Automated Strain Construction for Biosynthetic Pathway Screening in Yeast.

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-10-08 DOI:10.1021/acssynbio.5c00554

Maria C T Astolfi, Sam D Yoder, Marina Delfa-Lalaguna, Peter H Winegar, Sara K F Holm, Mengziang Lei, Xixi Zhao, Stephen E Tan, Randy Louie, Nathan J Hillson, Graham A Hudson, Jay D Keasling

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引用次数: 0

Abstract

Automation accelerates the Design-Build-Test-Learn (DBTL) cycle for synthetic biology; however, most strain construction pipelines lack robotic integration. Here, we present the workflow design and source code for a modular, integrated protocol that automates the Build step in Saccharomyces cerevisiae. We programmed the Hamilton Microlab VANTAGE to integrate off-deck hardware via its central robotic arm, enabling automated steps that increased throughput to 2,000 transformations per week. We developed a user interface with the Hamilton VENUS software to support on-demand parameter customization. As a proof of concept, we screened a gene library in an engineered yeast strain producing verazine, a key intermediate in the biosynthesis of steroidal alkaloids. Our pipeline rapidly identified pathway bottlenecks and genes that enhanced verazine production by 2.0- to 5-fold. This technical note provides resources for synthetic biologists designing yeast workflows for biofoundries to screen libraries for pathway discovery/optimization, combinatorial biosynthesis, and protein engineering.

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酵母生物合成途径筛选的自动化菌株构建。

自动化加速了合成生物学的设计-构建-测试-学习（DBTL）周期；然而，大多数应变施工管道缺乏机器人集成。在这里，我们展示了一个模块化集成协议的工作流程设计和源代码，该协议可以自动化酿酒酵母的构建步骤。我们对Hamilton Microlab VANTAGE进行了编程，通过其中央机械臂集成了甲板外的硬件，实现了自动化步骤，将吞吐量提高到每周2000次转换。我们开发了一个使用Hamilton VENUS软件的用户界面，以支持按需参数定制。为了证明这一概念，我们在一株生产维拉嗪的工程酵母菌株中筛选了一个基因库，维拉嗪是甾体生物碱生物合成的关键中间体。我们的管道快速确定了途径瓶颈和基因，将维拉嗪的产量提高了2.0至5倍。本技术说明为合成生物学家设计酵母工作流程提供了资源，用于生物foundry筛选文库，以进行途径发现/优化，组合生物合成和蛋白质工程。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.