Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA; DOE Center Advanced Bioenergy and Bioproducts Innovation, USA.
Department of Chemistry, University of Illinois, Urbana, IL, USA; DOE Center Advanced Bioenergy and Bioproducts Innovation, USA.
Metab Eng. 2023 May;77:21-31. doi: 10.1016/j.ymben.2023.02.012. Epub 2023 Mar 1.
The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by heterologous hosts is expression of a selective acyl-acyl carrier protein (ACP) thioesterase. However, few of these enzymes can generate a precise (greater than 90% of a desired chain-length) product distribution when expressed in a microbial or plant host. The presence of alternative chain-lengths can complicate purification in situations where blends of fatty acids are not desired. We report the assessment of several strategies for improving the dodecanoyl-ACP thioesterase from the California bay laurel to exhibit more selective production of medium-chain free fatty acids to near exclusivity. We demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was an effective library screening technique for identification of thioesterase variants with favorable shifts in chain-length specificity. This strategy proved to be a more effective screening technique than several rational approaches discussed herein. With this data, we isolated four thioesterase variants which exhibited a more selective FFA distribution over wildtype when expressed in the fatty acid accumulating E. coli strain, RL08. We then combined mutations from the MALDI isolates to generate BTE-MMD19, a thioesterase variant capable of producing free fatty acids consisting of 90% of C products. Of the four mutations which conferred a specificity shift, we noted that three affected the shape of the binding pocket, while one occurred on the positively charged acyl carrier protein landing pad. Finally, we fused the maltose binding protein (MBP) from E. coli to the N - terminus of BTE-MMD19 to improve enzyme solubility and achieve a titer of 1.9 g per L of twelve-carbon fatty acids in a shake flask.
定制由异源宿主合成的游离脂肪酸 (FFA) 的链长分布的主要策略是表达选择性酰基-酰基载体蛋白 (ACP) 硫酯酶。然而，当在微生物或植物宿主中表达时，这些酶中很少有能够产生精确的（大于所需链长的 90%）产物分布。在不希望混合脂肪酸的情况下，存在替代链长会使纯化复杂化。我们报告了几种策略的评估，这些策略旨在提高来自加利福尼亚月桂树的十二烷酰基-ACP 硫酯酶的选择性，以更接近专一地生产中链游离脂肪酸。我们证明基质辅助激光解吸/电离飞行时间质谱 (MALDI-ToF MS) 是一种有效的文库筛选技术，可用于鉴定具有有利链长特异性变化的硫酯酶变体。与本文讨论的几种合理方法相比，该策略被证明是一种更有效的筛选技术。有了这些数据，我们分离出了四个硫酯酶变体，当在脂肪酸积累大肠杆菌菌株 RL08 中表达时，它们表现出比野生型更具选择性的 FFA 分布。然后，我们将来自 MALDI 分离物的突变组合在一起，生成了 BTE-MMD19，这是一种能够产生由 90%的 C 产物组成的游离脂肪酸的硫酯酶变体。在赋予特异性变化的四个突变中，我们注意到三个突变影响了结合口袋的形状，而一个突变发生在带正电荷的酰基载体蛋白着陆垫上。最后，我们将来自大肠杆菌的麦芽糖结合蛋白 (MBP) 融合到 BTE-MMD19 的 N 端，以提高酶的溶解性，并在摇瓶中实现十二碳脂肪酸的 1.9 g/L 的产量。
