Department of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049, Madrid, Spain.
División de Tecnología Química y Nuevas Energías, Centro del Tecnología Química, Repsol S.A, 28935, Móstoles, Spain.
Sci Rep. 2018 Apr 3;8(1):5532. doi: 10.1038/s41598-018-23869-3.
Rubisco is an ancient, catalytically conserved yet slow enzyme, which plays a central role in the biosphere's carbon cycle. The design of Rubiscos to increase agricultural productivity has hitherto relied on the use of in vivo selection systems, precluding the exploration of biochemical traits that are not wired to cell survival. We present a directed -in vitro- evolution platform that extracts the enzyme from its biological context to provide a new avenue for Rubisco engineering. Precambrian and extant form II Rubiscos were subjected to an ensemble of directed evolution strategies aimed at improving thermostability. The most recent ancestor of proteobacteria -dating back 2.4 billion years- was uniquely tolerant to mutagenic loading. Adaptive evolution, focused evolution and genetic drift revealed a panel of thermostable mutants, some deviating from the characteristic trade-offs in CO-fixing speed and specificity. Our findings provide a novel approach for identifying Rubisco variants with improved catalytic evolution potential.
核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）是一种古老的、催化保守但缓慢的酶，在生物圈的碳循环中起着核心作用。为了提高农业生产力而对 Rubisco 进行设计，迄今为止一直依赖于使用体内选择系统，从而排除了对与细胞存活无关的生化特性的探索。我们提出了一种定向体外进化平台，该平台将该酶从其生物环境中提取出来，为 Rubisco 工程提供了新途径。前寒武纪和现存的 II 型 Rubisco 受到了一系列旨在提高热稳定性的定向进化策略的影响。可以追溯到 24 亿年前的最古老的细菌前体 - 对诱变负荷具有独特的耐受性。适应性进化、集中进化和遗传漂变揭示了一组耐热突变体，其中一些偏离了 CO 固定速度和特异性的特征权衡。我们的研究结果为鉴定具有改进的催化进化潜力的 Rubisco 变体提供了一种新方法。
