Systems metabolic engineering of Escherichia coli for production of the antitumor drugs violacein and deoxyviolacein
Systems metabolic engineering of Escherichia coli for production of the antitumor drugs violacein and deoxyviolacein
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DOI：
10.1016/j.ymben.2013.08.004
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发表时间：
2013-11-01
影响因子：
8.4
通讯作者：
Wittmann, Christoph
中科院分区：
文献类型：
--
作者：
Rodrigues, Andre L.;Trachtmann, Nathalie;Wittmann, Christoph
紫紫素和脱氧紫紫素是治疗致病菌和病毒以及肿瘤细胞的有趣药物。在目前的工作中，全系统代谢工程应用于大肠杆菌这一在制药生物技术中被广泛接受的重组宿主，以生产这些高价值产品。在araC诱导体系的控制下，表达紫色色杆菌vioABCE簇的基本生产者大肠杆菌dVio-1积累了180 mg L-1的脱氧紫色素。靶向细胞内代谢物分析随后确定了色氨酸支持途径的瓶颈，这是主要的产品构建块，用于丝氨酸，choris酸和色氨酸生物合成以及非氧化型葡萄糖-磷酸盐途径的综合工程。最终菌株大肠杆菌dVio-6在摇瓶培养中积累了320 mg L-1脱氧紫罗兰素。高通量色氨酸途径的创建底盘由紫肠杆菌的vioD基因的基因组整合补充，这使得紫色素的独家生产。在补料间歇过程中，产生的大肠杆菌Vio-4积累了710 mg L-1的所需产品。通过简单的肉汤提取和结晶，可以得到纯度为99.8%的紫堇素。这证明了大肠杆菌作为生产色氨酸治疗药物的平台的潜力。(C) 2013爱思唯尔公司版权所有。
Violacein and deoxyviolacein are interesting therapeutics against pathogenic bacteria and viruses as well as tumor cells. In the present work, systems-wide metabolic engineering was applied to target Escherichia coli, a widely accepted recombinant host in pharmaceutical biotechnology, for production of these high-value products. The basic producer, E coli dVio-1, that expressed the vioABCE cluster from Chromobacterium violaceum under control of the inducible araC system, accumulated 180 mg L-1 of deoxyviolacein. Targeted intracellular metabolite analysis then identified bottlenecks in tryptophan supporting pathways, the major product building block This was used for comprehensive engineering of serine, chorismate and tryptophan biosynthesis and the non-oxidative pentose-phosphate pathway. The final strain, E coli dVio-6, accumulated 320 mg L-1 deoxyviolacein in shake flask cultures. The created chassis of a high-flux tryptophan pathway was complemented by genomic integration of the vioD gene of Janthinobacterium lividum, which enabled exclusive production of violacein. In a fed-batch process, the resulting producer E coli Vio-4 accumulated 710 mg L-1 of the desired product. With straightforward broth extraction and subsequent crystallization, violacein could be obtained with 99.8% purity. This demonstrates the potential of E. coli as a platform for production of tryptophan based therapeutics. (C) 2013 Elsevier Inc. All rights reserved.