SnRK families in Chlamydomonas: promising targets for bioproduction

Abstract

Given the great world energy demand and the environmental costs associated to fossil fuels use, it is imperative to find a CO2 neutral, sustainable, and renewable energy source. Microalgae are one of the most studied biofuel feedstock, mainly because they produce considerable amounts of energetic compounds (TAG and starch) and other valuable secondary metabolites (such as pigments, vitamins, and bioplastic). Currently, a two-phase cultivation strategy including a stress imposition step is used to accumulate interesting compounds for biofuel production. However, microalgae cell growth is often reduced, requiring longer cultivation times, and stress imposition techniques are still expensive, which represent high costs for the microalgal biofuel production process. In order to make it profitable, a biorefinery approach must be used, combining the extraction of energetic molecules and high value-added by-products. However, biomass supply continues to represent a major limiting factor. To overcome this limitation, the study of the metabolic and regulatory networks involved in stress response is essential so that potential targets for bioengineering can be identified. This would allow either the maintenance of cell growth under stress conditions or the mimicking of a stress condition by coupling a gene of interest to a promoter induced by a simple stimulus, reducing production costs. Therefore, the model microalga Chlamydomonas reinhardtti was used to study the involvement of SnRK protein kinases in stress response. This family is highly associated to plant stress response mechanisms. A few studies also report its involvement in Chlamydomonas stress response, although little is known about it. We identified and classified Chlamydomonas SnRK based on sequence and domain structure similarities with the SnRK sequences described in Arabidopsis using bioinformatic tools. Moreover, its expression patterns were evaluated by RT-qPCR under a wide range of stress conditions in order to look for target genes that might be involved in Chlamydomonas stress response pathways. By using bioinformatic tools 19 SnRK genes coding for 20 proteins from 4 subfamilies (SnRK1, its regulatory subunits, and two groups of SnRK2 proteins) were identified. Surprisingly, the plant-specific SnRK3 subfamily was not found in Chlamydomonas. The analysis of SnRK expression patterns under a wide range of stresses by RT-qPCR identified SnRK2.9 as a potential candidate for future studies as its response was specific to heat stress. Also SnRK2.12 and SnRK2.7 seem to have an important role in mediating Iron deficiency and oxidative stress, respectively, according to the mining of available RNA-seq data. Furthermore, from the stresses studied, UV radiation showed interesting results as it led to lipid accumulation and it is a stimulus that can be applied inexpensively. This work represents a great advance in microalgal and stress biology research since that, although SnRK are a key group of protein kinases for biotechnology, this family was never described before in microalgae.