Metabolic Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare (SMESH) Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Research Division of Food Functionality, Korea Food Research Institute, Wanju-gun, Jeollabuk-do, Republic of Korea.
mBio. 2019 Jan 22;10(1):e02683-18. doi: 10.1128/mBio.02683-18.
Butanol production by is accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report development of several clostridial aldehyde/alcohol dehydrogenase (AAD) variants showing increased butanol selectivity by a series of design and analysis procedures, including random mutagenesis, substrate specificity feature analysis, and structure-based butanol selectivity design. The butanol/ethanol ratios (B/E ratios) were dramatically increased to 17.47 and 15.91 g butanol/g ethanol for AAD and AAD, respectively, which are 5.8-fold and 5.3-fold higher than the ratios obtained with the wild-type AAD. The much-increased B/E ratio obtained was due to the dramatic reduction in ethanol production (0.59 ± 0.01 g/liter) that resulted from engineering the substrate binding chamber and the active site of AAD. This protein design strategy can be applied generally for engineering enzymes to alter substrate selectivity. Renewable biofuel represents one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, clostridial strain improvement has been slower than improvement of other microorganisms. Furthermore, fermentation coproducing various by-products requires costly downstream processing for butanol purification. Here, we report the results of enzyme engineering of aldehyde/alcohol dehydrogenase (AAD) to increase butanol selectivity. A metabolically engineered strain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity.
在生产丁醇的过程中，会同时产生丙酮和乙醇，这会降低丁醇的产量并增加生产成本。在这里，我们报告了几种梭菌醛/醇脱氢酶（AAD）变体的开发情况，这些变体通过一系列设计和分析程序，包括随机诱变、底物特异性特征分析和基于结构的丁醇选择性设计，显示出更高的丁醇选择性。AAD 和 AAD 的丁醇/乙醇比（B/E 比）分别显著提高到 17.47 和 15.91 g 丁醇/g 乙醇，比野生型 AAD 获得的比值高 5.8 倍和 5.3 倍。获得的 B/E 比值大大提高是由于通过工程化 AAD 的底物结合腔和活性位点，导致乙醇产量显著降低（0.59±0.01 g/L）。这种蛋白质设计策略可以普遍应用于工程酶来改变底物选择性。可再生生物燃料是解决能源危机和气候变化问题的答案之一。然而，由于缺乏有效的遗传操作工具，梭菌菌株的改良速度比其他微生物的改良速度慢。此外，发酵联产各种副产物需要昂贵的下游处理来纯化丁醇。在这里，我们报告了醛/醇脱氢酶（AAD）的酶工程结果，以提高丁醇的选择性。表达工程化醛/醇脱氢酶基因的代谢工程化 菌株能够以高选择性生产丁醇。
