Fink Tina, Stevović Bojana, Verwaal René, Roubos Johannes A, Gaber Rok, Benčina Mojca, Jerala Roman, Gradišar Helena
Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000, Ljubljana, Slovenia.
DSM Biotechnology Center, DSM, Delft, The Netherlands.
AMB Express. 2020 May 24;10(1):97. doi: 10.1186/s13568-020-01031-5.
The clustering of biosynthetic enzymes is used in nature to channel reaction products and increase the yield of compounds produced by multiple reaction steps. The coupling of multiple enzymes has been shown to increase the biosynthetic product yield. Different clustering strategies have particular advantages as the spatial organization of multiple enzymes creates biocatalytic cascades with a higher efficiency of biochemical reaction. However, there are also some drawbacks, such as misfolding and the variable stability of interaction domains, which may differ between particular biosynthetic reactions and the host organism. Here, we compared different protein-based clustering strategies, including direct fusion, fusion mediated by intein, and noncovalent interactions mediated through small coiled-coil dimer-forming domains. The clustering of enzymes through orthogonally designed coiled-coil interaction domains increased the production of resveratrol in Escherichia coli more than the intein-mediated fusion of biosynthetic enzymes. The improvement of resveratrol production correlated with the stability of the coiled-coil dimers. The coiled-coil fusion-based approach also increased mevalonate production in Saccharomyces cerevisiae, thus demonstrating the wider applicability of this strategy.
生物合成酶的聚集在自然界中被用于引导反应产物，并提高多步反应产生的化合物的产量。已证明多种酶的偶联可提高生物合成产物的产量。不同的聚集策略具有特定优势，因为多种酶的空间组织可创建具有更高生化反应效率的生物催化级联反应。然而，也存在一些缺点，如错误折叠和相互作用结构域的可变稳定性，这在特定的生物合成反应和宿主生物体之间可能有所不同。在此，我们比较了不同的基于蛋白质的聚集策略，包括直接融合、内含肽介导的融合以及通过小的卷曲螺旋二聚体形成结构域介导的非共价相互作用。通过正交设计的卷曲螺旋相互作用结构域对酶进行聚集，比内含肽介导的生物合成酶融合在大肠杆菌中提高了白藜芦醇的产量。白藜芦醇产量的提高与卷曲螺旋二聚体的稳定性相关。基于卷曲螺旋融合的方法也提高了酿酒酵母中甲羟戊酸的产量，从而证明了该策略具有更广泛的适用性。
