Kay Jennifer E, Jewett Michael C
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA.
Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA.
Metab Eng Commun. 2019 Nov 20;10:e00114. doi: 10.1016/j.mec.2019.e00114. eCollection 2020 Jun.
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 , 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 . 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 ( 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.
对可持续、低成本生产生物能源和商品化学品的需求日益增加。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，基于无细胞或其他的方法可能提供一种替代方案。在此，我们评估了三类生长毒性化合物对基于粗提物的无细胞化学转化的影响。作为一个模型系统，我们在大肠杆菌裂解物中测试了一条将葡萄糖转化为2,3-丁二醇（2,3-BDO）的代谢途径。首先，我们用不同类别的抗生素对2,3-BDO的生产进行了表征，正如预期的那样，我们发现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（甲醇、正丁醇）的影响。我们观察到，随着添加醇类疏水性的增加，体积产率（克/升/小时）会降低。最后，我们研究了使用预处理的生物质水解物作为原料的影响，观察到由于香豆酰和阿魏酰酰胺的存在，2,3-BDO的产量降低了25%。总体而言，我们发现无细胞系统对于抗生素以及其他对细胞生长有毒但不会使相关酶变性或抑制相关酶的化合物的工作浓度具有耐受性。
