Construction Of An Efficient Biosynthesis System Of Tiancimycins And Discovery Of Yuanjiangmycins In S.sp.CB03234

The enediyne natural products are among the most cytotoxic small molecules known to date,and are classified into nine-membered and ten-membered enediynes.Anthraquinone-fused enediynes belong to ten-membered enediynes,which are ideal warheads for antibody-drug conjugates(ADCs).The yields of anthraquinone-fused enediynes in the original bacterial hosts are typically very low,which may be due to their extremely strong cytotoxicity.This seriously limits their biosynthetic research and drug development as warheads for ADCs.Tiancimycins(TNMs)are a new class of anthraquinone-fused enediynes discovered in Streptomyces sp.CB03234 through a novel genome mining technology.One of their salient features is that they exhibit rapid and complete cell killing ability to various cancer cell lines.The CB03234 strain can produce two enediyene congeners,i.e.,TNM-A and TNM-D,but their total yield is only 0.3 mg/L.Therefore,we designed and constructed an efficient biosynthetic system of TNMs in CB03234,by utilizing ribosome engineering,fermentation process optimization,and combined metabolic engineering strategies.In addition,ribosome engineering led to the activation of a silent biosynthetic gene cluster(BGC)in CB03234,and two new Pluramycin-family natural products Yuanjiangmycin(YJM)A and B with an usual 3,6-dideoxycycled sugar structure were isolated and characterized.The major facilitator superfamily(MFS)transporter Tnm T1 in TNMs BGC is responsible for the activation of YJMs in CB3234-S.The main research contents are as follows:(1)First,ribosome engineering on the wild-type strain CB03234 based on streptomycin-resistance was utilize to obtain a total of 71 mutants.A high-yield TNMs-producing strain CB03234-S with a double mutation of30 S ribosomal protein S12(rps L)and S-adenosylmethionine dependent16 S r RNA methyltransferase gene(rsm G)was screened out by a 96-well plate-based activity screening method.Through a systematic optimization of the culture media and fermentation engineering,the yield of TNMs increased from about 0.3 mg/L to 12.9 ± 0.3 mg/L.Furthermore,the biosynthetic capacity of TNMs in CB03234-S was significantly increased in a 30-L fermenter over the wild-type strain by a 109-fold increase of the total yield of TNMs.The yield of TNM-A and TNM-D reached to 13.7 ±0.3 and 19.2 ± 0.4 mg/L,respectively.Finally,the separation and purification processes of TNMs were optimized,with over 55% of their overall recovery rate.(2)Activation of a silent type II polyketide synthase BGC in CB03234-S to produce YJMs.Two new natural products YJM-A and B were discovered in CB03234-S,and their structures were determined through high-resolution mass spectrometry,as well as 1D and 2D nuclear magnetic resonance spectroscopy.YJM-A and B belong to Pluramycintype aromatic polyketides containing rare 3,6-dideoxycycled sugar structures.YJM-A had n M cytotoxicity against multiple tumor cell lines.In order to explore the activation mechanism of YJMs BGC in CB03234,we first constructed a rps L and rsm G double mutant strain in CB03234wild-type,and found that this mutant did not produce YJMs.However,YJMs BGC was unexpectedly activated when an MFS transporter gene tnm T1 belonging to TNMs BGC was overexpressed in CB03234,in order to explore the effect of tnm T1 on TNMs production.Gene knockout of tnm T1 in CB03234-S led to the abolishment of YJMs production.These data are consistent with the overexpression of tnm T1 in CB03234-S.Taken together,yjm BGC was directly activated by Tnm T1 from TNMs BGC.(3)Combined metabolic engineering strategies led to high-yield production of TNMs based on CB03234-S.Re-sequencing of CB03234-S genome revealed the deletion of a 583-kb DNA fragment,accounting for about 7.5% of its genome,in addition to the two previous ribosomal mutation sites.There are seven BGCs in the deleted DNA fragment.We further sequenced the transcriptome of both CB03234-S and CB03234wild-type strains.Based on the genome and transcriptome analyses,we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a highyield strain of TNMs based on CB03234-S.Inactivating seven potential competitive BGCs(including type I,II and III polyketide synthase BGCs and terpenoid BGCs)individually and jointly resulted in nine mutant strains.The mutant strain S1009 with the inactivation of both Tiancilactones and YJMs BGCs had a TNMs titer of28.2 ± 0.8 mg/L,which is 2.2-fold increase over that of CB03234-S.At the same time,we overexpressed five essential structural genes involved in TNMs biosynthesis,two genes responsible for post-translational modification,and three genes involved in their self-resistance,based on p SET152 and erm E*p strong promoter.Only mutants carrying core genes tnm E or tnm E10 showed the enhanced TNMs production.The yield of the mutant strain S1011 overexpressing tnm E reached to 27.7 ± 2.8 mg/L,which is about 2.1-fold of the original CB03234-S.The three mutants overexpressing the three resistance genes in the TNMs BGC significantly increased the tolerance of the host to TNM-A,but the yield of TNMs did not increase significantly.Finally,the overexpression of tnm E and tnm E10 in S1009 resulted in S1021 mutant,which produced 41.2 ± 2.7mg/L of TNMs in shaking flasks and 43.5 ± 2.4 mg/L of TNMs in a pilot scale 30 L fermenter.Therefore,the TNMs titer in S1021 was about 3.6-fold or 145-fold higher than that of CB03234-S and CB03234,which represented the highest reported titer among anthraquinone-fused enediynes so far.Moreover,a novel enediyne derivative TNM-W was also produced by S1021 under p H-stable fermentation conditions in the30-L fermenter.The structural characterization and preliminary biological studies indicated that TNM-W was the first anthraquinone-fused enediyne with a unique double bond fused to the enediyne carbocycle instead of a common epoxide moiety.In sum,efficient biosynthetic systems of enediyne TNMs from CB03234 have been designed and constructed.The titer of TNMs reached to 43.5 ± 2.4 mg/L in a pilot scale production,which is 145-fold higher than that of CB03234.This laid a solid foundation for further drug development of TNMs as ADC warheads,as well as biosynthetic studies of anthraquinone-fused enediynes and discovery of more effective derivatives.The systematic strategy to improve the yield of TNMs,including ribosome engineering,fermentation process optimization,and a combined metabolic engineering strategy,may be instrumental for improving the yield of other low-yield natural products in Streptomyces strains.In addition,two YJMs with a novel 3,6-dideoxycycled sugar structure was produced by an activated yjm BGC in CB03234-S,while an unusual activation mechanism based on an MFS transporter outside of yjm BGC was responsible for YJMs production.This represents a new strategy for the activation of silent BGCs in microorganisms.