Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Colleen McLoughlin - Boyce Thompson Institute / Cornell University
Co-Author(s): Shih-Chi Hsu and David Stern, Boyce Thompson Institute, Ithaca, NY
Biofuels are a source of renewable energy that are being investigated as a means to decrease greenhouse gas emissions. Some algal species have the ability to produce biofuels, including Botryococcus braunii, which makes botryococcenes, hydrocarbons that can be readily converted into gasoline, kerosene, and diesel through existing petroleum processing procedures. However, B. braunii grows slowly, so producing biofuels for practical applications is unrealistic. Unlike B. braunii, Chlamydomonas reinhardtii grows quickly and is a model species with a well characterized lifecycle, sequenced nuclear and organellar genomes, and genomic tools. Our goal is to transfer and optimize the botryococcene-producing biosynthetic capabilities of B. braunii into C. reinhardtii as a proof of concept. The synthesized botryococcene precursor IPP in C. reinhardtii chloroplasts will be used to complete botryococcene biosynthesis by the addition of three enzymes, SSL1, SSL3 and FPPS. Initially, the three requisite genes were introduced, with successful detection of the transgenic transcripts. Preliminary data indicated these transcripts were likely translated, however the protein products were not detected. Prompted by an earlier study showing that some amino acids at the N-terminal penultimate position destabilized transgenic proteins in tobacco chloroplasts (Apel et al., 2010), and given that the second codon of our transgenes encoded such amino acids, we hypothesized that these proteins were unstable in C. reinhardtii chloroplasts. We therefore created a series of new constructs that encode stabilizing amino acids, valine or glutamate, at the second position of each transgene to promote accumulation of the botryococcene biosynthesis enzymes. Following chloroplast transformation, I used DNA gel blots to confirm that 32 strains had correct transgene insertion. To determine the second codon of the transgenes in transformed cells, the Derived Cleaved Amplified Polymorphic Sequences (dCAPS) assay was used. My dCAPS and immunoblot results demonstrated that glutamate at the second position facilitates the accumulation of at least one transgenic protein. We will continue to study how the second codon in our transgenes affects protein stability, and use this information to optimize the abundance of botryococcene biosynthesis enzymes in Chlamydomonas chloroplasts.
Funder Acknowledgement(s): Funding was provided by the National Science Foundation EFRI REM grant, award number 1240478.
Faculty Advisor: David Stern,