Construction of industrial baker’s yeast with high level of cAMP

Abstract

Cyclic adenosine monophosphate (cAMP) plays an important role in modulating the activity of microbe cell. In this study, PKA (protein kinase A) activity was weakened through truncation of TPK2 promoter (−150 bp and −300 bp) and gene deletion of BCY1 (encodes the regulatory subunit of PKA), TPK1 and TPK3, generating strains BY9a‐T2‐150 and BY9a‐T2‐300, respectively. High‐performance liquid chromatography analysis showed cAMP levels in BY9a‐T2‐150 and BY9a‐T2‐300 were increased by 5‐ and 18‐fold, respectively, compared with that of parent strain, BY9a. The expression levels of TPK2 gene in two engineered strains were decreased by 95% and 97% compared with that of BY9a, respectively. The PKA activity reflected by heat resistance of engineered strains enhanced compared with parent strain BY9a. This study show a new method to increase the intracellular cAMP concentration in industrial yeast by fine‐tuning of PKA activity, without influence in growth and fermentation properties. Practical applications cAMP as the “second messenger,” is essential for plant, animal, and microorganisms and human life. But its synthesis is still limited by expensive cost and time‐consuming method. We constructed the industrial baker’s yeast with high level of cAMP and desired to be used to produce functional food for relaxing smooth muscle, expanding blood vessels, improving liver function, and promoting nerve regeneration and as a food additive for treating hyperthyreosis and hepatopathy. The methods of two step homologous recombination and backcross operated in this study eliminate the exogenous gene in engineered strains, made it safety to be used in food production. Fine‐tuning of PKA activity in engineered strains ensure produce high level of cAMP and exhibit normal growth performance in engineering strains. Therefore, this work is significant in functional foods product and has the potential to be used in practical application.

Crosstalk between protein kinase A and the HOG pathway under impaired biosynthesis of complex sphingolipids in budding yeast

“…cAMP was extracted from S. cerevisiae as described previously [64] with minor modifications. Briefly, cells (8 OD 600 U) were washed with 50 m m Tris/HCl, pH 7.5, and then suspended in 200 µL of 50 m m Tris/HCl, pH 7.5, and 40 µL of dimethyl sulfoxide.…”