Strategies to produce high levels of extracellular cAMP based on cAMP-PKA and purine synthesis pathway regulation in Saccharomyces cerevisiae
Shaolan Zou, Pingsheng Ma.
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
cAMP, an important compound involved in various biochemical reactions and a key second messenger, is widely used in animal food additives and pharmaceutical research. In yeast, cAMP concentration is tightly regulated because of the important role of the cAMP-PKA pathway in controlling a broad array of cellular processes. Up to now, lots of research has been focused on the cAMP-mediated regulatory pathway and the regulation of intracellular cAMP levels in Saccharomyces cerevisiae. In this study, the extracellular cAMP level and its regulation was investigated in detail and Saccharomyces cerevisiae was shown to be an excellent candidate for fermentative production of cAMP after manipulating the cAMP-PKA and purine synthesis pathways and optimizing the media. First, the cAMP dependent protein kinase (PKA)- encoding genes were deleted to remove the powerful feedback inhibition of PKA on cAMP synthesis and the lack of viability of tpk1 tpk2 tpk3 triple mutants was further suppressed by mutations such as yak1 or msn2/msn4. Second, the low-affinity cAMP phosphodiesterase Pde1 was inactivated to decrease the breakdown of cAMP. Third, Bas1p and Bas2p were fused and over-expressed by co-integrating into the genome to reinforce their cooperative interaction in purine synthesis pathway and thus the production of the cAMP precursors ADP and ATP. Bas1p is a Myb-related transcription factor. It acts together with the homeodomain-related Bas2p (Pho2p) to regulate purine and histidine biosynthesis genes in response to extracellular purine limitation. Fourth, the content of yeast extract and peptone in fermentation media were increased and the ratio of carbon to nitrogen source was optimized. As a result, the extracellular cAMP level could reach 6414.3 μmol/L.