Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates
Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates
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DOI：
10.1093/synbio/ysz003
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发表时间：
2019-01-01
影响因子：
3.2
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
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作者：
Dudley, Quentin M.;Nash, Connor J.;Jewett, Michael C.
异戊二烯类化合物是一类很有吸引力的代谢产物，可用于可再生底物的酶合成。然而，微生物代谢工程用于单萜类化合物的生产受到耗时的、通常是非直观的生物合成途径变异的组合调整以满足设计标准的需要的限制。为了缓解这一限制，这项工作的目标是建立一个模块化的，无细胞的平台，以香料和调味品分子柠檬烯为模型，构建和测试单萜途径。在这个平台中，多个大肠杆菌裂解物混合在一起，每个裂解物都富含一个过表达的途径酶，以构建完整的生物合成途径。首先，我们展示了从六种富含甲氧戊酸底物、三磷酸腺苷(ATP)来源和辅因子的裂解物中合成柠檬烯的能力。接下来，我们扩展了以葡萄糖为底物的途径，依赖于提取物中的天然代谢将葡萄糖转化为乙酰辅酶A，以及另外三种酶将乙酰辅酶A转化为甲氧戊酸。我们发现，天然的大肠杆菌法尼基二磷酸合成酶(ISPA)在裂解物中具有活性，并将通量从中间产物香叶基焦磷酸盐转移到法尼基焦磷酸酯和副产物法尼醇。通过调节辅因子NAD(+)、ATP和CoA的相对水平，该体系可在24 h内合成0.66 mM(90.2 mgL(-1))柠檬烯，产率为3.8 mgl(-1)h(-1)。我们的结果突出了粗裂解物维持复杂代谢的灵活性，通过激活包含20个生物合成步骤的9种异源酶激活葡萄糖到柠檬烯的途径，扩展了一种使用富含酶的裂解物来构建、表征和原型酶途径的方法。
Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often nonintuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD(+), ATP and CoA, the system can synthesize 0.66mM (90.2 mgl(-1)) limonene over 24 h, a productivity of 3.8mgl(-1) h(-1). Our results highlight the flexibility of crude lysates to sustain complex metabolismand, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.