Department of Molecular Biology, University of Potsdam, Potsdam, Germany.
Plant Signalling Group, Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany.
Methods Mol Biol. 2020;2205:277-303. doi: 10.1007/978-1-0716-0908-8_16.
For industry-scale production of high-value chemicals in microbial cell factories, the elimination of metabolic flux imbalances is a critical aspect. However, a priori knowledge about the genetic design of optimal production pathways is typically not available. COMPASS, COMbinatorial Pathway ASSembly, is a rapid cloning method for the balanced expression of multiple genes in biochemical pathways. The method generates thousands of individual DNA constructs in modular, parallel, and high-throughput manner. COMPASS employs inducible artificial transcription factors derived from plant (Arabidopsis thaliana) regulators to control the expression of pathway genes in yeast (Saccharomyces cerevisiae). It utilizes homologous recombination for parts assembly and employs a positive selection scheme to identify correctly assembled pathway variants after both in vivo and in vitro recombination. Finally, COMPASS is equipped with a CRISPR/Cas9 genome modification system allowing for the one-step multilocus integration of genes. Although COMPASS was initially developed for pathway engineering, it can equally be employed for balancing gene expression in other synthetic biology projects.
对于在微生物细胞工厂中进行高价值化学品的工业规模生产，消除代谢通量失衡是一个关键方面。然而，关于最佳生产途径的遗传设计的先验知识通常是不可用的。COMPASS（组合途径组装）是一种用于在生化途径中平衡表达多个基因的快速克隆方法。该方法以模块化、并行和高通量的方式生成数千个单独的 DNA 构建体。COMPASS 利用来自植物（拟南芥）调节剂的诱导型人工转录因子来控制酵母（酿酒酵母）中途径基因的表达。它利用同源重组进行部件组装，并采用阳性选择方案在体内和体外重组后识别正确组装的途径变体。最后，COMPASS 配备了 CRISPR/Cas9 基因组修饰系统，允许一步多基因多位点整合。尽管 COMPASS 最初是为途径工程开发的，但它同样可以用于平衡其他合成生物学项目中的基因表达。
