An inducible expression system in the alga Nannochloropsis gaditana controlled by the nitrate reductase promoter

Abstract

The marine microalgal species Nannochloropsis gaditana has emerged as a model organism for research into algal-derived biofuels due to its ability to produce large amounts of lipids. In addition, this species has biotechnological potential owing to its ease of transformation and availability of a sequenced and annotated genome. The ability to control expression of transgenes in an inducible manner is an important requirement for many genetic engineering approaches and as such, inducible promoters are an important component of the molecular toolkit for a host organism. We designed an expression vector containing a~1.3 kb region upstream of the endogenous nitrate reductase gene of N. gaditana and demonstrated this is capable of promoting the expression of a heterologous enhanced green fluorescent protein (eGFP) reporter. In the presence of ammonium, expression of the eGFP reporter was undetectable; however, in the presence of nitrate, eGFP expression is induced. In addition, we demonstrated how altering the nitrogen source of the growth media can be used to precisely control expression. We have shown that the nitrate reductase promoter can be used as a powerful molecular tool for heterologous protein expression in N. gaditana, further contributing to the development of this species as a candidate strain for biotechnology.

Development of a constitutive and an auto-inducible high-yield expression system for recombinant protein production in the microalga Nannochloropsis oceanica

“…Notably, our N. oceanica transformants remained stable even without selection pressure (unpubl. data), as reported for N. gaditana (Jackson et al 2018 ), which reduces the costs for industrial-scale cultivation.…”

“…6a and b ) had previously been described for P. tricornutum (Chu et al 2016 ) and in parallel studies for N. gaditana (Jackson et al 2018 ) and N. oceanica (Poliner et al 2020 ). Notably and contrary to our results, the NR promoter of N. gaditana retained residual and non-negligible activity in the presence of both ammonium and nitrate (Jackson et al 2018 ), preventing its application in an auto-induction system. Moderate concentrations of ammonium (up to 800 μM) fully repressed Venus expression for approx.…”

“…The LDSP promoter successfully expressed the hygromycin resistance gene in the pNoc ox vectors (Zienkiewicz et al 2017 ). The promoter of the NR gene is nitrate-inducible in higher plants and had so far been studied in C. reinhardtii (Loppes et al 1999 ), Chlorella vulgaris (Niu et al 2012 ), P. tricornutum (Chu et al 2016 ) and N. gaditana (Jackson et al 2018 ).…”

“…Previously, such high yields had been hardly reported for Nannochloropsis . Only based on a theoretical protein content per cell, a similarly high GFP yield of 1.5 ± 1.1% of TSP had been calculated for the NR promoter in N. gaditana (Jackson et al 2018 ). Interestingly, a recombinantly produced small antimicrobial peptide fused to red fluorescent protein accumulated to up to 4.3% of TSP in the cytosol of N. salina, when using a combined promoter of the heat shock protein 70A and RubisCO SSU from C. reinhardtii (Li and Tsai 2009 ).…”

Plastid Engineering of a Marine Alga, Nannochloropsisgaditana, for Co‐Expression of Two Recombinant Peptides

“…2017, Jackson et al. 2019). However, nuclear genetic engineering still remains challenging owing to the occurrence of gene silencing and position effects, resulting in poor expression of foreign genes (Li et al.…”