In Silico Cloning and Characterization of the Glycerol-3-Phosphate Dehydrogenase (GPDH) Gene Family in the Green Microalga Chlamydomonas reinhardtii

Abstract

Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) and NADH to glycerol-3-phosphate (G3P) and NAD(+). G3P is important as a precursor for glycerol and glycerolipid synthesis in microalgae. A GPDH enzyme has been previously purified from the green microalga Chlamydomonas reinhardtii, however, no genes coding for GPDH have been characterized before. In this study, we report the in silico characterization of three putative GPDH genes from C. reinhardtii: CrGPDH1, CrGPDH2, and CrGPDH3. These sequences showed a significant similarity to characterized GPDH genes from the microalgae Dunaliella salina and Dunaliella viridis. The prediction of the three-dimensional structure of the proteins showed the characteristic fold topology of GPDH enzymes. Furthermore, the phylogenetic analysis showed that the three CrGPDHs share the same clade with characterized GPDHs from Dunaliella suggesting a common evolutionary origin and a similar catalytic function. In addition, the K(a)/K(s) ratios of these sequences suggested that they are under purifying selection. Moreover, the expression analysis showed a constitutive expression of CrGPDH1, while CrGPDH2 and CrGPDH3 were induced in response to osmotic stress, suggesting a possible role for these two sequences in the synthesis of glycerol as a compatible solute in osmoregulation, and perhaps also in lipid synthesis in C. reinhardtii. This study has provided a foundation for further biochemical and genetic studies of the GPDH family in this model microalga, and also opportunities to assess the potential of these genes to enhance the synthesis of TAGs for biodiesel production.

Expression of Chlamydomonas reinhardtii CrGPDH2 and CrGPDH3 cDNAs in yeast reveals that they encode functional glycerol-3-phosphate dehydrogenases involved in glycerol production and osmotic stress tolerance

“…Those authors also showed that almost no glycerol accumulated under mild osmotic stress (e.g., 50 mM NaCl). In our previous study, we showed that a 200-mM NaCl treatment induced the expressions of CrGPDH2 and CrGPDH3, and in the absence of this treatment, the expression of both genes was barely detectable by end-point PCR (Herrera-Valencia et al 2012). We extended the expression analyses of these genes and found that both CrGPDH2 and CrGPDH3 were induced under all the NaCl treatments tested.…”

“…Based on in silico characterization, a phylogenetic analysis and an expression analysis, we had previously suggested that CrGPDH2 and CrGPDH3 encode GPDH enzymes that could be involved in glycerol production in response to NaCl stress in C. reinhardtii (Herrera-Valencia et al 2012). In the present study, we have demonstrated that both gene products have GPDH activity.…”

“…The cDNAs were synthesized using 10 μg total RNA and 200 U SuperScript III reverse transcriptase (Invitrogen) following the manufacturer's instructions. The PCR analyses were conducted as described by Herrera-Valencia et al (2012). The thermal cycling conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by final extension at 72°C for 10 min.…”

“…Recently, we reported the in silico and expression characterization of three GPDH genes from C. reinhardtii; CrGPDH1, CrGPDH2, and CrGPDH3 (Herrera-Valencia et al 2012). The expression analysis revealed constitutive expression of CrGPDH1, while CrGPDH2 and CrGPDH3 were expressed in response to 200 mM NaCl, suggesting that these two genes could be involved in glycerol production and osmotic stress tolerance as well as TAGs synthesis in this microalga (Herrera-Valencia et al 2012). In the present study, we have extended these previous findings by analyzing the expressions of CrGPDH2 and CrGPDH3 in response to lower NaCl concentrations and over time.…”

“…The relationship between glycerol biosynthesis and genetic expression of GPDH genes has been studied in these microalgae (He et al 2007(He et al , 2009Chen et al 2011). Recently, we reported the in silico and expression characterization of three GPDH genes from C. reinhardtii; CrGPDH1, CrGPDH2, and CrGPDH3 (Herrera-Valencia et al 2012). The expression analysis revealed constitutive expression of CrGPDH1, while CrGPDH2 and CrGPDH3 were expressed in response to 200 mM NaCl, suggesting that these two genes could be involved in glycerol production and osmotic stress tolerance as well as TAGs synthesis in this microalga (Herrera-Valencia et al 2012).…”