Targeted proteomics for metabolic pathway optimization: Application to terpene production
Targeted proteomics for metabolic pathway optimization: Application to terpene production
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DOI：
10.1016/j.ymben.2010.12.005
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发表时间：
2011-03-01
影响因子：
8.4
通讯作者：
Petzold, Christopher J.
中科院分区：
文献类型：
--
作者：
Redding-Johanson, Alyssa M.;Batth, Tanveer S.;Petzold, Christopher J.
成功的代谢工程依赖于有助于组装和优化微生物新途径的方法。许多不同的因素可能影响通路的性能，而由于mRNA丰度、蛋白质丰度或酶活性引起的问题可能无法通过监测产物滴度来发现。为此，合成生物学家和代谢工程师利用各种分析方法来确定限制生产的途径部分。在这项研究中，靶向蛋白质组学，通过选择反应监测（SRM）质谱法，被用于测量大肠杆菌菌株的蛋白质水平，这些菌株被改造成产生这些squiterpene， amorpha4,11-diene。从这一分析中，两个甲戊酸途径蛋白，即来自酿酒酵母的甲戊酸激酶（MK）和磷酰戊酸激酶（PMK），被确定为潜在的瓶颈。对MK和PMK编码基因进行密码子优化，并从一个较强的表达促进了MK和PMK蛋白水平的显著提高，最终定形蛋白4,11-二烯滴度提高了3倍以上（50 ~ 500 mg/L）。(C) 2011爱思唯尔公司版权所有。
Successful metabolic engineering relies on methodologies that aid assembly and optimization of novel pathways in microbes. Many different factors may contribute to pathway performance, and problems due to mRNA abundance, protein abundance, or enzymatic activity may not be evident by monitoring product titers. To this end, synthetic biologists and metabolic engineers utilize a variety of analytical methods to identify the parts of the pathway that limit production. In this study, targeted proteomics, via selected-reaction monitoring (SRM) mass spectrometry, was used to measure protein levels in Escherichia coli strains engineered to produce these squiterpene, amorpha-4,11-diene. From this analysis, two mevalonate pathway proteins, mevalonate kinase (MK) and phosphomevalonate kinase (PMK) from Saccharomyces cerevisiae, were identified as potential bottlenecks. Codon-optimization of the genes encoding MK and PMK and expression from a stronger promoterled to significantly improved MK and PMK protein levels and over three-fold improved final amorpha-4,11-diene titer(> 500 mg/L). (C) 2011 Elsevier Inc. All rights reserved.