Increased Acetate Ester Production of Polyploid Industrial Brewer’s Yeast Strains via Precise and Seamless “Self-cloning” Integration Strategy

Abstract

<strong>Background:</strong> Enhancing the industrial yeast strains ethyl acetate yield through a precise and seamless genetic manipulation strategy without any extraneous DNA sequences is an essential requisite and significant demand.<br /> <strong>Objectives:</strong> For increasing the ethyl acetate yield of industrial brewer's yeast strain, all the <em>ATF1</em> alleles were overexpressed through “self-cloning" integration strategy.<br /> <strong>Material and Methods:</strong> <em>Escherichia coli </em>strain DH5α was utilized for plasmid construction. <em>ATF1</em> alleles were overexpressed through a precise and seamless insertion of the <em>PGK1</em> promoter in industrial brewer's yeast strain S6. In addition, growth rates, <em>ATF1</em> mRNA levels, AATase activity, the fermentation performance of the engineered strains, and gas chromatography (GC) analysis was conducted.<br /> <strong>Results:</strong> The two engineered strains (S6-P-12 and S6-P-30) overexpressed all <em>ATF1</em> alleles but unaffected normal growth. The <em>ATF1</em> mRNA levels of the S6-P-12 and S6-P-30 were all 4-fold higher than that of S6. The AATase (Alcohol acetyl transferases, encoded by<em> ATF1</em> gene) activity of the two engineered strains was all 3-fold higher than that of the parent strain. In the beer fermentation at 10 ℃, the concentrations of ethyl acetate produced by the engineered strains S6-P-12 and S6-P-30 was increased to 23.98 and 24.00 mg L^-1, respectively, about 20.44% and 20.54% higher than that of S6.<br /> <strong>Conclusions:</strong> These results verify that the ethyl acetate yield could be enhanced by the overexpressed of <em>ATF1</em> in the polyploid industrial brewer's yeast strains via “self-cloning" integration strategy. The present study provides a reference for target gene modification in the diploid or polyploid industrial yeast strains.