Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli
Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli
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DOI：
10.1073/pnas.1515826113
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发表时间：
2016-03-22
影响因子：
11.1
通讯作者：
Ajikumar, Parayil Kumaran
中科院分区：
文献类型：
--
作者：
Biggs, Bradley Walters;Lim, Chin Giaw;Ajikumar, Parayil Kumaran
代谢工程的最新进展证明了利用生物化学合成复杂分子的潜力。到目前为止，大部分进展都利用越来越精确的遗传工具来控制酶的转录和翻译，以获得更好的生物合成途径性能。然而，将这些方法和原理应用于合成更复杂的天然产物将需要一套新的工具来实现体内各种类型的代谢化学(即环化、氧合、糖基化和卤化)。在这些不同的化学反应中，氧合作用是合成复杂天然产物最具挑战性和关键的化学方法之一。在这里，我们以紫杉醇为模型系统，利用自然界青睐的加氧酶--细胞色素P450，在大肠杆菌中进行高水平的氧合化学。P450的表达和上游途径酶的表达意外地耦合被发现并被确定为功能氧化化学的关键障碍。通过优化P450的表达、还原酶伴侣的相互作用和N末端修饰，我们在大肠杆菌中获得了报道的最高含氧紫杉烷效价(类似于570+/-45 mg/L)。总之，这项研究确立了大肠杆菌作为P450化学的易管理宿主，强调了合成生物学和代谢工程背景下蛋白质相互依赖的潜在规模，并指出了复杂化学实体的微生物合成的前景。
Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (similar to 570 +/- 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.