Abstract: A novel pathway for the biosynthesis of caffeine with xanthine as substrate has been found in microbes, which is an effective way to improve the efficiency of caffeine synthesis from xanthine produced from guanine catalyzed by guanine deaminase. This study aimed to clone the guanine deaminase gene and to construct a prokaryotic expression system to synthesize xanthine efficiently. The two guanine deaminase genes gud1 and egud were amplified from Saccharomyces cerevisiae and Escherichia coli by PCR, respectively. The target fragments were inserted into the pMAL-c5X vector and then the recombinant plasmid transformed into E. coli BL21(DE3) to induce protein expression. The expressed products were determined by high performance liquid chromatography (HPLC). Both the recombinant vectors pMAL-gud1 and pMAL-egud had the capability of synthesizing xanthine, and the efficiency of xanthine synthesis with GUD1 was higher as compared to EGUD. This study can enrich the theory of black tea processing technology and provide a theoretical foundation for constructing engineered strains that are capable of producing caffeine.

Key words: dark tea processing, caffeine, guanine deaminase, xanthine, biosynthesis, prokaryotic expression