Detection Of Free Cytosine In Microbes And Studies On Cytidine Monophosphate Hydrolase

Cytosine(C) is one of the four basic bases of nucleic acids.It mainly exists in the form of nucleosides or nucleotides in microorganisms,and free cytosine is rare.Take Blasticidin S(BS)for example,most of biosynthetic gene cluster of nucleoside antibiotics that utilize free cytosine as the precursor would encode a hydrolase that can hydrolyze cytidine monophosphate(CMP)into cytosine.While Gougerotin producer doesn't encodes a CMP hydrolase.The source of free cytosine in Gougerotin producing bacteria remains unclear.In this study,S.lividans WJ2,a BS heterologous producer along with other ten microbial strains commonly used in our laboratory were assayed for the existence of free cytosine,and explored the possible sources and production methods of free cytosine.In addition,we searched the potential CMP hydrolase in Streptomyces.In this study,bls M,previously assumed to be essential to BS biosynthesis was knocked out in Streptomyces lividans WJ2,unexpectedly,the resultant mutant WJ2?bls M still synthesizes BS and three other BS analogs,but the yield of each component is significantly lower than that of WJ2.The concentration of free cytosine was enriched from the 11 fractionated cell lysates and was measured by LC-MS.Among them,free cytosine was detected in Staphylococcus aureus,Amycolatopsis mediterranei and Bacillus subtilis and Streptomyces lividans.We detected CMP hydrolase activity in the cell-free extract(CFE)of S.lividans or S.coelicolor.Through tracking CMP hydrolytic activity in fractional protein precipitation by saturated ammonium sulfate solution and further resolution of this activity by FPLC,we identified potential hydrolases based on LC-MS/MS and bioinformatics analyses from the two Streptomyces spp.strains.In vitro and in vivo activity assay of these CMP hydrolase candidates were performed,and 8 putative CMP hydrolases were finally determined.In summary,we established a sensitive method for detecting free cytosine based on LC-MS,and proved the existence of free cytosine in Streptomyces lividans.We also found three bacteria that content high free cytosine.Moreover,the Bls M isoenzyme was purified from S.lividans and S.coelicolor.The study provides new insight into the pyrimidine metabolism pathway in microorganisms.It can also provide a reference to research the synergistic effect of primary metabolism on secondary metabolism of microorganisms.Through finding potential CMP hydrolase in S.lividans and S.coelicolor,this paper enriches the hydrolase pool and deepen our understanding of the two heterologous expression hosts.