Heterologous Expression of the Glutamine Synthetase Enzyme (GlnA) from Escherichia coli in Microalgae

Abstract

ABSTRACT: The use of microalgae for biofuel production has opened a new window in the biotechnology research [1]. To date, several genomes of different species of microalgae have been sequenced and, through them, the identification of genes involved in metabolic processes with biotechnological potential has been identified [2-4]. One of the most important and interesting processes in microalgae is the generation of biomass, where the nitrogen metabolism plays an essential role [5]. In previous studies, it has been reported that the enhancement of the glutamine synthetase activity in higher plants has generated substantial increases in their protein content [6]. Hence, the aim of this work was to implement a pCAMBIA1302 vector-based genetic engineering strategy for the heterologous expression of Glutamine Synthetase gene (glnA) from E. coli in Microalgae. Here, it has been proved that Scenedesmus spp is an ideal target system for the genetic manipulation of Microalgae, because: (1) its sensitivity to the antibiotics, (2) its compatibility with the pCAMBIA-CaMV35S promoter for the expression of the proteins, and (3) its responses for biomass production. In addition, a BioBrick molecular tool or intermediate pCAMBIA-derived plasmid, the pCAMBIA::Spec(+), was successfully achieved by genetic engineering of the commercial original plasmid pCAMBIA1302. The expression cloning of the glnA gene into pCAMBIA1302 plasmid was successfully achieved, by using a double-substitution cloning strategy, which can be straightforward performed and screened by chemical selection (Spec) and visual characterization (GFP). Moreover, the heterologous GlnA protein expression in Scenedesmus spp recombinant clones was achieved and detected by Western-Blot analysis. Finally, the major finding in this study was the construction of the intermediary vector pCAMBIA1302::Spec(+) and the production of the recombinant pCAMBIA::glnA Scenedesmus spp clones.