Heterologous Expression Of Cordyceps Militaris Cordycepin Synthesis Gene Cluster

Cordycepin,also known as 3’-deoxyadenosine,is a nucleoside analog with a wide range of pharmacological activities,including anti-tumor,anti-inflammatory,antioxidant,anti-diabetic,immunomodulatory,prevention and treatment of neurodegenerative diseases,anticoagulant,inhibition of blood vessel growth,anti-skin photoaging,et al.Thus cordycepin is considered as a precious raw material for the food,drug and cosmetic industries.However,the yield of cordycepin is limited by fungal fermentation,and the phase of chemical synthesis of cordycepin is long accompanying with high pollution to environment.Therefore,with the development of genetic engineering technology,it is possible to obtain recombinant strains with the capability to produce cordycepin by exogenously expressing the cordycepin biosynthesis gene cluster.In this study,the exogenous gene expression was used to express the cordycepin synthesis gene cluster in Escherichia coli and Saccharomyces cerevisiae,and the fermentation conditions were subsequently optimized to finally obtain an engineered strain of cordycepin.The main research results are as follows:1.Bioinformatics analysis of cordycepin biosynthesis gene clusterThe biosynthesis of cordycepin used adenosine or 3’-AMP as a substrate,and the gene clusters responsible for its biosynthesis mainly include cns3,cns2,and cns1 genes.It was found that the cns3,cns2,and cns1 genes encode 871,345,and 792 amino acids,respectively,and the corresponding encoded proteins Cns3,Cns2,and Cns1 have molecular weights of 96 k D,39 k D,and 88 k D.None of the three proteins has a transmembrane domain and signal peptide,and they are all non-secreted proteins.The results of physicochemical property analysis show that Cns3 protein is a stable protein,Cns2 and Cns1 are unstable proteins.Besides,domain analysis showed that,Cns3,Cns2,and Cns1 proteins contain phosphoribosylaminoimidazole succinylformamide synthase domain,phosphohydrolase domain and dehydrogenase domain,which belong to three categories: synthetases,hydrolases,and oxidoreductases,respectively.2.Construction of cordycepin-producing Escherichia coliWe performed two strategies for the expression of Cordyceps militaris biosynthesis gene cluster in Escherichia coli:（ⅰ）The key gene clusters cns3,cns2,and cns1 in the biosynthesis of cordycepin with E.coli codon preference were artificially synthesized into the p BAD24 plasmid.And the three single-gene recombinant plasmids were co-transformed into E.coli MG1655 competent cells to obtain E.coli MG1655（p BAD24-cns3,p BAD24-cns2,p BAD24-cns1）;（ⅱ）using p BAD24-cns3,p BAD24-cns2,p BAD24-cns1 single-gene recombinant plasmids as templates,design primers and add artificial regulatory element RBS to construct three-gene tandem expression vector p BAD24-cns3-cns2-cns1.And three-gene tandem expression vector p BAD24-cns3-cns2-cns1 was also transformed into E.coli MG1655 competent cells E.coli MG1655（p BAD24-cns3-cns2-cns1）.After induced of E.coli engineering strains,it was found that protein bands with corresponding molecular weights could be correctly expressed by SDS-PAGE detection.It was further detected by high performance liquid chromatography（methanol:water=15:85;chromatographic column diamonsil C18;detection wavelength 260 nm;column temperature 30℃;flow rate 1 m L/min）found that E.coli engineering strain MG1655（p BAD24-cns3,p BAD24-cns2,p BAD24-cns1）was fermented with 10 m M L-arabinose and 0.75 m M adenosine for 24 h,and the maximum yield of cordycepin was 4.52±0.03 mg/L.E.coli engineering strain MG1655（p BAD24-cns3-cns2-cns1）under the conditions of adding 15 m M L-arabinose and 0.75 m M adenosine for 32 h fermentation,the maximum yield of cordycepin reached up to5.31±0.05 mg/L,which was 17.5% higher than that of E.coli engineering strain MG1655（p BAD24-cns3,p BAD24-cns2,p BAD24-cns1）.3.Construction of cordycepin-producing Saccharomyces cerevisiaeThe key genes cns3,cns2,and cns1 for the biosynthesis of cordycepin with codon preference in S.cerevisiae were artificially synthesized.And the single-gene yeast expression vector p YES3/CT-cns3 and the dual-gene co-expression vector p ESC-LEU-cns2-cns1 were constructed.The two recombinant expression vectors were transformed into S.cerevisiae INVSC1 strain to obtain S.cerevisiae engineering strain INVSC1（p YES3/CT-cns3,p ESC-LEU-cns2-cns1）.After induction of S.cerevisiae engineered strain,western blot analysis showed that the cns2 and cns1 genes could be expressed normally,but cns3 failed to express.However,S.cerevisiae engineered strain INVSC1（p YES3/CT-cns3,p ESC-LEU-cns2-cns1）that successfully expressed Cns2 and Cns1 proteins can produce cordycepin.It is speculated that the Cns2 and Cns1 proteins can directly utilize the 3’-AMP produced by m RNA degradation in S.cerevisiae to synthesize cordycepin.In addition,after optimization of the amount of added inducer,fermentation culture time and added adenosine,it was found that under the conditions of adding 10 g/L galactose and 0.5 m M adenosine for 36 h fermentation,S.cerevisiae engineered strain INVSC1（p ESC-LEU-cns2-cns1）cordycepin yield reached up to 3.92±0.03 mg/L.In conclusion,in this study,two engineered cordycepin-producing E.coli strains and one engineered cordycepin-producing S.cerevisiae strain were successfully obtained by means of genetic engineering,and the fermentation conditions of the obtained engineered strains were optimized.Finally,the cordycepin yields of the three engineered strains were in the range of 3-6 mg/L,among which the E.coli engineering strain MG1655（p BAD24-cns3-cns2-cns1）had the highest cordycepin yield,which reached up to5.31±0.05 mg/L.This study lays a theoretical foundation for the researches on improving the biological source of cordycepin,and also provides a reference for the in vivo synthesis of other active substances in E.coli and S.cerevisiae.