Research Of Related Biosynthetic Genes For The Antitumor Natural Products CC-1065 And YM-216391

CC-1065 produced by Streptomyces Zelensvs NRRL 11183 was found by the Upjohn Company,USA,in 1978,which possesses anti-fungal and anti-tumor activity.CC-1065 belongs to the same family with Yatakemycin,Duocarmycin A,DuocarmycinS with the skeleton of one indole and two pyrrol indoles connected through two amide bonds.The cyclopropane benzodiazepine pyrrolediyl,the function group of CC-1065,could directly bind DNA to form a stable structure in the cells of the G2 and M phases of mitosis,and the structure is difficult for helicase to unwind DNA,strongly inhibiting the tumor cells to propagate.Although the CC-1065 has a strong anti-tumor activity,the clinical application is limited for its poor water-solubility,liver and kidney toxicity.We want to understand its biosynthesis mechanisms so as to change the structures and develop new,less toxic,higher-potency compounds.On the basis of complete CC-1065 gene cluster obtained by Dr Jian Xiaohong,the gene cluster was detailedly analyzed,containing a total of 38.626 kb,29 ORFs.In addition,we speculate the most likely function of each gene in the CC-1065 biosynthesis,and give a reasonable assumption of CC-1065 biosynthesis mechanisms.This work will focus on the function analysis of 10 genes in CC-1065 gene cluster.By knocking-out,c10V,c10K,c10E,c10S and c10O gene mutants were found not to produce CC-1065 by HPLC analysis,while c10W,c10D and c10R6 gene mutants could still produce CC-1065.When the resistance gene c10R5 was knocked out,CC-1065 productivity of the deletion mutation strain AR5 reduced 17%compared with the wild-type,and the recovered mutant increased by 5%,suggesting the self-protection mechanism of the resistance.In c10A deletion experiments,a new compound with m/z = 432(M + NH4)was accumulated,and the identification of the new compound may be usful to reveal the CC-1065 biosynthetic mechanism.The preliminary study of those genes laid the foundation for us to know CC-1065 biosynthetic pathway,as well as the related enzyme catalysis research to create new compounds.In addition,our group has studied the representative of the cyclic peptide YM-216391.Cyclopeptide YM-216391 contains multioxazolines-thiazole ring and has a good anti-tumor activity.Previously,our laboratory has cloned the biosynthetic gene cluster,and this study will explore the heterologous synthesis of cyclic peptide YM-216391.The results showed that YM-216391b could be heterologously expressed in several different Streptomyces hosts,of which the highest yield was in the Streptomyces albus.On this basis,through the medium formulation improvements,YM-216391 yield was raise to 5.664 ± 1.176 mg/L,providing a basis for follow-up research to produce new compounds by combinational biosynthesis.