Genetic Engineering of Algal Producer Strains for Advanced Biofuels

Strategic Energy Research Consortium (SERC)

Background

Algae are a promising source of biofuels. They can produce high amounts of oils and potentially have less demands on land use than plant-based biofuel crops, such as corn. Botryococcus braunii is a green microalga that is found in fresh and brackish water (Figure 1) and is notable for production of high levels of long-chain hydrocarbons. One type of B. braunii that produces the triterpene C30-37 botryococcene and methylated squalene, has been reported to accumulate botryococcenes up to 86% of its dry weight (Metzger et al., 1987; Wolf et al., 1985). Some of these long chain hydrocarbons (>C34) appear to be present in the extracellular pools where they are readily extracted with gentler methods that do not damage cells, such that the cells can continue to be used for growth and hydrocarbon production. Overall, the extraction procedure simplifies an otherwise difficult-to-scale step in the production of algal biofuels and makes the process more feasible economically. These unique features of B. braunii present major advantages compared to current algal producer strains that could significantly reduce the cost of next-generation biofuels.

Project Goals

This project aims to overcome some of the challenges in making biofuel from algae commercially competitive. One drawback of B. braunii relative to other oil producing algae is its slow growth rate. Efforts to improve the growth rate of B. braunii (doubling time >2 days) and hydrocarbon production have been limited to optimization of environmental conditions but have not leveraged molecular level understanding of the organism. Until now, foundational knowledge is missing to optimize B. braunii as a biofuel producer strain and/or transfer its genes for hydrocarbon biosynthesis or export to engineer new producer strains. This project aims to tackle some of the barriers to harnessing B. braunii’s unique biological features to engineer optimized biofuel producer strains.

Approach

The major knowledge gap in the use of B. braunii hydrocarbons is the identification of genes required for hydrocarbon biosynthesis and export and its regulation. In this work the researchers will annotate the genome based on homology with previously characterized genes with a particular focus on identifying genes potentially involved in the synthesis, modification, binding, and transport of hydrocarbons. In addition to annotating the genome for putative functions based on homology to known genes, they will also conduct transcriptome profiling to obtain functional information about genes. A second challenge is to establish robust genetic engineering methods for B. braunii. Unlike microalgae that have been genetically transformed such as Chlamydomonas reinhardtii and Nannochloropsis, B. braunii cells have a rigid outer cell wall and reside in a dense cell matrix that are significant physical barriers for the introduction of DNA.

Team Members

Ellen Yeh 
Ellen Yeh is the Associate Professor of Pathology and of Microbiology and Immunology. Ellen Yeh's research focuses on the apicoplast, a prokaryotically-derived plastid organelle unique to Plasmodium (and other pathogenic Apicomplexa parasites) and a key anti-malarial drug target. The Yeh laboratory's goal is to elucidate apicoplast biology, function, and role in pathogenesis with the ultimate goal of realizing the potential of the apicoplast as a therapeutic target.

Arthur Grossman
Arthur Grossman has been a Staff Scientist at The Carnegie Institution for Science, Department of Plant Biology since 1982, and holds a courtesy appointment as Professor in the Department of Biology at Stanford University. He has performed research across fields ranging from plant biology, microbiology, marine biology, ecology, genomics, engineering and photosynthesis and initiated large scale algal genomics by leading the Chlamydomonas genome project (sequencing of the genome coupled to transcriptomics). During his tenure at Carnegie, he mentored more than fifteen PhD students and approximately 40 post-doctoral fellows (many of whom have become very successful independent scientists at both major universities and in industry). In 2002 he received the Darbaker Prize (Botanical Society of America) for work on microalgae, in 2009 received the Gilbert Morgan Smith Medal (National Academy of Sciences) for the quality of his publications on marine and freshwater algae and in 2024 received the ASPB Lawrence Bogorad Prize for excellence in plant biology. In 2015 he was Vice Chair of the Gordon Research Conference on Photosynthesis and in 2017 was Chair of that same conference (Photosynthetic plasticity: From the environment to synthetic systems). He also gave the Arnon endowed lecture on photosynthesis in Berkeley in March of 2017, has given numerous plenary lectures and received a number of fellowships throughout his career, including the Visiting Scientist Fellowship - Department of Life and Environmental Sciences (DiSVA), Università Politecnica delle Marche (UNIVPM) (Italy, 2014), the Lady Davis Fellowship (Israel, 2011) and most recently the Chaire Edmond de Rothschild (to work IBPC in Paris in 2017-2018). He has been Co-Editor in Chief of Journal of Phycology and has served on the editorial boards of many well-respected biological journals including the Annual Review of Genetics, Plant Physiology, Eukaryotic Cell, Journal of Biological Chemistry, Molecular Plant, and Current Genetics. He has also reviewed innumerable papers and grants, served on many scientific panels that has evaluated various programs for granting agencies [NSF, CNRS, Marden program (New Zealand)] and private companies. He has also served in a scientific advisory capacity for both nonprofit and for profit companies including Phoenix Bioinformatics, Excelixis, Martex, Solazyme/TerraVia, Checkerspot, Phycoil and CarbonDrop. Recently he has worked with Francis-Andre Wollman, Susan Dutcher and Ursula Goodenough to complete a highly expanded third edition of the Chlamydomonas Sourcebook (Elsevier, 2023).

Other Team Members:
Petra Redekop, Postdoctoral Researcher, Carnegie Institution for Science
Lev Tsypin, Stanford Energy Postdoctoral Fellow, Stanford University
Monika Kajal, Postdoctoral Fellow, Carnegie Institution for Science