Question

ExxonMobil and Synthetic Genomics Inc. announced a new phase in their joint algae biofuel research program in March that could lead to the technical ability to produce 10,000 barrels of algae biofuel per day by 2025. This is based on a study reported last June in Nature Biotechnology that described how a targeted mutation introduced in the algal genome resulted in doubling the lipid output of the organism.

Outline TWO different models for how a mutation in the algae might possibly lead to an increase in lipid synthesis. You should include diagrams as well as text for each of your models.

Answer 1

Successful genetic transformation has been reported for the green (Chlorophyta), red (Rhodophyta), and brown (Phaeophyta) algae; diatoms; euglenids; and dinoflagellates. More than 30 different strains of microalgae have been transformed successfully to date. In many cases, transformation resulted in stable expression of transgenes, from either the nucleus or the plastid, but in some cases only transient expression was observed. Methods developed primarily with C. reinhardtii demonstrate that the stability of expression can be improved through proper codon usage, the use of strong endogenous promoters, and inclusion of species-specific 5?, 3?, and intron sequences. The efficiency of transformation seems to be strongly species dependent, and the method of transformation has to be carefully selected and optimized for each microalga. A variety of transformation methods have been used to transfer DNA into microalgal cells, including agitation in the presence of glass beads or silicon carbide whiskers, electroporation, biolistic microparticle bombardment, and Agrobacterium tumefaciens-mediated gene transfer.

Efficient isolation of genetic transformants is greatly facilitated by the use of selection markers, including antibiotic resistance and/or fluorescent/biochemical markers. Several different antibiotic resistance genes have been used successfully for microalgal transformant selection, including bleomycin, spectinomycin, streptomycin, paromomycin, nourseothricin and others.

While increasing the expression of genes involved in fatty acid synthesis has had small successes, with regard to increasing the total amount of seed oils, some interesting results have been achieved through the overexpression of genes involved in TAG assembly. One of the most successful attempts to increase the amount of seed lipid is the overexpression of a cytosolic yeast, glycerol-3-phosphate dehydrogenase (G3PDH), in the seeds of B. napus, which resulted in a 40% increase in lipid content. G3PDH catalyzes the formation of glycerol-3-phosphate, which is needed for TAG formation. This interesting result suggests that genes involved in TAG assembly are of importance for total seed oil production. This is further supported by several other studies in which overexpression of TAG assembly genes resulted in increases in seed oil content. For example, overexpression of glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, or diacylglycerol acyltransferase (DAGAT) all result in significant increases in plant lipid production (79, 80, 96, 186, 215, 218). Due to the fact that enzymes such as these seem to be good candidates for overexpression strategies with the goal of increasing storage lipid content, an attempt has also been made to use directed evolution to increase the efficiency of one of these enzymes, DAGAT (172).

Another possible approach to increasing the cellular lipid content is blocking metabolic pathways that lead to the accumulation of energy-rich storage compounds, such as starch. For example, two different starch-deficient strains of C. reinhardtii, the sta6 and sta7 mutants, have disruptions in the ADP-glucose pyrophosphorylase or isoamylase genes, respectively. Wang et al. (197) have shown that these mutants accumulate increased levels of TAG during nitrogen deprivation. Another starchless mutant of Chlorella pyrenoidosa has also been shown to have elevated polyunsaturated fatty acid content