High-Throughput Part Characterization and Tool Development to Advance Chloroplast Synthetic Biology

Plant synthetic biology holds promise for developing climate-resilient crop varieties particularly through advancements in engineering the chloroplast genome. However, the field faces limitations due to the scarcity of genetic tools and the long generation times of photosynthetic eukaryotes. To address these challenges, we established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology, by significantly advancing plastome engineering tools. We developed an automation workflow that enabled the generation, handling, and analysis of 3,156 transplastomic Chlamydomonas strains and expanded the repertoire of selection markers for chloroplast transformation in Chlamydomonas to five, alongside establishing six new reporter genes. Moreover, we characterized over 140 regulatory parts, including promoters, UTRs, and intercistronic expression elements, all integrated within the Phytobrick cloning framework, for which we demonstrated a broad range of gene expression strength. Additionally, we explored library-based approaches to overcome the throughput limitations of biolistic chloroplast transformation. Finally, we demonstrated the utility of all tools by introducing a chloroplast-based synthetic photorespiration pathway. The high conservation of the chloroplast genetic system suggests that our findings in Chlamydomonas could be translated to advance chloroplast engineering in land plants and provides a prototyping platform for future crop improvements to address the challenges posed by a changing global climate.

植物合成生物学在培育抗气候胁迫作物品种领域展现出巨大潜力，其中叶绿体基因组工程技术的进步更是关键突破口。然而，该领域目前仍面临两大核心局限：遗传工具储备匮乏，以及光合真核生物的世代周期漫长。为破解上述挑战，我们通过优化质体工程工具体系，建立了以莱茵衣藻（Chlamydomonas reinhardtii）作为叶绿体合成生物学原型底盘的研究平台。我们开发了一套自动化工作流程，可完成3156株转质体莱茵衣藻菌株的构建、培养与分析；同时将衣藻叶绿体转化可用的筛选标记扩充至5种，并确立了6个全新的报告基因。此外，我们对超过140种调控元件（包括启动子、非翻译区（Untranslated Region，UTR）以及顺反子间表达元件）进行了功能表征，所有元件均整合至Phytobrick克隆框架中，并展现出跨度广泛的基因表达调控强度。我们还探索了基于文库的研究策略，以突破基因枪介导的叶绿体转化的通量限制。最后，我们通过引入基于叶绿体的合成光呼吸途径，验证了整套工具的实用价值。叶绿体遗传系统具有高度的进化保守性，这意味着我们在莱茵衣藻中取得的研究成果可推广应用于陆生植物的叶绿体工程改造，同时也为未来应对全球气候变化挑战的作物遗传改良提供了可靠的原型研发平台。