Metabolic and transcriptional response to the induction of a heterologous poly-3-hydroxybutyrate pathway in Phaeodactylum tricornutum

The marine diatom Phaeodactylum tricornutum is an emerging host for metabolic engineering, but little is known about how introduced pathways are integrated into the existing metabolic framework of the host and how this influences trans- and native-gene expression. In this study we used episomal vectors to express the heterologous poly-3-hydroxybutyrate (PHB) pathway in diatoms, which produces precursors for polymeric bioplastics from acetyl coenzyme-A (AcCoA). By experimentally perturbing cultivation conditions, we gained insight into the regulation of the endogenous metabolism in transgenic lines under various environmental scenarios. Biosynthesis of PHB led to distinct shifts in the metabolome of the host, and further analysis revealed a condition-dependent relationship between endogenous and transgenic metabolic pathways. Under N-limitation, which induced a significant increase in neutral lipid content, both metabolic and transcriptomic data suggest that AcCoA was preferentially shunted into the endogenous metabolism over the transgenic PHB pathway. In contrast, supply of organic carbon in the form of glycerol supported both fatty acid and PHB biosynthesis, suggesting cross-talk between cytosolic and plastidial AcCoA precursors. This is the first study to investigate the transcriptomic and metabolomic response of diatom cell lines expressing a heterologous multi-gene pathway under different environmental conditions, providing useful insights for future engineering attempts for pathways based on the precursor AcCoA. Overall design: RNA-seq

海洋硅藻三角褐指藻（Phaeodactylum tricornutum）是一类新兴的代谢工程宿主，但目前对于外源导入的代谢通路如何整合至宿主现有代谢框架，以及该过程如何影响转基因与内源基因的表达，仍知之甚少。本研究通过附加体质粒（episomal vectors）在硅藻中表达异源聚-3-羟基丁酸酯（poly-3-hydroxybutyrate, PHB）通路——该通路可利用乙酰辅酶A（acetyl coenzyme-A, AcCoA）合成可聚合生物塑料的前体物质。通过实验调控培养条件，我们解析了不同环境场景下转基因藻株的内源代谢调控机制。PHB的生物合成会使宿主代谢组发生显著改变，进一步分析发现内源与转基因代谢通路间存在环境依赖的互作关系。在氮限制条件下（该条件可显著提升中性脂质含量），代谢组与转录组数据均显示，乙酰辅酶A更倾向于流向内源代谢通路而非转基因PHB合成通路。与之相反，以甘油形式提供的有机碳可同时支持脂肪酸与PHB的生物合成，这表明细胞质与质体来源的乙酰辅酶A前体间存在交互调控。本研究首次针对表达异源多基因通路的硅藻细胞系，探究了其在不同环境条件下的转录组与代谢组响应，为后续基于乙酰辅酶A前体的代谢通路工程化改造提供了重要参考。实验整体设计：RNA测序（RNA-seq）