Biosynthetic engineering and fermentation media development leads to gram-scale production of spliceostatin natural products in Burkholderia sp.
Biosynthetic engineering and fermentation media development leads to gram-scale production of spliceostatin natural products in Burkholderia sp.
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DOI：
10.1016/j.ymben.2015.11.003
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发表时间：
2016-01-01
影响因子：
8.4
通讯作者：
Koehn, Frank E.
中科院分区：
文献类型：
--
作者：
Eustaquio, Alessandra S.;Chang, Li-Ping;Koehn, Frank E.
天然产物药物发现的一个关键挑战是化合物供应。需要数百克的纯化材料来推进天然产品的临床前开发。剪接体抑制素是与剪接体结合的聚酮-非核糖体肽天然产物，剪接体是癌症治疗的新兴靶点。野生型细菌伯克霍尔德氏菌FERM BP-3421产生一套具有不同生物活性和生理稳定性的spliceostatin同源物。半缩酮化合物如FR 901464是第一个被描述的。由于其改进的性质，我们特别感兴趣的羧酸前体类似物，首次报道从伯克霍尔德氏菌属MSMB 43，并命名为thailanstatin A。铁/α-酮戊二酸依赖性双加氧酶基因fr 9 P的失活已显示阻断半缩酮生物合成。然而，泰兰他汀A的4-脱氧同源物是在双加氧酶突变体中观察到的主要产物。我们在这里表明，细胞色素P450基因fr 9 R的表达是一个代谢瓶颈，因为使用t-阿拉伯糖诱导系统导致4-脱氧类似物几乎完全转化为靶分子。通过将发酵培养基开发方法与生物合成工程相结合，我们能够将目标化合物的生产滴度提高> 40倍，从开始的类似60 mg/L提高到2.5 g/L，并实现主要是单一组分的生产概况。这些改进有助于使spliceostatin类似物作为化疗的临床前开发成为可能。(C)2015年国际代谢工程学会。爱思唯尔公司出版All rights reserved.
A key challenge in natural products drug discovery is compound supply. Hundreds of grams of purified material are needed to advance a natural product lead through preclinical development. Spliceostatins are polyketide-nonribosomal peptide natural products that bind to the spliceosome, an emerging target in cancer therapy. The wild-type bacterium Burkholderia sp. FERM BP-3421 produces a suite of spliceostatin congeners with varying biological activities and physiological stabilities. Hemiketal compounds such as FR901464 were the first to be described. Due to its improved properties, we were particularly interested in a carboxylic acid precursor analog that was first reported from Burkholderia sp. MSMB 43 and termed thailanstatin A. Inactivation of the iron/oc-ketoglutarate-dependent dioxygenase gene fr9P had been shown to block hemiketal biosynthesis. However, a 4-deoxy congener of thailanstatin A was the main product seen in the dioxygenase mutant. We show here that expression of the cytochrome P450 gene fr9R is a metabolic bottle neck, as use of an t-arabinose inducible system led to nearly complete conversion of the 4-deoxy analog to the target molecule. By integrating fermentation media development approaches with biosynthetic engineering, we were able to improve production titers of the target compound > 40-fold, going from the starting similar to 60 mg/L to 2.5 g/L, and to achieve what is predominantly a single component production profile. These improvements were instrumental in enabling preclinical development of spliceostatin analogs as chemotherapy. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.