Discovery and detection of free cytosine in Streptomyces lividans

Abstract:

[Background] Cytosine is one of the four basic bases of nucleic acids. Cytosine is firstly synthesized in the form of cytosine triphosphate. There is no specific primary pathway for the formation of free cytosine in nucleic acid primary metabolism. The biosynthetic pathways of blasticidin S and gougerotin both utilize free cytosine as the precursor. The biosynthetic gene cluster of blasticidin S contains a hydrolase BlsM that can hydrolyze cytidine monophosphate into cytosine, while gougerotin producer doesn’t encodes a homolog to BlsM. [Objective] To detect the existence of free cytosine in different bacteria, and to explore whether there is an isoenzyme or a new pathway to produce free cytosine. [Methods] blsM was knocked out in blasticidin S heterologous producer Streptomyces lividans WJ2, its fermentation products of the mutant strain and WJ2 were detected by high performance liquid chromatography. Free cytosine in the supernatant of 10 fractionated cell lysates was measured by LC-MS. [Results] The mutant WJ2?blsM strain still synthesizes blasticidin S, but the yield of each component is significantly lower than that of WJ2. In addition to Streptomyces lividans, free cytosine was detected in Staphylococcus aureus, Amycolatopsis mediterranei and Bacillus subtilis. [Conclusion] WJ2?blsM still produces blasticidin S, indicating that wild-type Streptomyces lividans has an uncovered pathway for synthesis of free cytosine to meet blasticidin S synthesis. The content of free cytosine varies in different microorganisms.