Deploying Top-Down and Bottom-Up Strategies for Genetic Engineering of Auxenochlorella protothecoides for the Production of Sustainable Biofuels
Derrick Chuang1, Shivani Upadhyaya1, Krishna K. Niyogi1,2, Setsuko Wakao2, Melissa S. Roth1* (Mroth@berkeley.edu), and Sabeeha Merchant1
1University of California–Berkeley; and 2Lawrence Berkeley National Laboratory
Auxenochlorella protothecoides, a Trebouxiophyte oleaginous alga, is a reference organism for discovery and a platform for synthetic biology driven by photosynthesis. Researchers will expand transformation markers, regulatory sequences and reporter genes, improve transformation efficiency, and develop RNP-mediated gene-editing methods for genome modification. Systems analyses and metabolic modeling approaches will inform genome modifications for rational improvement of photosynthetic carbon fixation and strain engineering to produce cyclopropane fatty acids. The team will identify regulatory factors and signaling pathways responsible for activating fatty acid and triacylglycerol biosynthesis and will manipulate them to increase lipid productivity. Non-photochemical quenching and a regulatory circuit for maintaining photosynthesis under Cu limitation, both of which are absent in A. protothecoides, will be introduced to improve photosynthetic resilience, and the performance of engineered strains will be modeled. Here, researchers focus on two genetic engineering strategies, improving core photosynthesis and upregulating de novo fatty acid biosynthesis.
Microalgae can play an integral role in a sustainable bioeconomy by helping to meet the rising demand for energy and products. Microalgae use solar energy to capture and convert CO2 into biomass and achieve rapid growth without competing with food crops for land and water. However, there are considerable practical limitations in the photosynthetic production of biofuels from microalgae, resulting in low productivity and high costs. A. protothecoides has a highly flexible metabolism with rapid growth reaching high density under photoautotrophic and heterotrophic conditions, the latter in which cells accumulate large amounts of triacylglycerol (TAG). However, as with all photosynthetic organisms, one of the challenges that ultimately limits the theoretical yield is the low efficiency of photosynthetic energy conversion. Photosynthesis works efficiently when light is limiting. Under moderate to high light intensities, carbon fixation becomes limiting, and photoprotection mechanisms known as non-photochemical quenching (NPQ) are induced to minimize the generation of reactive oxygen species and photoinhibition. To enhance photosynthesis efficiency, the team will increase sedoheptulose-bisphosphatase activity to relieve the rate-limiting step of the Calvin-Benson (CB) cycle, thereby enhancing carbon fixation and decreasing the dissipation of light energy through the induction of NPQ.
cis-regulatory elements, regions of non-coding DNA, play critical roles in transcriptional regulation in algae and plants and can be used in genetic engineering approaches to increase the expression of fatty acid biosynthesis (FAS) genes. Here, researchers established a pipeline to extract transcription factors based on Interproscan IDs from the available A. protothecoides 0710 genome. Using available transcriptomic data, the team identified novel putative transcription factors involved in regulation of de novo FAS and glycolysis, which produces the precursor for FAS, pyruvate, and then tested candidate transcription factors using time-resolved qPCR analysis to confirm their upregulation during TAG accumulation. Researchers are also using systems biology tools such as cis-regulatory element discovery using MEME suite (Bailey et al. 2015) to identify key regulatory motifs potentially involved in regulation of FAS. Simultaneously, researchers will generate and integrate transcriptomic and proteomic data to create gene regulatory networks and identify hub transcription factors and use DAP-Seq (Bartlett et al. 2017) to find and validate binding targets in promoters, untranslated regions (UTR) and introns. Altogether, this work will inform the engineering of strains to improve total lipid accumulation in A. protothecoides.
Strains with engineered SBPase will be characterized for their growth, biomass, and photosynthetic capacities to test whether they have enhanced carbon fixation. Metabolomics analysis of polar metabolites will inform researchers on how the flux through the CB cycle has changed and offer new strategies for strain improvement. Similarly, strains engineered with the transcription factors will be analyzed for lipid accumulation. The metabolome data will be fed to metabolic flux modeling to iteratively improve the engineering strategies through the DBTL cycle. While characterization of each of these strategies will be interesting and important to understand the regulation of each biochemical pathway, combining both in a single strain will likely be necessary to optimize lipid production.
Bailey, T. L., et al. 2015. “The MEME Suite.” Nucleic acids research 43(W1), W39–49.
Bartlett, A., et al. 2017. “Mapping Genome-Wide Transcription-Factor Binding Sites Using DAP-Seq.” Nature protocols 12(8), 1659–72.
This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, under Award Number DE-SC0023027.