Novel promoters for constitutive and inducible expression of transgenes in the diatom Phaeodactylum tricornutum under varied nitrate availability

Abstract

Recent developments in the genetic engineering of biotechnologically important microalgae—among them the diatom Phaeodactylum tricornutum—promise rapid progress in strain improvements for producing various valuable commodities. Nitrogen (N) depletion and starvation are commonly employed strategies to induce or enhance the formation of secondary carotenoids and storage lipids in microalgal biotechnology. The manipulation of gene expression for improving or rerouting the metabolic pathways requires the development of suitable stage-specific promoters to drive gene expression under desired conditions. Here, we describe the isolation, cloning, and characterization of novel P. tricornutum promoters that are either constitutively active in the presence or absence of nitrate as the sole N source, or upregulated under N starvation. An enhanced green fluorescent protein (eGFP), fused to a plasma membrane-anchoring sequence at the C terminus, was expressed under the control of several endogenous promoters, which were chosen based on previously reported gene expression levels under nitrate-limited or deprived conditions. Among the four promoters tested in this work, the ammonium transporter gene (AMT, PHATRDRAFT_27877) promoter strongly induced eGFP accumulation under N starvation, and the purine permease gene (PUP, PHATRDRAFT_52619) promoter strongly and constitutively drove eGFP accumulation, regardless of nitrate availability. The Act2 (actin-like 2, PHATRDRAFT_29136) and previously characterized DGAT1 gene promoters had relatively weak constitutive and late-inducible expression, respectively, under N starvation. The fucoxanthin-chlorophyll a/c-binding protein gene (fcpA) promoter used as a control was downregulated under N starvation. A comparison of quantitative real-time PCR gene expression data with western blot results suggested that controlled transcriptional regulation is important for efficient transgenic protein accumulation under N starvation. The results are of interest for the expression of transgenes in P. tricornutum for engineering oil production capacity and accumulating recombinant proteins.