J
Inoue
T
T
D002262:Carboxy-Lyases / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D003931:Diacetyl / Q000378:metabolism; D005285:Fermentation; D005800:Genes, Fungal; D005822:Genetic Vectors; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D002262:Carboxy-Lyases; C023746:acetolactate decarboxylase; D003931:Diacetyl