Mateljak Ivan, Rice Austin, Yang Kevin, Tron Thierry, Alcalde Miguel
Department of Biocatalysis , Institute of Catalysis, CSIC , Cantoblanco , 28049 Madrid , Spain.
Division of Chemistry and Chemical Engineering , California Institute of Technology, CALTECH , Pasadena , California 91125-4100 , United States.
ACS Synth Biol. 2019 Apr 19;8(4):833-843. doi: 10.1021/acssynbio.8b00509. Epub 2019 Apr 1.
Fungal laccases are biotechnologically relevant enzymes that are capable of oxidizing a wide array of compounds, using oxygen from the air and releasing water as the only byproduct. The laccase structure is comprised of three cupredoxin domains sheltering two copper centers-the T1Cu site and the T2/T3 trinuclear Cu cluster-connected to each other through a highly conserved internal electron transfer pathway. As such, the generation of laccase chimeras with high sequence diversity from different orthologs is difficult to achieve without compromising protein functionality. Here, we have obtained a diverse family of functional chimeras showing increased thermostability from three fungal laccase orthologs with ∼70% protein sequence identity. Assisted by the high frequency of homologous DNA recombination in Saccharomyces cerevisiae, computationally selected SCHEMA-RASPP blocks were spliced and cloned in a one-pot transformation. As a result of this in vivo assembly, an enriched library of laccase chimeras was rapidly generated, with multiple recombination events simultaneously occurring between and within the SCHEMA blocks. The resulting library was screened at high temperature, identifying a collection of thermostable chimeras with considerable sequence diversity, which varied from their closest parent homologue by 46 amino acids on average. The most thermostable variant increased its half-life of thermal inactivation at 70 °C 5-fold (up to 108 min), whereas several chimeras also displayed improved stability at acidic pH. The two catalytic copper sites spanned different SCHEMA blocks, shedding light on the recognition of specific residues involved in substrate oxidation. In summary, this case-study, through comparison with previous laccase engineering studies, highlights the benefits of bringing together computationally guided recombination and in vivo shuffling as an invaluable strategy for laccase evolution, which can be translated to other enzyme systems.
真菌漆酶是具有生物技术相关性的酶，能够利用空气中的氧气氧化多种化合物，并仅释放水作为唯一的副产物。漆酶结构由三个铜蓝蛋白结构域组成，其中包含两个铜中心——T1Cu位点和T2/T3三核铜簇——通过高度保守的内部电子传递途径相互连接。因此，在不影响蛋白质功能的情况下，很难从不同的直系同源物中产生具有高序列多样性的漆酶嵌合体。在这里，我们从三个蛋白质序列同一性约为70%的真菌漆酶直系同源物中获得了一个功能嵌合体的多样化家族，这些嵌合体表现出更高的热稳定性。在酿酒酵母中同源DNA重组的高频率辅助下，通过计算选择的SCHEMA-RASPP模块在一锅转化中进行拼接和克隆。由于这种体内组装，迅速产生了一个丰富的漆酶嵌合体文库，在SCHEMA模块之间和内部同时发生了多个重组事件。在高温下对所得文库进行筛选，鉴定出一系列具有相当序列多样性的热稳定嵌合体，它们与最接近的亲本同源物平均相差46个氨基酸。最耐热的变体在70°C下的热失活半衰期增加了5倍（长达108分钟），而几个嵌合体在酸性pH下也表现出更高的稳定性。两个催化铜位点跨越不同的SCHEMA模块，这有助于了解参与底物氧化的特定残基的识别。总之，通过与之前的漆酶工程研究进行比较，这个案例研究突出了将计算指导的重组和体内改组结合起来作为漆酶进化的宝贵策略的好处，这一策略也可以应用于其他酶系统。
