Eric E. Jarvis

Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances

Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20-50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization.

GENETIC TRANSFORMATION OF THE DIATOMS CYCLOTELLA CRYPTICA AND NAVICULA SAPROPHILA

Two species of diatoms were genetically transformed by introducing plasmid vectors containing the Escherichia coli neomycin phosphotransferase II (nptII)gene. Expression of the bacterial nptII gene in the diatoms was achieved using the putative promoter and terminator sequences from the acetyl‐CoA carboxylase gene from the centric diatom Cyclotella cryptica T13L Reimann, Lewin, and Guillard. The vectors were introduced into C. cryptica and the pennate diatom Navicula saprophila NAVIC1 Lange‐Bertalot and Bonik by microprojectile bombardment. Putative transformants were selected based on their ability to grow in the presence of the antibiotic G418, and production of the neomycin phosphotransferase protein by the transformed cells was confirmed by western blotting. The foreign DNA integrated into one or more random sites within the genome of the transformed algal cells, often in the form of tandem repeats. This is the first report of reproducible, stable genetic transformation of a chlorophyll c‐containing alga.

Manipulation of microalgal lipid production using genetic engineering

Genetic transformation of two species of diatoms has been accomplished by introducing chimeric plasmid vectors containing a bacterial antibiotic resistance gene driven by regulatory sequences from the acetyl-CoA carboxylase (ACCase) gene from the diatom Cyclotella cryptica. The recombinant DNA integrated into one or more random sites within the algal genome and the foreign protein was produced by the algal transformants. This is the first report of genetic transformation of any chlorophyll c-containing microalgal strain. We are using this system to introduce additional copies of the ACCase gene into diatoms in an attempt to manipulate lipid accumulation in transformed strains.

Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea

SummaryWe report here on the development of a transient expression system for Chlorella ellipsoidea using a heterologous gene, firefly luciferase. Cells of this unicellular green alga were converted to protoplasts and treated with plasmid pDO432, which bears luciferase under the control of the CaMV 35S promoter. This treatment resulted in detectable luciferase activity in cell extracts. Expression required Cellulysin treatment, active cell metabolism, and the addition of carrier DNA and polyethylene glycol. Linearization of the luciferase plasmid did not significantly alter the activity. A time course of expression showed that luciferase is made rapidly, within about 7 h after addition of DNA, but that the activity disappears over the course of a few days. These experiments represent an important first step in the development of a Chlorella transformation system.

Efficient anaerobic digestion of whole microalgae and lipid-extracted microalgae residues for methane energy production

The primary aim of this study was to completely investigate extensive biological methane potential (BMP) on both whole microalgae and its lipid-extracted biomass residues with various degrees of biomass pretreatment. Specific methane productivities (SMP) under batch conditions for non-lipid extracted biomass were better than lipid-extracted biomass residues and exhibited no signs of ammonia or carbon/nitrogen (C/N) ratio inhibition when digested at high I/S ratio (I/S ratio⩾1.0). SMP for suitably extracted biomass ranged from 0.30 to 0.38LCH4/gVS (volatile solids). For both whole and lipid-extracted biomass, overall organic conversion ranged from 59.33 to 78.50 as a measure of %VS reduction with greater percentage biodegradability in general found within the lipid-extracted biomass. Higher production levels correlated to lipid content with a linear relationship between SMP and ash-free lipid content being developed at a R(2) of 0.814.

DNA nucleoside composition and methylation in several species of microalgae

Total DNA was isolated from 10 species of microalgae, including representatives of the Chlorophyceae (Chlorella ellipsoidea, Chlamydomonas reinhardtii, and Monoraphidium minutum), Bacillariophyceae (Cyclotella cryptica, Navicula saprophila, Nitzschia pusilla, and Phaeodactylum tricornutum), Charophyceae (Stichococcus sp.), Dinophyceae (Crypthecodinium cohnii), and Prasinophyceae (Tetraselmis suecica). Control samples of Escherichia coli and calf thymus DNA were also analyzed. The nucleoside base composition of each DNA sample was determined by reversed‐phase high performance liquid chromatography. All samples contained 5‐methyldeoxycytidine, although at widely varying levels. In M. minutum, about one‐third of the cytidine residues were methylated. Restriction analysis supported this high degree of methylation in M. minutum and suggested that methylation is biased toward 5′‐CG dinucleotides. The guanosine + cytosine (GC) contents of the green algae were, with the exception of Stichococcus sp., consistently higher than those of the diatoms. Monoraphidium minutum exhibited an extremely high GC content of 71%. Such a value is rare among eukaryotic organisms and might indicate an unusual codon usage. This work is important for developing strategies for transformation and gene cloning in these algae.

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose

Genes encoding l‐arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on l‐arabinose was dependent on a functioning l‐arabinose transporter, or by screening a differential display library, respectively. These transporters also transport d‐xylose and were designated KmAXT1 (arabinose–xylose transporter) and PgAXT1, respectively. Transport assays using l‐arabinose showed that KmAxt1p has Km 263 mm and Vmax 57 nm/mg/min, and PgAxt1p has Km 0.13 mm and Vmax 18 nm/mg/min. Glucose, galactose and xylose significantly inhibit l‐arabinose transport by both transporters. Transport assays using d‐xylose showed that KmAxt1p has Km 27 mm and Vmax 3.8 nm/mg/min, and PgAxt1p has Km 65 mm and Vmax 8.7 nm/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed Km 371 mm and Vmax 341 nm/mg/min for l‐arabinose, and Km 25 mm and Vmax 76 nm/mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both l‐arabinose and d‐xylose, one scenario for the complete usage of biomass‐derived pentose sugars would require only the low‐affinity, high‐throughput transporter Gal2p and one additional high‐affinity general pentose transporter, rather than dedicated d‐xylose or l‐arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Accession Nos: KmAXT1: GZ791039; PgAXT1: GZ791040 Copyright

New method for discovery of starch phenotypes in growing microalgal colonies.

To identify algal strains with altered starch metabolism from a large pool of candidates of growing algal colonies, we have developed a novel, high-throughput screening tool by combining gaseous bleaching of replica transferred colonies and subsequent iodine staining to visualize starch. Screening of healthy growing colonies of microalgae has not been possible previously because high levels of chlorophyll make the detection of starch with an iodine stain impossible. We demonstrated that chlorine dioxide (ClO(2)) removes essentially all chlorophyll from the colonies and enables high-throughput screening of, for example, a population of mutagenized cells or a culture collection isolated in a bioprospecting project.