Biofuels

Methods of modification of algal DNA for increasing synthetic bio-fuel production

Chris Moeseneder Bronte Everson Dylan Jones

Biofuels from algae

Biofuels from algae seem to be a promising alternate energy source
Not economically viable compared to current fossil fuel industry Scientists are attempting to increase economic viability by modifying algal genomes to increase lipid production (Georgianna et al., 2012)

Georgianna, D.R. et al., 2012. Nature, 488: 329-335

Biological concepts
Increasing permeability of cell membranes to allow DNA constructs to be integrated into the cell Manipulation of DNA and multiple loci sites during recombination
Utilising the natural power of photosynthesis for energy production

Key points
Through electroporation, the Nannochloropsis sp. cells in a high electric field, the cell wall can be penetrated in order for DNA fragments to be integrated into the original DNA The chloroplast genome of Chlamydomonas reinhardtii was altered in order to better utilise the power of photosynthesis Methods of DNA modification to improve lipid production in microalgae can have complex and sometimes do not produce feasible results

An exogenous chloroplast genome for complex sequence manipulation in algae ONeill et al. (2011)

Aim was to find a system for the cloning and modification of the chloroplast genome in Chlamydomonas reinhardtii (ONeill et al., 2011)

Genetic manipulation of the algal species was limited due to the lack of homologous recombination and a heavily annotated genome nuclear sequence (ONeill et al., 2011)

O'Neill et al., 2011, Nucleic Acids Research, 40: 2782-2792

High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.Killian et al. (2011)
Aimed to develop an efficient transformation method for Nannochloropsis sp. (Killian et al., 2011). Electroporation of the Nannochloropsis sp. cells allowed DNA transformants to penetrate the cell wall (Killian et al., 2011) Gene knockout constructs used to test for homologous recombination, with nitrite reductase (NiR) and nitrate reductase (NR) genes targeted Disruption of these genes can be detected as the alga is known to grow on nitrate and nitrite based sources (Killian et al., 2011)

Killian et al., Proc. Natl Acad. Sci. USA, 108: 21265-21269

From Killian et al., 2011

ONeill et al. (2011) Findings

Exogenous modification was possible through the combination of algal DNA sequences and bacterial selection markers. DNA sections were amplified using PCR in order for the modification of algal DNA to occur (ONeill et al., 2011) The modified algal DNA could then be assembled in a yeast hybrid and transplanted back into algal cells

From ONeill et al., 2011

Killian et al. (2011) Findings

Linear amount of DNA used in electroporation relative to the amount transformants obtained. Shows that more DNA fragments are needed for efficient transformation (Killian et al., 2011) Successful gene knockout of NR / NiR in one transformation step ( 94%) discovered that Nannochloropsis is haploid (Killian et al., 2011, Table 2) Haploid nature allows for easy insertion of transformants through homologous recombination

From Killian et al., 2011

Judgement and Evalution

Both research groups pursued a similar goal, but using very different methods Killian et al. (2011): a simple method, but had no suggestions for applications ONeill et al. (2011): a very complex methodology, but showed that it could be used for sequence deletions, rearrangement and minimization

Neither of the studies clearly show how their findings will lead to an increase in oil production

Ways of economically producing biofuels from algae are important

However, these research papers dont shed light on non- CO2 emitting alternative energy sources.
The concentration of specific model algal species and strains for biofuel production will cause a reduction in the variability of the gene pool, hence leading to a lack of disease resistance

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