Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Engineering Auxenochlorella protothecoides: Artificial Chromosomes, Regulators of Lipid Biosynthesis, and Improving Photosynthesis

Authors:

Jeffrey Moseley1* (jlmoseley@berkeley.edu), Marco Duenas1, Rory Craig1, Derrick Chuang1, Setsuko Wakao2, Shivani Upadhyaya1, Melissa Roth1, Carrie Nicora3, Sam Purvine3, Mary Lipton3, Krishna Niyogi1, Sabeeha Merchant1

Institutions:

1University of California–Berkeley; 2Lawrence Berkeley National Laboratory; 3Pacific Northwest National Laboratory

Goals

Facile gene targeting in the nuclear genome makes Auxenochlorella protothecoides, a unicellular, freshwater Trebouxiophyte, useful as a reference organism for discovery and a platform for synthetic biology. The team aims to expand the molecular genetic toolkit with additional neutral integration sites, transformation markers, regulatory sequences and reporter genes, along with improving transformation efficiency and developing RNP-mediated gene-editing methods for genome modification. Researchers are employing systems analyses and metabolic modeling approaches to inform engineering of the Calvin-Benson cycle for improved photosynthetic carbon (C) fixation, and to identify signaling pathways and regulators responsible for controlling fatty acid and triacylglycerol biosynthesis. Genome modifications predicted from these analyses to increase lipid productivity will be combined with strain engineering to produce cyclopropane fatty acids. Nonphotochemical 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.

Abstract

Researchers used PacBio long-read sequencing to generate a gapless, telomere-to- telomere, phased diploid nuclear genome, and fully resolved the circular organelle genomes of Auxenochlorella protothecoides UTEX 250. This well-annotated, 45 Mb diploid nuclear genome resembles a genetic hybrid, with extensive inter- and intra-chromosomal recombination, and two instances of trisomy. Chromosome 3 trisomy was confirmed by knock-in of a Venus reporter at one allele of Ammonium Transporter 1B (AMT1B), and activation of the AMT1B promoter by nitrogen depletion in heterotrophic cells resulted in increased Venus fluorescence. The team will exploit this redundant chromosome as a landing pad for transgene integration, and putative centromere sequences will be tested for their ability to allow maintenance of stable centromeric plasmids.

Photosynthgreen algae can utilize sunlight to power photosystems for C fixation. In the daytime, algae are exposed to dynamic light conditions ranging from high-to-low or dark-changing light conditions have significant impacts on their growth, biomass and production. Researchers propose two strategies to improve C capture and growth under a field-like setting for A. protothecoides: (1) engineering a rate-limiting enzyme of the Calvin-Benson cycle (CBC), sedoheptulose 1,7-bisphosphatase (SBPase); and (2) introducing a photoprotective nonphotochemical quenching (NPQ) protein (LHCSR) to allow for robust growth under fluctuating light. Taking advantage of homologous recombination, researchers have generated strains to test both strategies. Preliminary data indicate that overexpression of SBPase leads to a growth benefit, and the introduction of a well characterized algal LHCSR improves NPQ kinetics. Molecular characterization of the engineered strains is in progress to understand the changes in the metabolic flux through CBC and the regulation of the newly introduced NPQ and to determine whether these modifications confer growth advantages under dynamic light conditions.

Transcription factors play critical roles in transcriptional regulation of fatty acid biosynthesis (FAS) genes and can be used in genetic engineering approaches to increase the expression of the FAS pathway. Researchers established a pipeline to extract transcription factors based on InterProScan IDs from the A. protothecoides UTEX 250 genome. In parallel, the team conducted a proteomics experiment under lipid accumulating conditions (nitrogen;N depletion and glucose addition). This analyses identified novel putative transcription factors that are upregulated in lipid accumulating cells and therefore are candidates for involvement in regulation of acclimation to N starvation, glycolysis and de novo fatty acid synthesis. Researchers are currently generating mutants to test the roles of these candidate transcription factors. Altogether, this work will inform the engineering of strains to increase total lipid accumulation in A. protothecoides.

Funding Information

This work was supported by the DOE Office of Science, BER program under award no. DE-SC0023027.