Cloning and heterologous expression of nitrate reductase genes from Dunaliella salina

Abstract

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.

A duplicated carbonic anhydrase 1 (DCA1) promoter mediates the nitrate reductase gene switch of Dunaliella salina

“…The native nitrate reductase (NR) of D. salina shares common structural features with NRs from higher plants and algae: Mo-MPT domain, the heme-Fe domain, and the FAD domain (Xie et al 2007). It has been reported that expression of the NR gene is induced by nitrate but repressed by ammonium, and that NR enzyme triggers the toxicity response of chlorate in several eukaryotic organisms, including D. salina (Solomonson and Vennesland 1972;Li et al 2007a, b).…”

Characterization of novel nitrate reductase-deficient mutants for transgenic Dunaliella salina systems

“…Previous study have shown that NR from D. salina shares common structural features with NR from higher plants and other green algae including the Mo-MPT domain with the nitrate-reducing site between amino acids 82 and 311, the heme-Fe domain between amino acids 543 and 616, and the FAD domain between amino acids 661 and 757 (Xie et al, 2007). Mutation analysis of the sequences herein suggests that three kinds of critical mutations led to a loss of all NR catalytic activity including property changes of some amino acid residues, stop codon introduction, and frameshift mutations.…”

“…NR-deficient mutants of D. salina were identified by detecting NR activity according to methods described by Xie et al, (2007). Briefly, mutant cells grown in urea-containing medium and wild-type cells grown in nitrate-containing medium (approximately 1-2 x 10 6 cells/mL) were harvested and washed with nitrogen-free medium twice.…”

“…Our previous work has shown that a full-length cDNA (3694 bp) of NR from D. salina can be heterologously expressed in E. coli as a fusion protein (Xie et al, 2007), that NR transcripts are induced by nitrate but repressed by ammonium, and that NIA1 has a strong inducible promoter that can be used to control heterologous gene expression in transgenic D. salina . While the isolation and characterization of NR-deficient mutants is important in the development of a novel D. salina transformation system using NIA1 as a selectable marker, the isolation of D. salina mutants with nitrate assimilation defects has not yet been explored.…”

Paving the Road to Algal Biofuels with the Development of a Genetic Infrastructure

“…Highly active endogenous promoter and 3'-untranslated region (UTR) pairs are of particular interest and significance to expressing transgenes in Dunaliella. Recent publications describing the use of the actin, rbcS, carbonic anhydrase, and ubiquitin promoters Walker et al, 2004;Chen et al, 2009) and nitA 3'-UTR Xie et al, 2007) are the basis for many of the pioneering attempts to genetically engineer D. salina, including the work presented hereafter.…”