Abstract
cAMP (cyclic adenosine-3',5'-monophosphate) has extensive physiological functions and nutritional value for living organisms, and it regulates cellular metabolism mainly by modulating PKA (protein kinase A) activity. The current yields of cAMP synthesized by microbial fermentation are still low, which is arousing interest in developing high-yield cAMP strains. In this work, two baker's yeasts with high cAMP content were constructed by knocking out BCY1, TPK3, and TPK2 genes, and truncating the promoter of the TPK1 gene. The content of cAMP in BN5-126 and BN5-310 (with the TPK1 gene promoter truncated by 126 and 310 bp in BN5) was improved by 30- and 9-fold, respectively, relative to the wild strain. The TPK1 gene mRNA levels of BN5-126 and BN5-310 were decreased by 18% and 40%, respectively, without significant changes in growth performance. The results of heat shock tolerance of engineered strains also reflected the enhanced PKA activity. This work demonstrates a novel strategy for regulating gene expression to boost cAMP biosynthesis in yeast, providing a promising platform for producing nutritionally enriched and functional fermented products.