FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No.968 up to 19 mg/L in a pilot industry-scale batch fermentation.Here we report a genomics-guided discovery of Burkholderia tha...FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No.968 up to 19 mg/L in a pilot industry-scale batch fermentation.Here we report a genomics-guided discovery of Burkholderia thailandensis MSMB43 as a new and significantly better source of FK228.The genome of B.thailandensis MSMB43 was found to contain a functional biosynthetic gene cluster highly homologous to that of FK228 in C.violaceum No.968,and the bacterium indeed produces authentic FK228.By simple fermentation in shaking flasks in a preferred M8 medium,B.thailandensis MSMB43 produced FK228 up to 67.7 mg/L;by fedbatch fermentation in a 20-L fermentor in M8 medium,B.thailandensis MSMB43 produced FK228 up to 115.9 mg/L,which is 95 fold higher than that of C.violaceum No.968 under the same laboratory fermentation conditions.RT-PCR analysis indicated that the high FK228 yield of B.thailandensis MSMB43 was due to high expression of biosynthetic genes,represented by Bth_depA,during the fermentation process.Further genetic manipulation resulted in a recombinant strain,B.thailandensis MSMB43/pBMTL3-tdpR,which harbors a broad host-range vector expressing the thailandepsin biosynthetic pathway regulatory gene tdpR.This engineered strain produced up to 168.5 mg/L of FK228 in fed-batch fermentation in a 20-L fermentor in M8 medium.Therefore,the wild-type B.thailandensis MSMB43 or its engineered derivative could potentially be a good starting point for an industrial process to improve FK228 production for its expanding use in therapy.展开更多
Antibody-mediated rejection(AMR)is one of the major causes of graft loss after transplantation.Recently,the regulation of B cell differentiation and the prevention of donor-specific antibody(DSA)production have gained...Antibody-mediated rejection(AMR)is one of the major causes of graft loss after transplantation.Recently,the regulation of B cell differentiation and the prevention of donor-specific antibody(DSA)production have gained increased attention in transplant research.Herein,we established a secondary allogeneic in vivo skin transplant model to study the effects of romidepsin(FK228)on DSA.The survival of grafted skins was monitored daily.The serum levels of DSA and the number of relevant immunocytes in the recipient spleens were evaluated by flow cytometry.Then,we isolated and purified B cells from B6mouse spleens in vitro by magnetic bead sorting.The B cells were cultured with interleukin-4(IL-4)and anti-clusters of differentiation 40(CD40)antibody with or without FK228 treatment.The immunoglobulin G1(IgG1)and IgM levels in the supernatant were evaluated by enzyme-linked immunosorbent assay(ELISA).Quantitative reverse transcription-polymerase chain reaction(RT-q PCR)and western blotting were conducted to determine the corresponding levels of messenger RNA(mRNA)and protein expression in cultured cells and the recipient spleens.The results showed that FK228 significantly improved the survival of allogeneic skin grafts.Moreover,FK228 inhibited DSA production in the serum along with the suppression of histone deacetylase 1(HADC1)and HDAC2 and the upregulation of the acetylation of histones H2A and H3.It also inhibited the differentiation of B cells to plasma cells,decreased the transcription of positive regulatory domain-containing 1(Prdm1)and X-box-binding protein 1(Xbp1),and decreased the expression of phosphorylated inositol-requiring enzyme 1α(p-IRE1α),XBP1,and B lymphocyte-induced maturation protein-1(Blimp-1).In conclusion,FK228 could decrease the production of antibodies by B cells via inhibition of the IRE1α-XBP1 signaling pathway.Thus,FK228 is considered as a promising therapeutic agent for the clinical treatment of AMR.展开更多
基金the University of Wisconsin-Milwaukee Research Foundation and a Public Health Service grant(CA152212)the National Cancer Institute to YQC,the National Science Foundation of China(31430002,31770055,31570031)+1 种基金the Fundamental Research Funds for the Central Universities(22221818014)the Major Basic Program of the Natural Science Foundation of Shandong Province(ZR2017ZB0206)。
文摘FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No.968 up to 19 mg/L in a pilot industry-scale batch fermentation.Here we report a genomics-guided discovery of Burkholderia thailandensis MSMB43 as a new and significantly better source of FK228.The genome of B.thailandensis MSMB43 was found to contain a functional biosynthetic gene cluster highly homologous to that of FK228 in C.violaceum No.968,and the bacterium indeed produces authentic FK228.By simple fermentation in shaking flasks in a preferred M8 medium,B.thailandensis MSMB43 produced FK228 up to 67.7 mg/L;by fedbatch fermentation in a 20-L fermentor in M8 medium,B.thailandensis MSMB43 produced FK228 up to 115.9 mg/L,which is 95 fold higher than that of C.violaceum No.968 under the same laboratory fermentation conditions.RT-PCR analysis indicated that the high FK228 yield of B.thailandensis MSMB43 was due to high expression of biosynthetic genes,represented by Bth_depA,during the fermentation process.Further genetic manipulation resulted in a recombinant strain,B.thailandensis MSMB43/pBMTL3-tdpR,which harbors a broad host-range vector expressing the thailandepsin biosynthetic pathway regulatory gene tdpR.This engineered strain produced up to 168.5 mg/L of FK228 in fed-batch fermentation in a 20-L fermentor in M8 medium.Therefore,the wild-type B.thailandensis MSMB43 or its engineered derivative could potentially be a good starting point for an industrial process to improve FK228 production for its expanding use in therapy.
基金supported by the National Natural Science Foundation of China(Nos.81873511 and 81471587)。
文摘Antibody-mediated rejection(AMR)is one of the major causes of graft loss after transplantation.Recently,the regulation of B cell differentiation and the prevention of donor-specific antibody(DSA)production have gained increased attention in transplant research.Herein,we established a secondary allogeneic in vivo skin transplant model to study the effects of romidepsin(FK228)on DSA.The survival of grafted skins was monitored daily.The serum levels of DSA and the number of relevant immunocytes in the recipient spleens were evaluated by flow cytometry.Then,we isolated and purified B cells from B6mouse spleens in vitro by magnetic bead sorting.The B cells were cultured with interleukin-4(IL-4)and anti-clusters of differentiation 40(CD40)antibody with or without FK228 treatment.The immunoglobulin G1(IgG1)and IgM levels in the supernatant were evaluated by enzyme-linked immunosorbent assay(ELISA).Quantitative reverse transcription-polymerase chain reaction(RT-q PCR)and western blotting were conducted to determine the corresponding levels of messenger RNA(mRNA)and protein expression in cultured cells and the recipient spleens.The results showed that FK228 significantly improved the survival of allogeneic skin grafts.Moreover,FK228 inhibited DSA production in the serum along with the suppression of histone deacetylase 1(HADC1)and HDAC2 and the upregulation of the acetylation of histones H2A and H3.It also inhibited the differentiation of B cells to plasma cells,decreased the transcription of positive regulatory domain-containing 1(Prdm1)and X-box-binding protein 1(Xbp1),and decreased the expression of phosphorylated inositol-requiring enzyme 1α(p-IRE1α),XBP1,and B lymphocyte-induced maturation protein-1(Blimp-1).In conclusion,FK228 could decrease the production of antibodies by B cells via inhibition of the IRE1α-XBP1 signaling pathway.Thus,FK228 is considered as a promising therapeutic agent for the clinical treatment of AMR.
