Titer improvement of iso-migrastatin in selected heterologous Streptomyces hosts and related analysis of mRNA expression by quantitative RT-PCR.
iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3-18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO(3) to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT-PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies.
Department of Chemical and Biological Engineering, Institute of Bioengineering, Zhejiang University, Hangzhou, Zhejiang, China.
Dong
D
Yang
Xiangcheng
X
Zhu
Xueyun
X
Wu
Zhiyang
Z
Feng
Lei
L
Huang
Ben
B
Shen
Zhinan
Z
Xu
D000970:Antineoplastic Agents / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D020869:Gene Expression Profiling; D018942:Macrolides / Q000378:metabolism*; D010881:Piperidones / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048170:Streptomyces lividans / Q000235:genetics* / Q000378:metabolism*
D000970:Antineoplastic Agents; D018942:Macrolides; D010881:Piperidones; D012333:RNA, Messenger; C455261:isomigrastatin