Engineered Protein Nano-Compartments for Targeted Enzyme Localization

Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis.

酶与底物的区室化共定位，是工程化细胞与生物催化领域中实现多酶合成的极具吸引力的策略。对酶与底物进行隔离封存，不仅可大幅提升反应效率，还能保护工程化宿主细胞免受潜在有毒反应中间体的侵害。部分细菌可形成基于蛋白质的多面体微区室，用于封存功能相关的酶，并调控酶与底物及其他小分子代谢物的接触。这类细菌微区室可被改造为基于蛋白质的纳米生物反应器，前提是其能在非天然宿主细胞中组装，且异源酶与底物可被靶向导入该工程化区室。本研究证实，在大肠杆菌（E. coli）中重组表达肠炎沙门氏菌（Salmonella enterica）乙醇胺利用（eut）细菌微区室的外壳蛋白，可形成多面体蛋白质外壳。纯化后的重组外壳在形态上与从肠炎沙门氏菌中纯化得到的天然Eut微区室相似。令人意外的是，仅需重组表达单一种外壳蛋白（EutS），即可成功构建边界清晰的区室，且该蛋白为必要条件。与EutS共表达，还可促进携带推定Eut外壳靶向信号序列的增强绿色荧光蛋白（EGFP）被包裹进入该重组外壳。本研究还证实，异源酶β-半乳糖苷酶（β-galactosidase）可被靶向定位于该重组外壳并保持催化活性。综上，本研究结果为用于生物合成与生物催化的蛋白质纳米区室的工程化改造提供了概念验证。