Biosynthetic Studies Of Desmethyl-C-1027 And Iso-Migrastatin From Metabolically Engineered Streptomyce Strains

Biosynthetic studies of desmethyl-C-1027 and iso-Migrastatin from metabolically engineered Streptomyce strains were carried out in the present work.C-1027, a potent anticancer agent, is a chromoprotein natural product produced by Streptomyces globisporus and belongs to the enediyne family of antitumor antibiotics. The sgcD4 gene was successfully inactivated in the C-1027 producer S. globisporus to generate engineered mutant strain SB1052 for the production of desmethyl-C-1027, and the C-1027 production could be restored by expressing sgcD4 in trans. These findings not only support the previous functional assignment of sgcD4 to encode an O-methyl transferase, but also significantly highlight the wisdom to generate structural diversity of complex natural products, such as enediynes, by rational engineering of their biosynthetic pathways. Among C-1027 analogues reported, only desmethyl-C-1027 could induce DNA damage response similar to its parent compounds. Subsequently, fermentation optimization for desmethyl-C-1027 production in the SB 1052 strain is described. With systematic comparison and evaluation, the final optimized fermentation medium was determined. The optimized yield of desmethyl-C-1027 chromoprotein in flask or the bench-top fermentor was-95 mg/L, which is 7-fold higher than the original medium, and reached the level of the parent compound C-1027 produced by the wild-type strain (50～100 mg/L). This study represents an effective strategy combining the contemporary combinatorial biosynthesis methods and traditional fermentation optimization skills to improve titers of a designer metabolite in an engineered strain, which provides us a viable means to expand natural product libraries for drug discovery.iso-Migrastatin (iso-MGS) has emerged and been actively pursued recently as an outstanding candidate of antimetastasis agents. With characterized iso-MGS biosynthetic gene cluster, the heterologous production of iso-MGS was successfully carried out in five engineered Streptomyces hosts. However, under otherwise similar fermentation conditions, the iso-MGS titer in the engineered strains are significantly lower than that in the native producer Streptomyces platensis NRRL 18993. To explore the capacity of hosts to produce iso-MGS, a thorough examination of these five engineered strains and three fermentation media for iso-MGS production was undertaken. The comparison of hosts revealed that SB 11001 and SB 11002 are better candidates to support iso-MGS heterologous production. Subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract are optimal components 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 SB11001 was significantly improved to 186.7 mg/L by hybrid media strategy. And with the addition of 2 g/L NaHCO3, the final highest titer of iso-MGS was 213.8 mg/L, about 4.9-folds higher than the original one. Choosing SB 11001 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 had displayed the connection between gene expression and iso-MGS production for the first time. It seemed that the synchronous expression of the whole gene cluster rather than the individual gene is more crucial to affect the iso-MGS production.These exciting results have revealed the potential of the heterologous production and the details of the biosynthetic mechanism of iso-MGS, and provided the primary data for better improvement of the heterologous host, to realize the large scale production of iso-MGS and its further clinical developments.