Automated Construction of a Yeast-Based Multigene Library via Homologous Recombination in a Biofoundry Workflow

Efficiently
building metabolic pathways via multigene assembly
has long been constrained by the limitations of traditional cloning
techniques, necessitating a breakthrough in gene assembly methods.
Notably, various in vitro gene assembly methods have been developed
to simplify the construction of an expression-tunable library. However,
in vitro gene assembly requires a tedious multistep construction process,
making it time-consuming and labor-intensive. Therefore, in this study,
we developed an automated one-step multigene assembly method for constructing
an expression-tunable library based on in vivo homologous recombination.
We optimized the shuttle vector for in vivo homologous recombination
to improve the assembly efficiency. We also scaled down the whole
assembly method for a high-throughput gene assembly. Finally, the
developed method demonstrated the construction of the expression-tunable
multigene library in the biofoundry. Therefore, this study offers
a versatile strategy for parallel and high-throughput genetic engineering
in synthetic biology.

长期以来，借助多基因组装高效构建代谢通路的研究始终受限于传统克隆技术的固有局限，亟需基因组装方法领域的突破性革新。值得注意的是，学界已开发出多种体外基因组装（in vitro gene assembly）方法，以简化表达可调文库（expression-tunable library）的构建流程。然而，体外基因组装往往需要繁琐的多步骤构建流程，不仅耗时冗长，且劳动强度较大。有鉴于此，本研究基于体内同源重组（in vivo homologous recombination）技术，开发了一种自动化的一步法多基因组装方法，用于构建表达可调文库。我们针对体内同源重组优化了穿梭载体（shuttle vector），以提升组装效率；同时将整套组装流程进行小型化适配，以满足高通量基因组装的需求。最终，所开发的方法在生物铸造厂（biofoundry）中成功实现了表达可调多基因文库的构建。综上，本研究为合成生物学（synthetic biology）领域内的并行化、高通量基因工程研究提供了一种通用策略。