Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK.
CONAHCyT, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), CDMX 04510, México.
Plant Cell Physiol. 2024 Sep 3;65(8):1298-1309. doi: 10.1093/pcp/pcae063.
Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterization of genetic elements would make heterologous gene expression more predictable in this test bed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S × 2) provided the highest yield of proteins, although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia genes for ETHYLENE RESPONSE FACTOR 1 and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER protein drove expression to higher levels across all tissues without a growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed RUBY, a polycistronic betalain synthesis cassette linked by P2A sequences, to demonstrate coordinated expression of metabolic enzymes. A heat-shock-inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing tool kit for gene expression in Marchantia and provided new resources for the Marchantia research community.
地钱已成为比较研究和合成生物学的重要模式系统。遗传元件的系统特征将使异源基因表达在基因电路组装和生物生产的这个试验台上更具可预测性。然而，地钱的遗传元件工具包仅包括少数几个组成型启动子，需要进行基准测试以评估其效用。我们比较了先前表征和新的组成型启动子的表达模式。我们发现，使用花椰菜花叶病毒 35S 启动子（pro35S×2）的双增强子版本驱动表达可以提供最高的蛋白质产量，尽管它也会抑制转化体的生长。相比之下，源自地钱基因的 ETHYLENE RESPONSE FACTOR 1 和 CLASS II HOMEODOMAIN-LEUCINE ZIPPER 蛋白的启动子在不影响生长的情况下，在所有组织中均可驱动表达至更高水平，并可提供中等水平的基因表达。此外，我们表明，细胞质是靶向异源蛋白以实现更高表达水平而不增加显著生长负担的最佳亚细胞区室。为了展示这些启动子在地钱中的潜力，我们表达了 RUBY，这是一个通过 P2A 序列连接的多顺反子甜菜红素合成盒，以证明代谢酶的协调表达。使用热休克诱导型启动子进一步减轻与大量甜菜红素积累相关的生长负担。我们已经扩展了地钱中基因表达的现有工具包，并为地钱研究社区提供了新的资源。
