Evaluation of novel starch-deficient mutants of Chlorella sorokiniana for hyper-accumulation of lipids

When green algae are exposed to physiological stresses such as nutrient deprivation, growth is arrested and the cells channel fixed carbon instead into storage compounds, accumulating first starch granules and then lipid bodies containing triacylglycerides. In recent years there has been significant interest in the commercial exploitation of algal lipids as a sustainable source of biodiesel. Since starch and lipid biosynthesis involves the same C3 precursor pool, it has been proposed that mutations blocking starch accumulation should result in increased lipid yields, and indeed several studies have supported this. The fast-growing, thermotolerant alga Chlorella sorokiniana represents an attractive strain for industrial cultivation. We have therefore generated and characterized starch-deficient mutants of C. sorokiniana and determined whether lipid levels are increased in these strains under stress conditions. One mutant (ST68) is shown to lack isoamylase, whilst two others (ST3 and ST12) are defective in starch phosphorylase. However, we find no significant change in the accumulation or profile of fatty acids in these mutants compared to the wild-type, suggesting that a failure to accumulate starch per se is not sufficient for the hyper-accumulation of lipid, and that more subtle regulatory steps underlie the partitioning of carbon to the two storage products

The 88 KD DEBRANCHING ENZYME MISSING IN GLYCOGEN ACCUMULATING MUTANTS OF CLAMYDOMONAS REINHARDTII DISPLAYS AN ISOAMYLASE-TYPE SPECIFICITY

International audienceTo investigate the functions of debranching enzymes in starch biosynthesis, we have partially purified and characterized these activities from wild-type and mutant sta7 Clamydomonas Reinhardtii. Two distinct debranching enzymes of 95 and 88 kD were detected. The 88 kD enzyme behaved as a part of a very large homo and heteromultimeric complex containing a minimum of 4 subunits. The 95 kD debranching enzyme cleaved -1,6 linkages separated by as few as 3 glucose residues while the multimeric complex containing the 88 kD hydrolase was unable to do so. Both enzymes were able to debranch amylopectin efficiently while the -1,6 linkages of glycogen were completely debranched by the 88 kD hydrolase only. Therefore the 95 and 88 kD debranching enzymes display respectively the limit-dextrinase (pullulanase) and isoamylase-type specificities. Various mutations in the STA7 locus caused the loss of the 88 kD isoamylase. At variance with the results obtained from maize and rice, however, the isoamylase deficiency did not result in any qualitative or quantitative difference in pullulanase activity. Morever, because the isoamylase activity accounted for over 95% of the total debranching enzyme activity we believe that the relative abundance of both types of debranching enzymes differs markedly from that found in vascular plants. The consequences of these findings with respect to the recently proposed mechanism for plant amylopectin synthesis are discussed

From raw microalgae to bioplastics: Conversion of Chlorella vulgaris starch granules into thermoplastic starch

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