Characterisation of novel regulatory sequences compatible with modular assembly in the diatom Phaeodactylum tricornutum

Abstract

Phaeodactylum tricornutum is a model pennate diatom for molecular studies due to its sequenced genome, genetic tractability, and capacity to produce a myriad of compounds of biotechnological interest. While tools for genetic engineering and heterologous gene expression have been developed for this species, the available set of characterised promoters remains limited. A diverse and well-understood set of genetic regulatory elements is necessary to enable fine tuning of gene expression for metabolic engineering and synthetic biology approaches. In this study, we investigated the expression profile of four endogenous constitutive promoter and terminator pairs Nub (Phatr3_J14260), SVP (Phatr3_J16798), 45582 (Phatr3_J45582), and Pbt (Phatr3_J54958) selected from a multi-experiment, integrated transcriptomics database under a variety of growth conditions, including various light regimes and nitrogen (N) limitation. Expression of the fluorescent reporter mVenus was driven from non-integrative episomes and monitored via flow cytometry. Gene expression was validated on a transcript and protein level using RT-qPCR and western blot, respectively. We found that all four promoter and terminator pairs exhibited stable expression throughout exponential growth, with distinct expression patterns and reduced light dependency compared to the widely-used FcpB promoter. Furthermore, the activity of these promoters was less impacted by N depletion than for FcpB. Our results indicate these novel promoters and terminators are promising tools for fine tuning single or multiple gene expression in P. tricornutum.