Evaluation of strategies to narrow the product chain-length distribution of microbially synthesized free fatty acids
Evaluation of strategies to narrow the product chain-length distribution of microbially synthesized free fatty acids
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DOI：
10.1016/j.ymben.2023.02.012
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发表时间：
2023-03-10
影响因子：
8.4
通讯作者：
Pfleger, Brian F.
中科院分区：
文献类型：
--
作者：
Jindra, Michael A.;Choe, Kisurb;Pfleger, Brian F.
调整异源宿主合成的游离脂肪酸（FFA）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，这些酶很少能产生精确的（大于所需链长的 90%）产物分布。在不需要脂肪酸混合物的情况下，替代链长的存在会使纯化变得复杂。我们报告了对改进来自加州月桂树的十二酰基-ACP硫酯酶的几种策略的评估，以表现出更具选择性地生产中链游离脂肪酸，以达到接近排他性。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，可用于鉴定链长特异性发生有利变化的硫酯酶变体。事实证明，该策略是比本文讨论的几种合理方法更有效的筛选技术。利用这些数据，我们分离了四种硫酯酶变体，当在脂肪酸积累的大肠杆菌菌株 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 MALDI 分离株的突变组合起来，生成 BTE-MMD19，这是一种硫酯酶变体，能够产生由 90% 的 C12 产物组成的游离脂肪酸。在赋予特异性转变的四个突变中，我们注意到三个影响了结合袋的形状，而一个发生在带正电的酰基载体蛋白着陆垫上。最后，我们将大肠杆菌的麦芽糖结合蛋白（MBP）融合到BTE-MMD19的N端，以提高酶的溶解度，并在摇瓶中实现每升十二碳脂肪酸1.9 g的效价。
The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by het-erologous 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 C12 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.