Microalgae lipid fuel production
Background
The use of photosynthetic microorganisms as sources of renewable energy has recently been the subject of much research and debate. Notably, microalgae lipid production has been considered as a potential source of biofuels that would not compete with food products. Other advantages include the atmospheric capture CO2 by algae, which might help neutralize the effects of later hydrocarbon combustion. Algae biofuel is also non-toxic and biodegradable and renewable.
Research Problem and Goals
There are many limitations to producing and extracting oil from algae, such as optimizing algae growth in bioreactors; lipid production and characteristics; extraction efficiency; and economic sustainability. We would like to improve the efficiency of algae energy production. Specifically, we will focus on the up-regulation of lipid biosynthesis, leading to increased fuel yield. High yields might help dispel initial cost worries. By using available genome sequence information from a variety of algae, as discoveries from other microorganism systems, we could select a point in the micro-algae lipid synthesis pathway to modify. Some options would be to increase expression of anabolic enzymes, or decrease inhibitory regulation mechanisms. Another possibility could be to add transform algae strains so as to extend the biosynthetic pathway of lipids in such a way that the biomass produced contains lipids that are closer to those that are used for combustion.
Overexpressing the YEAST G3P dehydrogenase in microalgae: Since modifications of the TAG assembly pathway have given some of the best increases in lipid production in terrestial photosynthetic organisms, we wish to transform Chlamydomonas reinhardtii via over-expression of the yeast G3PDH gene.
Project details and methods
Model system: Chlamydonomas reinhardtii
-eukaryotic, single celled greed microalgae. -extensively studied and characterized (Gene bank data); most microalgae research has been constructed in this strain - can be used as a model platform to design modifications transferrable to other strains, OR could be engineered into a biofuel super source.
- Targeting the fatty acid production pathway in the chloroplast/plastid had thus far been unsuccessful. However, Vigeolas et. al. were able to increase TAG production by 40% by upregulating the TAG assembly pathway in the cytosol. **
Two parts:
- Transforming oil-seed rape by overexpression of yeast gpd1 gene under napin promoter
-This results in increased expression of glycerol-3-phosphate dehydrogenase (Gly3PDH). > This enzyme catalyzes the conversion of the glycolytic intermediate dihydroxyacetone phosphate (DHAP) into glycerol-3-phosphate, which along with fatty acids, is one of the co-limiting substrates for tryacylglycerol (TAG) assembly. -This study showed some important findings: a. b. c. d.
As of April 2010, this method has not yet been tried in photosynthetic algae. We wish to transfer this kno
- Microalgae transformation methods.
1. Grow an arg- Chlamydomonas reinhardtii strain at 25°C under constant illumination. 2. Transform using electroporation in presence of the gpd1, Arg7, Kan+ containing plasmid.
Methods:
- Bioreactor necessary?
- How to test for efficient transformation: markers
- How to measure change in lipid production?
Projected Outcomes
If everyting works well:
If nothing works:
In between case:
Needed Resources
Societal Impact
- By increasing lipid yield per liter of microalgae, we could make algae biofuels a more economically feasible source of biofuels
- Alternative to other biofuels sources that compete with food consumption
- Alternative to petroleum and other fossil fuels, whose drawbacks include unstable costs and foreign sources
Sources
Radakovits R, Jinkerson RE, Darzins A, Posewitz1 MC. Eukar Cell 2010 (9,4):486–501.
This review collects the current research regarding algae biofuels and summarizes the challenges that need to be addressed in order to make algae a viable source of biofuels. Some of the approaches proposed in this paper include: upgrading the direct biosynthesis of lipids, altering lipid characteristics so as to make them a more relevant fuel source, optimizing the conversion of carbohydrates to lipids, and improving photosynthetic efficiency. From this paper, which was just published this month, we gathered that there is great interest in developing algae biofuels as an energy source. As a consequence of this, there is ample opportunity for innovation and genetic optimization. This paper also contains numerous references that can serve as starting points for research ideas and tools
- Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M. Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 229: 873-883.
-This paper describes the manipulation of foreign genes and the plasmid methods necessary for transforming our desired microalgal strain.
Necessary steps include:
1. Taking the yeast gene gpd1 (S. cerviciae GenBank Z24454, Larsson et.al. 1993), and optimizing for C. reinhardtii codon usage. Amplify.
1a. Artificiallu divide the gene into two exons using recombinant PCR and restriction enzyme methods.
2.Inserting endogenous introns 1,2,3 (in order) from the C. reinhardtii gene RCBS2 : these introns facilitate nuclear transformation and cytosolic expression of our enzyme.
3. Fusing into plasmid expression vectors : transferring into pHsp70A/RbscS2-Chlamy, then fusion with the pUC-Arg7-lox-B plasmid via Cre/lox mediated site-specific recombination.
-This paper also describes the growth and transformation of the algae:
(ENTER CONTENT)
- Vigeolas H, Waldeck P, Zank T, Geigenberger P. Increasing seed oil content in oil-seed rape (Brassica napus L.) by over-expression of yeast glycerlo-3-phosphate dehydrogenase under the control of a seed-specific promoter. Plant biotechnol. J. 5: 431-441.
- This article decribes a successful increase of TAG production in oil-seed rape. We want to reproduce the results in algae. -Describes that gas chromatography was used to measure TAG content.
- Heitzer M, Zschoernig B (2007) Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system. Biotechniques 43:324, 326, 328
-This article describes the expression vector we will use to transform our C. reinhardtii.
- Leon-Banares R, Gonzalez-Ballester D, Galvan A, Fernandez E. Transgenic microalgae as green cell-factories. Trends Biotechnol. 2004; 22: 45-52.
-Useful markers, summary of transformation methods, drawback.
- Larsson K, Ansell R, Eriksson P, Adler L. A gene encoding sn-glycerol-3-phosphate dehydrogenase (NAD+) complements a osmosensitive mutant of Saccharomyces cervisiae. Mol. Microbiol. 1993; 10: 1101-1111.
-Here is the article about the yeast gpd1 gene we will be using.
- Some interesting presentations:
Engineering Challenges in Algae Energy
- Useful webpages:
UCSD Center for Algae Techology
- Potentially useful articles:
-Hu, Q., M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, and A. Darzins. 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54:621–639.
