A cell-free system for production of 2,3-butanediol is robust to growth-toxic compounds
A cell-free system for production of 2,3-butanediol is robust to growth-toxic compounds
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DOI：
10.1016/j.mec.2019.e00114
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发表时间：
2020-06-01
影响因子：
5.2
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
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作者：
Kay, Jennifer E.;Jewett, Michael C.
对生物能源和大宗化学品的可持续、低成本生产的需求正在不断增加。不幸的是，全细胞催化剂满足这一需求的工程潜力可能会受到细胞毒性的阻碍。当此类瓶颈限制全细胞发酵的商业可行性时，基于无细胞或体外的方法可能提供替代方案。在这里，我们评估了三类生长有毒化合物对基于粗提取物的无细胞化学转化的影响。作为模型系统，我们测试了大肠杆菌裂解物中葡萄糖转化为 2,3-丁二醇 (2,3-BDO) 的代谢途径。首先，我们对不同类别抗生素的 2,3-BDO 生产进行了表征，结果正如预期的那样，该系统不受通过细胞壁复制以及 DNA、RNA 和蛋白质合成来阻止细胞生长的化合物的抑制。其次，我们考虑了添加极性溶剂（例如甲醇、正丁醇）的影响。我们观察到，随着添加醇的疏水性增加，体积生产率（g/L/h）减慢。最后，我们研究了使用预处理的生物质水解产物作为原料的效果，观察到由于香豆酰和阿魏酰酰胺，2,3-BDO 产量减少了 25%。总体而言，我们发现无细胞系统对抗生素和其他对细胞生长有毒的化合物的工作浓度具有鲁棒性，但不会使相关酶变性或抑制。
The need for sustainable, low-cost production of bioenergy and commodity chemicals is increasing. Unfortunately, the engineering potential of whole-cell catalysts to address this need can be hampered by cellular toxicity. When such bottlenecks limit the commercial feasibility of whole-cell fermentation, cell-free, or in vitro, based approaches may offer an alternative. Here, we assess the impact of three classes of growth toxic compounds on crude extract-based, cell-free chemical conversions. As a model system, we test a metabolic pathway for conversion of glucose to 2,3-butanediol (2,3-BDO) in lysates of Escherichia coli. First, we characterized 2,3-BDO production with different classes of antibiotics and found, as expected, that the system is uninhibited by compounds that prevent cell growth by means of cell wall replication and DNA, RNA, and protein synthesis. Second, we considered the impact of polar solvent addition (e.g., methanol, n-butanol). We observed that volumetric productivities (g/L/h) were slowed with increasing hydrophobicity of added alcohols. Finally, we investigated the effects of using pretreated biomass hydrolysate as a feed stock, observing a 25% reduction in 2,3-BDO production as a result of coumaroyl and feruloyl amides. Overall, we find the cell-free system to be robust to working concentrations of antibiotics and other compounds that are toxic to cell growth, but do not denature or inhibit relevant enzymes.