Zhang Jiawei, Huang Haolong, Zhang Zhengyu, Tang Jiawei, Gui Jiali, Yang Songbai, Chen Shaoxin
National Key Laboratory of Lead Druggability Research, China State Institute of Pharmaceutical Industry, Shanghai Institute of Pharmaceutical Industry, 285 Gebaini Road, Pudong, Shanghai, 201203, P. R. China.
Department of Biological Medicines & Shanghai Engineering Research Center of Immunotherapeutics, Fudan University School of Pharmacy, 826 Zhangheng Road, Pudong, Shanghai, 201203, P. R. China.
World J Microbiol Biotechnol. 2025 Jun 25;41(7):224. doi: 10.1007/s11274-025-04410-x.
Tacrolimus, an important immunosuppressive drug widely used in clinical practice, is produced by Streptomyces tsukubaensis. In order to enhance the strain productivity, a high-yield mutant strain, 2108N-1-4, was obtained through natural isolation, screening and physical mutagenesis form S. tsukubaensis NRRL 18488. Transcriptome analysis was conducted to identify the potential modification targets, among which the overexpression of regulatory gene fkbN was found to significantly improve tacrolimus production. To further optimize the expression of fkbN through altering promoter strength and increasing the gene copy number, a strain 2108N-1-4/DfkbN carried three copies of fkbN were constructed. The engineered strain achieved a tacrolimus yield of 1342 mg/L. Additionally, it was found that in the late fermentation phase, high concentration of tacrolimus inhibits both the growth of the strain and the expression of genes within the tacrolimus biosynthetic gene cluster. To address this issue, adsorbents such as LX-60 and β-cyclodextrin were added in the fermentation medium to reduce the tacrolimus concentration. This approach increased the tacrolimus yield to 3746 mg/L in flask shake culture and 3639 mg/L when up-scaled to a 5 L fermenter. This study significantly improves tacrolimus production through strain mutation breeding, genetic engineering and process optimization, achieving the highest production yield reported to date. These findings provide valuable insights for the industrial-scale production of tacrolimus.
他克莫司是一种广泛应用于临床的重要免疫抑制药物，由筑波链霉菌产生。为提高菌株的生产力，通过自然分离、筛选及物理诱变，从筑波链霉菌NRRL 18488中获得了高产突变菌株2108N-1-4。进行转录组分析以确定潜在的修饰靶点，其中发现调节基因fkbN的过表达显著提高了他克莫司的产量。为通过改变启动子强度和增加基因拷贝数进一步优化fkbN的表达，构建了携带三个fkbN拷贝的菌株2108N-1-4/DfkbN。该工程菌株的他克莫司产量达到了1342 mg/L。此外，还发现发酵后期高浓度的他克莫司会抑制菌株的生长以及他克莫司生物合成基因簇内基因的表达。为解决这一问题，在发酵培养基中添加了LX-60和β-环糊精等吸附剂以降低他克莫司的浓度。此方法在摇瓶培养中将他克莫司产量提高到了3746 mg/L，扩大到5 L发酵罐培养时产量为3639 mg/L。本研究通过菌株诱变育种、基因工程和工艺优化显著提高了他克莫司的产量，达到了迄今为止报道的最高产量。这些发现为他克莫司的工业化生产提供了有价值的见解。
