Tools and Targets: Engineering Modified Fatty Acids and Improved Photosynthetic Resilience in Auxenochlorella protothecoides
Jeffrey L. Moseley1* (firstname.lastname@example.org), Dimitrios J. Camacho1, Rory J. Craig1, Marco A. Duenas1, Sean D. Gallaher1, Suzanne M. Kosina2, Kyle J. Lauersen3, Yang-Tsung Lin1, Radhika Mehta1, Crysten E. Blaby-Haas2, Trent R. Northen2, and Sabeeha S. Merchant1
1University of California–Berkeley; 2Lawrence Berkeley National Laboratory; and 3King Abdullah University of Science and Technology
Auxenochlorella protothecoides, a Trebouxiophyte oleaginous alga, is a reference for discovery and a platform for photosynthesis-driven synthetic biology and sustainable bio-production. 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. Regulatory factors and signaling pathways responsible for activating fatty acid and triacylglycerol biosynthesis will be identified, and researchers 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.
Researchers have established transformation and targeted gene-replacement by homologous recombination in A. protothecoides and now seek to engineer strains with improved photosynthesis that produce modified fatty acids. As an essential preliminary step, researchers have resolved the organellar genomes and generated a gapless, ~22 Mbp (haploid size), phased diploid nuclear genome of strain UTEX 250. Iso-seq and RNA-seq analyses facilitated the annotation of >7500 gene models on 12 chromosomes. Elemental analysis by ICP-MS was used to optimize the defined growth medium, enabling luxury consumption of essential nutrients. Subsequent systems analyses will be carried out to identify the full complement of metalloproteins encoded in the genome and to understand how metal nutrients are allocated. Two biosafe transformation markers, Arabidopsis THIC, restoring thiamine prototrophy, and Saccharomyces SUC2, enabling sucrose assimilation, are in routine use, along with the nptII gene, conferring resistance to the aminoglycoside antibiotic, G418. Additional herbicide resistance transformation markers are under development, along with counter-selectable recyclable marker genes that will allow for scarless and marker-free integrations. Strong promoters from genes encoding an ammonium transporter, stearoyl-ACP desaturase, methionine synthase, RuBisCO small subunit, photosystem I and light-harvesting complex components have been demonstrated to activate expression of codon-optimized transgenes. Chlamydomonas BKT1, encoding beta-carotene ketolase, Gaussia princeps luciferase, Venus and mCherry have been used as quantifiable reporter genes for evaluating promoter strength, and plastid targeting of the fluorescent proteins was demonstrated. Researchers have also investigated the optimal arrangement of ORFs in polycistronic constructs; the results are consistent with more balanced translation when the shorter of the two ORFs is upstream. This is compatible with the finding that ORFs in endogenous polycistronic genes across the green lineage usually have the shorter ORF upstream.
One stated goal is to modify Auxenochlorella fatty acid and lipid biosynthesis to produce medium (mid)-chain length cyclopropane fatty acids (CPFAs) suitable as precursors for jet fuel. This will require chain-length control to increase mid-chain fatty acids, control of saturation level, since cyclopropane fatty acid synthases (CPS) compete with endogenous microsomal desaturases for mono-unsaturated substrates, and increased exchange between phospholipids (the site of CPFA synthesis) and Kennedy pathway intermediates in TAG biosynthesis (so that CPFAs are incorporated into storage lipids). Initial experiments established that accumulation of C12:0 and C14:0 fatty acids increased in strains expressing the Cuphea wrightii FATB2 thioesterase gene, driven by a SAD2 promoter that is activated during N-starvation and lipid production. C16:0 levels were increased by expressing FATB3 from Brassica juncea. Researchers will test whether mid-chain levels can be enhanced further by co-expressing CwFATB2 with a beta-ketoacyl-ACP synthase gene (CwKASA1) from the same species. To generate CPFAs the team intend to first make Auxenochlorella strains with reduced Δ12-desaturase activity, thus removing the most significant competing activity for CPS. These strains will simultaneously overexpress phosphatidylcholine: diacylglycerol cholinephosphotransferase (PDCT), lysophosphatidylcholine acyltransferase (LPCAT), and lysophosphatidic acid acyltransferase (LPAAT) from Sterculia foetida and Litchi chinensis; both plant species that accumulate high amounts of CPFAs in their seed oils. The team will then screen CPS from E. coli and CPFA-accumulating plants or marine bacteria to identify the most active enzymes in Auxenochlorella.
Researchers will also introduce a regulatory circuit into Auxenochlorella to maintain photosynthetic resilience in response to Cu-deficiency. The major sink for Cu in plants is the thylakoid lumen protein plastocyanin, which transfers electrons from the cyt b6/f complex to oxidized photosystem I. Consequently, Cu limitation, which is common in many environments, severely reduces plant photosynthesis and growth. Some algae and cyanobacteria acclimate to Cu deficiency by substituting a heme-containing cytochrome that performs the same electron carrier function as plastocyanin, thereby maintaining high rates of photosynthetic electron transfer and reducing their cellular Cu quota. In Chlamydomonas the CYC6 gene, encoding Cyt c6, is regulated by a copper-sensing transcription factor CRR1. A potential CRR1 homolog is identified in the Auxenochlorella genome, and preliminary RNA-seq analysis indicates that a putative copper transporter gene, CTR1, is activated during Cu starvation. The team will use the CTR1 promoter to activate expression of a synthetic, codon-optimized CYC6 gene in Cu-deficient Auxenochlorella cells, and test for improved photosynthetic performance and growth. Efficient import of heterologous Cyt c6 into the Auxenochlorella thylakoid lumen may require replacement of the native bipartite transit peptide with the transit peptide of an endogenous lumen targeted protein, such as Auxenochlorella plastocyanin. This work will establish A. protothecoides as a powerful photosynthetically driven cell chassis for sustainable bioproduction of fuels and specialty products.
This work was supported by the DOE Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-SC0023027.