Activation Of Enediyne Biosynthetic Gene Cluster In Streptomyces Sp. CB02130 And Enediyne Titer Improvement In Streptomyces Sp. CB03234

Enediynes are some of the most toxic natural products ever discovered,such as calicheamicin and C-1027 with half maximal inhibitory concentrations between 0.001~10 p M for selected cancer cell lines.All enediynes contain a core unit consisting of two acetylenic groups conjugated to a double bond,which may form a transient benzenoid diradical through reductive activation.The benzenoid diradical causes DNA double-strand breaks and inter-strand cross-links by abstracting hydrogen atoms from the DNA strand.Due to their exquisite mechanism of action and extraordinary cytotoxicity,enediyne natural products are excellent warheads for antibody-drug conjugates.Ten-membered enediyne calicheamicin was the warhead of Mylotarg and Besponsa used for the treatment of acute myeloid leukemia and acute lymphoblastic leukemia,respectively.Fourteen enediynes have been structurally characterized to date.With the development of microbial genomics and the deeper understanding of enediyne biosynthesis,the large gap between the enediyne biosynthetic gene clusters(BGCs)and the known enediyne natural products suggested that most enediyne BGCs are silent under conventional culture conditions.In order to obtain new enediyne natural products,new methods are needed to activate these silent BGCs.Tiancimycin is the anthraquinone-fused enediyne with potent antitumor activity against melanoma and breast cancer,suggesting that it is an ideal payload for antibody-drug conjugates.The titer of tiancimycin was only 0.3 mg/L in Streptomyces sp.CB03234,suggesting the need for its titer improvement.In this thesis,we activated a nine-membered enediyne BGC in Streptomyces sp.CB02130 and also studied titer improvement of tiancimycin in CB03234.The main research content is as follows:Activation and biosynthetic study of enediyne BGC in Streptomyces sp.CB02130.Firstly,Bioinformatics analysis revealed seven enediyne BGCs with a conserved regulatory cassette.The wuliangshan(wls)enediyne BGC in S.sp.CB02130 was prioritized as a prototype BGC,since CB02130 can produce all trans-heptaene(1).The regulatory genes in wls BGC were systematically knocked-out or overexpressed.When we inactivated the Tet R-family regulatory gene wls R3,the resulting mutant strain produced the caffeic acid derivatives 8-9 and pentaene polyols 5-7.Compounds 5-7 have two vicinal diols that are different from the previously reported end products of enediyne polyketide synthases,such as 1.After further knock-out of the polyketide synthase gene wls E and thioeaterase gene wls E10 in Δwls R3,5-7 disappeared.In addition,sgc E,responsible for C-1027 core biosynthesis,could successfully complemented the wls E null mutant,which suggests that 5-7 are relevant to the biosynthesis of 9-membered enediyne core.We further characterized WlsC4 as an ammonia lyase by in vitro enzymatic assay,in vivo knockdown,and substrate feeding.WlsC4 has catalytic activity on both L-phenylalanine and L-tyrosine,showing certain degree of substrate tolerate.The comparison of WlsC4 homology model with Sgc C4 revealed that WlsC4 has different amino residues in the Out-loop region.It is consistent with the highly homologous Sgc C4 and WlsC4 with identical catalytic residues,while they exhibit distinct catalytic activity.Finally,we used wls PDH,the unique gene of wls-type enediyne BGC,as the in silico probe to mine 32 new enediyne BGCs in Genbank.A co-evolutionary relationship between wls PDH homologs and enediyne BGCs was proposed.Metabolic engineering improved the production of anthraquinone-fused enediynes tiancimycin.Firstly,the mutant CB03234-R-16 was obtained by ribosome engineering and its yield could reach to 1.18 mg/L.In order to avoid the degradation of tiancimycin in the fermentation and relieve tiancimycin toxicity in the producing strain,six different types of microporous resins were evaluated;HP2MGL resins were selected and added to the production medium.When 1.5% HP2MGL(m/v)resins were added,the titer of tiancimycin reached to 7.3 mg/L.Through the analysis of mycelium-time and p H-time curves of CB03234 and CB03234-R-16,we found that the mycelium decreased sharply with the sharp increase of the p H of supernatant around 120 h.It indicated that the lack of nutrients in the medium led to the gradual death of culturing strain.When fourfold of the original production medium was used for tiancimycin production,its yield reached to 22.5 mg/L.A scale-up fermentation of CB03234-R-16 on a 15-L fermenter was explored for the influence of inoculation volume,dissolved oxygen,p H,and resin amounts.The yield of tiancimycin in the 15-L fermenter was 14 mg/L.Deciphering of the biosynthetic mechanism of enediyne core remains a significant challenge.In this study,three all-trans pentaene polyols related to enediyne core biosynthesis were discovered,distinct from all other all-trans polyenes reported in the past 15 years,through the activation of enediyne BGC in CB02130.It reveals potential oxidative tailoring steps for construction of nine-membered enediyne macrocycles.By fermentation optimization in both shaking-flasks and 15-L fermenters,the titer of tiancimycin was 22.5 and 14 mg/L,respectively,which was 75 or 47-fold that of the wild-type strain CB03234,laying a solid foundation for development of tiancimycin into promising anticancer drugs.