Mysterious Metabolism: Understanding the Biosynthesis of Zeaxanthin from β-Carotene in Diatoms
Abstract Diatoms are ecologically important and industrially attractive brown microalgae. Much is unknown about what enzymes catalyze key steps in their carotenoid pigment biosynthesis pathway. For example, diatoms do not have the enzyme β-carotene hydroxylase (BCH), which catalyzes the conversion of β-carotene to zeaxanthin in green microalgae and plants. The diatom enzyme LUT-like (LTL) is hypothesized to catalyze the reaction instead, but this has never been experimentally demonstrated. The goal of my summer research is to experimentally test the hypothesis by knocking down the two LTL-encoding genes present in the model diatom Thalassiosira pseudonana, and assessing the impact on pigment composition.
Research Question Diatoms are a type of brown microalgae characterized by a silicate cell wall. They have been a major part of the carbon cycle on Earth for billions of years, and have contributed to geological deposits of diatomaceous earth and oil.1 They are responsible for roughly a third of marine primary production, and sink a large fraction of the atmospheric carbon dioxide yearly.2 From 1978 to 1996, the US Department of Energy conducted a broad comparative study of microalgae and their utility for fuel production, which showed that diatoms are prime candidates.3 Diatoms are easily induced to high levels of lipid production by nutrient starvation, and their lipids can be easily converted to fuel oil through chemical processes. They also have numerous pigments and metabolic products that are of commercial and medicinal interest.1 Diatoms are photosynthetic, like plants and green algae, but differ in the types of light-harvesting and photoprotective pigments they utilize. The major light-harvesting and photoprotective pigments in diatoms are the end products of the carotenoid biosynthesis pathway. The early steps in the pathway are conserved between diatoms and plants/green algae. However, the later diatom-specific steps, and specifically which enzymes catalyze them in diatoms, are not well understood. 2,4,5 What I plan to research over the summer is the conversion of β-carotene to zeaxanthin in diatoms. Zeaxanthin plays a role in photoprotection in plants and green algae, and is present and able to function in that capacity in diatoms, but is secondary to the main diatom photoprotective mechanism and is only found in trace amounts, as a precursor to the biosynthesis of other diatom pigments. In plants and green algae, zeaxanthin biosynthesis from β-carotene is catalyzed by the enzyme β-carotene hydroxylase (BCH). There are two known types of BCH, however diatoms possess neither. An enzyme called LTL (LUT-like) is hypothesized to function in place of the BCH in the diatom carotenoid biosynthesis pathway. LUT is a protein that catalyzes the hydroxylation of α-carotene in the model plant Arabidopisis thaliana. Diatom LTLs bear similarity to the LUTs, yet diatoms do not make α-carotene. The significance of their similarity is that similar proteins likely have similar functions. Since diatoms do not have BCH, something else must be catalyzing the hydroxylation of β-carotene. The involvement of LTLs is an attractive hypothesis, but has yet to be demonstrated experimentally.2
Methods and Theoretical Framework I will be working with the model diatom Thalassiosira pseudonana, for which genomic, transcriptomic, and physiological data, as well as sophisticated techniques for genetic manipulation, are available, which make it a good candidate for exploring carotenoid biosynthesis.6,7,8,9 Bioinformatic analysis has found two copies of the LTL gene in T. pseudonana. My summer research will consist of simultaneously knocking down both of the potential LTL genes to see what effect this has on pigment production. This will support or refute the involvement of LTLs in diatom carotenoid biosynthesis, and in doing so, broaden what is known about the metabolism of these ubiquitous, commercially-appealing organisms. If the LTLs function as hypothesized, the knockdown lines should accumulate more β-carotene, and have less of the downstream pigments, than wild-type T. pseudonana. A change other than that would indicate that the LTLs are involved in the diatom carotenoid biosynthesis pathway, but in an unanticipated manner. If there is no change in pigment composition with the knockdown, the involvement of LTLs in β- carotene hydroxylation will be contradicted. Gene knockdowns in T. pseudonana are a routine procedure in the lab.9 Gateway cloning, also well-established in the lab, will be used for generating the knockdown construct, targeting both LTLs at the same time.9 Wild-type T. pseudonana will be transformed by tungsten particle bombardment. Successful transformants will be selected for using resistance to nourseothricin (NAT)10. PCR will be used to confirm the presence of the knockdown construct. I will allow four clones, as well as a wild-type control, to acclimate to two different irradiance levels in order to induce different levels of pigment production, harvest the cultures using filtration, then asses the pigment composition with High Performance Liquid Chromatography (HPLC)11.
Preliminary Work and Experience
I currently volunteer in the Hildebrand lab and work alongside a PhD student, helping to elucidate other parts of diatom pigment metabolism. I have gained relevant experience with preparing growth media, primer design, PCR, transformation of E. coli, Gateway cloning, plasmid minipreps, and soon will be learning how to generate transgenic algae. The lab experience I will have already gained will allow me to approach my project without a steep learning curve.
Projected Timeline June 13th – July 5th: 3 weeks to generate the knockdown construct using Gateway cloning July 5th – July 11th: 1 week to transform T. pseudonana using tungsten particle bombardment July 11th – August 1st: 3 weeks to select for transformants and screen with PCR for the presence of construct August 1st – August 22nd: 3 weeks for differential light exposure to induce different pigment expression, harvesting for pigment analysis August 22nd – August 29th: 1 week to analyze the pigment composition using High Performance Liquid Chromatography
During down time, I will assist with other projects, continuing to gain more experience and learning new methods and techniques. I also plan to research what is known about the active site chemistry of several other enzymes being studied in the lab, which may help in elucidating their functions, and happens to be a particular interest of mine.
IRB approval will not be required.
References 1. Mark Hildebrand, Aubrey K Davis, Sarah R Smith, Jesse C Traller & Raffaela Abbriano (2012) The place of diatoms in the biofuels industry, Biofuels, 3(2): 221-240 2. Martine Bertrand (2010) Carotenoid biosynthesis in diatoms, Photosynth Res 106:89-102, 3. Sheehan J, Dunahay T, Benemann J, Roessler P. (1998) A look back at the U.S. Department of Energy’s Aquatic Species Program: biodiesel from algae; close-out report. United States. Doi:10.2172/15003040. 4. Coesel S, Obornik M, Varela J, Falciatore A, Bowler C. (2008) Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms. PLoS One 3(8):e2896. 5. Dambeck M, Eilers U, Breitenbach J, Steiger S, Buchel C, Sandmann G. (2012) Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum. J Exp Bot 63(15):5607-5612. 6. Armbrust EV, Berges JA, Bowler C, Green BR, Martinez D et al. (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306: 79-86. 7. Smith et al. under revision – remind me to look up full citation for you 8. Shrestha RP, Tesson B, Norden-Krichmar T, Federowicz S, Hildebrand M, Allen AE. (2012) Whole transcriptome analysis of the silicon response of the diatom Thalassiosira pseudonana. BMC Genomics 13:499. 9. Trentacoste EM, Shrestha RP, Smith SR, Gle C, Hartmann AC, Hildebrand M, Gerwick WH. (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. PNAS 110(49):19748-19753 10. Nicole Poulsen, Patrick M. Chesley, and Nils Kröger (2006) MOLECULAR GENETIC MANIPULATION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE). Journal of Phycology 42(5): 1059-1065 11. Kozlowski WA, Deutschman D, Garibotti I, Trees C, Vernet M. (2011) An evaluation of the application of CHEMTAX to Antarctic coastal pigment data. Deep-Sea Res Pt I 58:350-364.
Personal Statement My name is Dylan Mills. I am from the San Francisco Bay Area, and just transferred to UCSD in the summer of 2015. I’m a junior, working on a B.S. in Biochemistry. Before transferring, I attended Chabot College in Hayward, California, where I served as a tutor and laboratory section assistant for the Chemistry Department. During the summer of 2015 I attended a talk by a member of the UCSD faculty, Dr. Stephen Mayfield, about the use of algae for biofuels and biomanufacturing of therapeutic substances. This piqued my interest in algae laboratories, because my long term plan is operating a company which develops and applies biomanufacturing techniques. Shortly thereafter, I applied to volunteer at Mark Hildebrand’s laboratory at SIO, where diatoms, a type of brown microalgae, are studied and developed for a variety of applications, including biofuels and nanotechnology. I have been volunteering since October 2015, which has allowed me to learn some standard molecular biology and microalgal culture techniques. The focus of my education is to learn how biological systems catalyze reactions that are useful to humans. Part of this goal is to develop a detailed understanding of the way protein structure affects catalysis, which would enable the design of custom enzymes for cost restrictive steps in industrial scale chemical synthesis. Another part is to learn how to metabolically engineer amenable organisms to function as chemical factories, enabling biofuel and drug synthesis in living platforms that have modest nutritional requirements, such as light and vitamins. Yet another part is to explore the use of engineered biomaterials in nanotechnology. Life naturally has the capacity to manipulate matter and energy at a fundamental, quantum level, and it is this power that I wish to harness. An inspirational analogy for the type of work I would like to do is that of Dr. Craig Venter. In 2007 Dr. Venter’s institute assembled a synthetic bacterial genome over a million base pairs in length and inserted that genome into a bacterium which had its DNA removed. The bacterium was able to replicate and divide normally. This was a major step in the pursuit of creating a basic template of bacterial life, on top of which chemical biosynthesis pathways can be introduced. While this was a major advance in synthetic biology, with promise for biomanufacturing, there are inherent limits to biomanufacturing in bacteria. For example, bacteria cannot properly fold or modify some, more complex, eukaryotic proteins. Eukaryotic microalgae have the metabolic tools to regulate proper synthesis and folding of a much wider array of eukaryotic proteins, which is what makes them so interesting to me. The proposed summer research will give me further firsthand experience with the tools and concepts used in manipulating microalgal metabolism. It will allow continued development of my skills in molecular biology and generation of transgenic microalgae, which are crucial to what I hope to accomplish. Finally, it will afford me the opportunity to contribute to what is known about diatom metabolism, an exciting prospect in and of itself.