Department of Biological Medicines & Shanghai Engineering Research Center of Immunotherapeutics, Fudan University School of Pharmacy, 826 Zhangheng Road, Pudong, Shanghai, 201203, People's Republic of China.
State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, 285 Gebaini Road, Pudong, Shanghai, 201203, People's Republic of China.
Appl Microbiol Biotechnol. 2023 Sep;107(18):5701-5714. doi: 10.1007/s00253-023-12661-7. Epub 2023 Jul 22.
Staurosporine is the most well-known member of the indolocarbazole alkaloid family; it can induce apoptosis of many types of cells as a strong protein kinase inhibitor, and is used as an important lead compound for the synthesis of the antitumor drugs. However, the low fermentation level of the native producer remains the bottleneck of staurosporine production. Herein, integration of multi-copy biosynthetic gene cluster (BGC) in well characterized heterologous host and optimization of the fermentation process were performed to enable high-level production of staurosporine. First, the 22.5 kb staurosporine BGC was captured by CRISPR/Cas9-mediated TAR (transformation-associated recombination) from the native producer (145 mg/L), and then introduced into three heterologous hosts Streptomyces avermitilis (ATCC 31267), Streptomyces lividans TK24 and Streptomyces albus J1074 to evaluate the staurosporine production capacity. The highest yield was achieved in S. albus J1074 (750 mg/L), which was used for further production improvement. Next, we integrated two additional staurosporine BGCs into the chromosome of strain S-STA via two different attB sites (vwb and TG1), leading to a double increase in the production of staurosporine. And finally, optimization of fermentation process by controlling the pH and glucose feeding could improve the yield of staurosporine to 4568 mg/L, which was approximately 30-fold higher than that of the native producer. This is the highest yield ever reported, paving the way for the industrial production of staurosporine. KEYPOINTS: • Streptomyces albus J1074 was the most suitable heterologous host to express the biosynthetic gene cluster of staurosporine. • Amplification of the biosynthetic gene cluster had obvious effect on improving the production of staurosporine. • The highest yield of staurosporine was achieved to 4568 mg/L by stepwise increase strategy.
星孢菌素是吲哚咔唑生物碱家族中最著名的成员；作为一种强大的蛋白激酶抑制剂，它可以诱导多种类型的细胞凋亡，并且被用作合成抗肿瘤药物的重要先导化合物。然而，天然产生菌的低发酵水平仍然是星孢菌素生产的瓶颈。在此，通过 CRISPR/Cas9 介导的 TAR（转化相关重组）从天然产生菌（145mg/L）中捕获 22.5kb 的星孢菌素 BGC，并将其引入三种异源宿主链霉菌阿维霉素（ATCC 31267）、变铅青链霉菌 TK24 和白色链霉菌 J1074 中，以评估星孢菌素的生产能力。在白色链霉菌 J1074 中获得了最高的产量（750mg/L），并用于进一步的生产改进。接下来，我们通过两个不同的 attB 位点（vwb 和 TG1）将另外两个星孢菌素 BGC 整合到菌株 S-STA 的染色体中，导致星孢菌素产量增加了两倍。最后，通过控制 pH 和葡萄糖补料优化发酵过程可以将星孢菌素的产量提高到 4568mg/L，比天然产生菌提高了约 30 倍。这是迄今为止报道的最高产量，为星孢菌素的工业化生产铺平了道路。关键点： • 白色链霉菌 J1074 是表达星孢菌素生物合成基因簇的最适异源宿主。 • 生物合成基因簇的扩增对提高星孢菌素的产量有明显的效果。 • 通过逐步增加策略，星孢菌素的最高产量达到 4568mg/L。
