Improvement of FK506 Production in Streptomyces tsukubaensis by Genetic Enhancement of the Supply of Unusual Polyketide Extender Units via Utilization of Two Distinct Site-Specific Recombination Systems
Improvement of FK506 Production in Streptomyces tsukubaensis by Genetic Enhancement of the Supply of Unusual Polyketide Extender Units via Utilization of Two Distinct Site-Specific Recombination Systems
复制标题
通过利用两种不同的位点特异性重组系统来遗传增强不寻常的聚酮化合物延伸单元的供应，从而提高筑波链霉菌中 FK506 的产量
DOI：
10.1128/aem.00450-12
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发表时间：
2012-08-01
影响因子：
4.4
通讯作者：
Liu, Wen
中科院分区：
文献类型：
--
作者：
Chen, Dandan;Zhang, Qi;Liu, Wen
FK 506是一种有效的免疫抑制剂，具有广泛的临床应用。它的23元大环支架，主要与聚酮化合物的起源，具有两个甲氧基在C-13和C-15和一个烯丙基侧链在C-21，由于区域特异性掺入两个不寻常的扩展单元，分别来自甲氧基丙二酰-酰基载体蛋白（ACP）和烯丙基丙二酰-辅酶A（CoA）。它们的细胞内形成是否会成为FK 506生产的瓶颈仍然是难以捉摸的。在这项研究中，我们报告了FK 506产量的提高生产菌株Streptomyces tsukubaensis的两套途径特异性基因的重复，分别编码这两个扩展单元的生物合成，从而提供了一个有前途的方法来产生高FK 506生产菌株通过遗传操作。利用S. tsukubaensis是适合两个放线菌噬菌体（ΦC31和VWB）整合酶介导的重组系统，我们遗传增强甲氧基丙二酰-ACP和烯丙基丙二酰-CoA的生物合成，如转录分析所示。随着葡萄糖补充量的优化，最终FK 506的最大滴度与原始菌株相比增加了约150%。这里描述的工程不寻常的扩展单元的生物合成的策略可能适用于提高生产的其他聚酮化合物或非核糖体肽天然产物，含有途径特异性的积木。
ABSTRACT FK506 is a potent immunosuppressant that has a wide range of clinical applications. Its 23-member macrocyclic scaffold, mainly with a polyketide origin, features two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, due to the region-specific incorporation of two unusual extender units derived from methoxymalonyl-acyl carrier protein (ACP) and allylmalonyl-coenzyme A (CoA), respectively. Whether their intracellular formations can be a bottleneck for FK506 production remains elusive. In this study, we report the improvement of FK506 yield in the producing strain Streptomyces tsukubaensis by the duplication of two sets of pathway-specific genes individually encoding the biosyntheses of these two extender units, thereby providing a promising approach to generate high-FK506-producing strains via genetic manipulation. Taking advantage of the fact that S. tsukubaensis is amenable to two actinophage (ΦC31 and VWB) integrase-mediated recombination systems, we genetically enhanced the biosyntheses of methoxymalonyl-ACP and allylmalonyl-CoA, as indicated by transcriptional analysis. Together with the optimization of glucose supplementation, the maximal FK506 titer eventually increased by approximately 150% in comparison with that of the original strain. The strategy of engineering the biosynthesis of unusual extender units described here may be applicable to improving the production of other polyketide or nonribosomal peptide natural products that contain pathway-specific building blocks.