Highly Active C(8)-Acyl-ACP Thioesterase Variant Isolated by a Synthetic Selection Strategy.
Highly Active C(8)-Acyl-ACP Thioesterase Variant Isolated by a Synthetic Selection Strategy.
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DOI：
10.1021/acssynbio.8b00215
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发表时间：
2018-09-21
影响因子：
4.7
通讯作者：
Pfleger BF
中科院分区：
文献类型：
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作者：
Hernández Lozada NJ;Lai RY;Simmons TR;Thomas KA;Chowdhury R;Maranas CD;Pfleger BF
微生物代谢是生产油脂化学工业中使用的中链长脂肪酸（例如辛酸）的有吸引力的途径。这种策略的一个挑战是缺乏在微生物宿主中具有高活性并且对具有所需链长的底物具有选择性的酶。在脂肪酸生物合成的许多步骤中，硫酯酶是用于控制链长的最广泛使用的酶。硫酯酶水解脂肪酸和酰基载体蛋白（ACP）或辅酶A（CoA）辅因子之间的硫酯键。硫酯酶的功能作用在生物体之间（即细菌与植物）不同，因此底物特异性也不同。因此，利用异源硫酯酶的微生物生物催化剂产生高滴度的具有混合链长的脂肪酸或低滴度的具有窄链长分布的产物。为了寻找选择性水解辛酰-ACP的高活性酶，我们开发了基于大肠杆菌的硫辛酸需求的遗传选择。我们使用的选择，以确定在随机诱变库的C8特异性的沼泽萼距花FatB 1硫酯酶的变体。优化硫酯酶的表达条件后，E.大肠杆菌培养物从该硫酯酶的单个染色体拷贝产生1.7g/L辛酸，特异性>90%。体外研究证实，突变硫酯酶具有15倍的kcat增加相比，其天然序列。高水平的比活性允许低水平的表达，同时保持脂肪酸滴度。低表达要求将允许代谢工程师使用更多的细胞资源来解决途径中的其他限制并最大化整体生产力。
Microbial metabolism is an attractive route for producing medium chain-length fatty acids, e.g. octanoic acid, used in the oleochemical industry. One challenge to this strategy is the lack of enzymes that are both highly active in a microbial host and selective towards substrates with desired chain-length. Of the many steps in fatty acid biosynthesis, the thioesterase is the most widely used enzyme for controlling chain length. Thioesterases hydrolyze the thioester bond between fatty acids and the acyl-carrier protein (ACP) or coenzyme A (CoA) co-factor. The functional role of thioesterases varies between organisms (i.e. bacteria vs. plant) and therefore so do the substrate specificities. As a result, microbial biocatalysts that utilize a heterologous thioesterase either produce high titers of fatty acids with mixed chain-lengths or low titers of products with a narrow chain-length distribution. To search for highly active enzymes that selectively hydrolyze octanoyl-ACP, we developed a genetic-selection based on the lipoic acid requirement of Escherichia coli. We used the selection to identify variants in a randomly mutagenized library of the C8-specific Cuphea palustris FatB1 thioesterase. After optimizing expression of the thioesterase, E. coli cultures produced 1.7 g/L of octanoic acid with >90% specificity from a single chromosomal copy of this thioesterase. In vitro studies confirmed the mutant thioesterase possessed a 15-fold increase in kcat compared to its native sequence. The high level of specific activity allowed for low levels of expression while maintaining fatty acid titer. The low expression requirement will allow metabolic engineers to use more cellular resources to address other limitations in the pathway and maximize overall productivity.