A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.基因文库（gene library）构建领域的核心挑战之一，在于保障其具备充足的功能规模与多样性，从而大幅提升筛选获得不同功能蛋白质变体的概率。采用三核苷酸混合物（trinucleotides mixtures）进行可控随机化，不仅可获得更优异的文库多样性，还能够精准设定每个位点的氨基酸（amino acid）种类与分布模式。本研究以超嗜热菌海栖热袍菌（Thermotoga maritima）的tHisF蛋白作为骨架（scaffold），构建了一款高多样性基因文库；我们将多种理性设计准则与随机化思路相结合，针对该tHisF基因序列中选定的26个密码子（codon）开展可控程度的随机化改造。我们开发了一种全新的方法，通过小型亚文库（sub-library）的组合组装来构建全长基因文库：高多样性的全长基因文库可便捷地按需由多样性更低的小型亚文库组装得到，从而规避了高多样性噬菌体（phage）或质粒（plasmid）集合长期存档与反复扩增这一长期以来棘手的难题。我们还开发了一款通用型软件工具，用于突变基因序列的分析，可为文库多样性分析提供高效辅助支持；最后，我们通过评估文库成员的构象稳定性（conformational stability），并从中分离获得具有HisF活性的蛋白质变体，从原理上验证了该文库的实际应用价值。本研究方案将核酸合成化学（nucleic acids synthetic chemistry）、生物化学（biochemistry）与分子遗传学（molecular genetics）的多项技术优势融为一体，形成了一套连贯统一、灵活易用且稳定性优异的组合式基因合成方法。