Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany.
Nat Chem Biol. 2015 Feb;11(2):148-55. doi: 10.1038/nchembio.1715. Epub 2015 Jan 5.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the fixation of CO2 in photosynthesis. Despite its pivotal role, Rubisco is an inefficient enzyme and thus is a key target for directed evolution. Rubisco biogenesis depends on auxiliary factors, including the GroEL/ES-type chaperonin for folding and the chaperone RbcX for assembly. Here we performed directed evolution of cyanobacterial form I Rubisco using a Rubisco-dependent Escherichia coli strain. Overexpression of GroEL/ES enhanced Rubisco solubility and tended to expand the range of permissible mutations. In contrast, the specific assembly chaperone RbcX had a negative effect on evolvability by preventing a subset of mutants from forming holoenzyme. Mutation F140I in the large Rubisco subunit, isolated in the absence of RbcX, increased carboxylation efficiency approximately threefold without reducing CO2 specificity. The F140I mutant resulted in a ∼55% improved photosynthesis rate in Synechocystis PCC6803. The requirement of specific biogenesis factors downstream of chaperonin may have retarded the natural evolution of Rubisco.
核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）催化光合作用中 CO2 的固定。尽管 Rubisco 具有关键作用，但它是一种低效的酶，因此是定向进化的关键目标。Rubisco 的生物发生依赖于辅助因子，包括用于折叠的 GroEL/ES 型伴侣蛋白和用于组装的伴侣蛋白 RbcX。在这里，我们使用依赖 Rubisco 的大肠杆菌菌株对蓝细菌形式 I Rubisco 进行了定向进化。GroEL/ES 的过表达提高了 Rubisco 的可溶性，并倾向于扩大允许突变的范围。相比之下，特定的组装伴侣蛋白 RbcX 通过阻止一部分突变体形成全酶，对可进化性产生了负面影响。在没有 RbcX 的情况下分离出的大亚基中的突变 F140I 增加了羧化效率约三倍，而不会降低 CO2 特异性。F140I 突变体使 Synechocystis PCC6803 的光合作用速率提高了约 55%。伴侣蛋白下游特定生物发生因子的需求可能阻碍了 Rubisco 的自然进化。
