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Optimization and Engineering of Fatty Acid Photodecarboxylase for Substrate Specificity
ChemCatChem ( IF 3.8 ) Pub Date : 2021-07-12 , DOI: 10.1002/cctc.202100840
Paul Santner 1 , László Krisztián Szabó 2 , Santiago Nahuel Chanquia 2 , Aske Høj Merrild 3 , Frank Hollmann 4 , Selin Kara 2 , Bekir Engin Eser 5
Affiliation  

Fatty acid photodecarboxylase (FAP) is one of the few photoenzymes in nature. The ability of FAP to convert fatty acids into alka(e)nes without the need for reducing equivalents put this enzyme into spotlight for biocatalytic applications. Although it has been discovered only a few years ago, many studies already emerged demonstrating its potential in areas from biofuel production and enzymatic kinetic resolution to being a critical component of multi-enzyme cascades. While there have been few protein engineering studies for modulating activity of FAP towards very short chain fatty acids, no study has yet addressed substrate selectivity within the medium to long chain fatty acid range, where FAP shows great promise for the synthesis of drop-in biofuels from ubiquitous fatty acids with chain lengths from C12 to C18. Here, after determining optimum expression and assay conditions for FAP, we screened 22 rationally designed mutant enzymes towards four naturally abundant fatty acid substrates; C12 : 0, C16 : 0, C18 : 0 and C18 : 1. Depending on the type of the exchanged amino acid, we observed selectivity shifts towards shorter or longer chains, compared to wild type enzyme. Notably, we obtained two groups of mutants; one group with high selectivity towards only C18 : 0, and another group that is selective towards C12 : 0 substrate. Moreover, we measured light and thermal stability of the wild type enzyme as well as the light stability of a mutant engineered for selectivity.

中文翻译:

用于底物特异性的脂肪酸光脱羧酶的优化和工程化

脂肪酸光脱羧酶 (FAP) 是自然界中为数不多的光酶之一。FAP 无需还原当量即可将脂肪酸转化为烷 (e) 烯的能力使这种酶成为生物催化应用的焦点。尽管它仅在几年前才被发现,但许多研究已经表明其在从生物燃料生产和酶动力学解析到成为多酶级联的关键组成部分等领域的潜力。虽然很少有蛋白质工程研究用于调节 FAP 对极短链脂肪酸的活性,但还没有研究解决中长链脂肪酸范围内的底物选择性,其中 FAP 在合成生物燃料方面显示出巨大的希望来自普遍存在的脂肪酸,链长从 C12 到 C18。这里,在确定 FAP 的最佳表达和检测条件后,我们针对四种天然丰富的脂肪酸底物筛选了 22 种合理设计的突变酶;C12:0、C16:0、C18:0 和 C18:1。根据交换氨基酸的类型,我们观察到与野生型酶相比,选择性向更短或更长的链转移。值得注意的是,我们获得了两组突变体;一组仅对 C18:0 具有高选择性,另一组对 C12:0 底物具有选择性。此外,我们测量了野生型酶的光稳定性和热稳定性,以及为选择性设计的突变体的光稳定性。根据交换氨基酸的类型,与野生型酶相比,我们观察到选择性向更短或更长的链转移。值得注意的是,我们获得了两组突变体;一组仅对 C18:0 具有高选择性,另一组对 C12:0 底物具有选择性。此外,我们测量了野生型酶的光稳定性和热稳定性,以及为选择性设计的突变体的光稳定性。根据交换氨基酸的类型,与野生型酶相比,我们观察到选择性向更短或更长的链转移。值得注意的是,我们获得了两组突变体;一组仅对 C18:0 具有高选择性,另一组对 C12:0 底物具有选择性。此外,我们测量了野生型酶的光稳定性和热稳定性,以及为选择性设计的突变体的光稳定性。
更新日期:2021-09-17
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