Department of Chemical and Biological Engineering, Institute of Bioengineering, Zhejiang University, Hangzhou, Zhejiang, China.
Appl Microbiol Biotechnol. 2011 Mar;89(6):1709-19. doi: 10.1007/s00253-010-3025-1. Epub 2010 Dec 4.
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.
异美登他汀（iso-MGS）最近作为一种优秀的抗转移剂候选药物受到了广泛关注。从其天然产生菌链霉菌 platensis NRRL 18993 中鉴定出异美登他汀生物合成基因簇后，我们最近成功地在五种选定的异源链霉菌宿主中生产出了异美登他汀，尽管产量较低，未能达到预期，并对该新技术在大规模生产中的应用提出了质疑。为了进一步探索和利用这些宿主的生产能力，我们对这五种用于异美登他汀生产的工程菌株进行了彻底的研究，并使用三种发酵培养基。发现白色链霉菌 J1074 和变铅青链霉菌 K4-114 是较好的异源宿主，随后对碳源和氮源的分析表明，蔗糖和酵母提取物是异美登他汀生产的理想选择。在初步优化后，在优化的 R2YE 培养基中，所有五种宿主的异美登他汀产量都提高了 3-18 倍。此外，通过混合培养基策略，白色链霉菌 J1074（pBS11001）的异美登他汀产量提高到 186.7mg/L。最后，向后者中添加 NaHCO3，最终得到优化的异美登他汀产量为 213.8mg/L，比最初报道的系统提高了约 5 倍。以白色链霉菌 J1074（pBS11001）为模型宿主，通过定量 RT-PCR 技术系统研究了异美登他汀基因簇在四种不同培养基中的表达情况。首次比较了基因表达与异美登他汀生产之间的相关性；整个基因簇的同步表达对于异美登他汀的最佳生产至关重要。这些结果揭示了异美登他汀在异源宿主中的生物合成机制的新见解，并为实现异美登他汀的大规模生产以进行进一步的临床前研究提供了初步数据。
