Domesticating Cyanobacteria Through Development of New Genome Engineering Tools, and Isolation of New Bio-Prospected Model Strains
Authors:
Max Schubert1* (max_schubert@hms.harvard.edu), Tzu-Chieh Tang1, Isabella Goodchild-Michelman1, Krista Ryon2, James Henriksen3, Paola Quatrini4, Marco Milazzo4, Gabriele Turco4, Davide Spatafora4, Braden Tierney2, Christopher Mason2, and George M. Church1
Institutions:
1Harvard University; 2Weill Cornell Medical College; 3Colorado State University; and 4Palermo University
URLs:
Goals
Cyanobacteria are facile models of photosynthesis and chassis organisms for carbon-negative bioproduction. To help advance the development of cyanobacteria biotechnology, a team of researchers is both developing new genetic tools for cyanobacteria and bio-prospecting novel cyanobacteria model strains. New genetic tools aim both toward genome-scale study of cyanobacterial genomes, including applications like recoding and directed evolution of bacterial genomes, toward improved performance in bioproduction. Novel cyanobacterial model strains possess unique differences to common model strains that further understanding of and improve photosynthetic microbial bioproduction, further enabling the carbon-neutral bioeconomy.
Abstract
Facile genome engineering tools further the ability to explore and apply biology, and previous work generating recoded and/or biocontained organisms, or performing directed evolution in bacteria, shows that recombineering is a critical tool (Mandell et al. 2015; Lajoie et al. 2013; Schubert et al. 2021). Recombineering is enabled by phage sequential structure alignment program (SSAP) proteins, which both produce and stabilize single-stranded DNA recombination intermediates and recruit them to the replisome (Filsinger et al. 2021; Caldwell et al. 2019). These proteins are known to have host-specific activity, and finding SSAPs that function efficiently in a specific clade of bacteria is a first step toward improving tools for precision genome engineering (Filsinger et al. 2021; Wannier et al. 2020, 2021). Researchers have identified 22 candidate SSAPs within both protein databases and metagenomic databases occurring within cyanobacteria or their phages. These proteins could extend recombineering approaches enabling multiplex engineering, recoding, and genome-scale remodeling into cyanobacteria and help form a roadmap for identifying efficient SSAPs in new model microbes.
Recombineering is one tool for engineering genomes which is well-suited to rational approaches. In contrast, transposon mutagenesis and Transposon Insertion Sequencing (TnSeq) have been successful at improving understanding of genomes through irrational approaches and fast generation of large pools of genetic diversity (Gray et al. 2015). This project demonstrates that simple modifications to existing transposon mutagenesis procedures result in many random transposon insertions within cyanobacterial genomes. The resulting strains with large numbers of inactivated and/or upregulated genes suggest strategies for directed evolution and genome streamlining.
The project is also seeking to apply these technologies in unreported cyanobacterial model strains that could improve photosynthetic-based bioproduction. Indeed, various cyanobacterial model strains are used in the literature for these efforts as well as studying the fundamental processes of photosynthesis (Goodchild-Michelman et al. 2023). Together with the Two Frontiers project (twofrontiers.org) which aims to explore microbial communities in extreme environments, including high CO2 environments, researchers have isolated two closely related cyanobacterial strains with promising growth phenotypes from seawater in the photic zone off the coast of Sicily. The two strains have ~99% nucleotide identity with each other with 32,897 single nucleotide polymorphisms differing between the strains and ~98% identity to the closest relative with genome sequence available, Cyanobacterium aponium PCC10605. Comparative genomics reveal each novel strain possesses 50–60 unique genes differing between both these strains and PCC10605, and 108 shared genes differing between them and PCC10605. One novel strain remarkably grows to a higher density in batch growth than Synechococcus sp. PCC11901, which holds the published record for high density batch biomass growth in cyanobacteria (Włodarczyk et al. 2020; Mills et al. 2022). Research shows that larger and more dense cells than common model strains may improve the economics of dewatering cyanobacterial biomass, and thus production of bioproducts. The second strain possesses unique characteristics such as programmed formation of large aggregates and phototactic motility. In sum, these strains obtained from a CO2-emitting volcanic vent are a promising new model for studies in cyanobacteria and possibly for photosynthetic bioproduction.
References
Caldwell, B. J., et al. 2019. “Crystal Structure of the Redβ C-Terminal Domain in Complex with λ Exonuclease Reveals an Unexpected Homology with λ Orf and an Interaction with Escherichia coli Single Stranded DNA Binding Protein,” Nucleic Acids Research 47, 1950–63.
Filsinger, G. T., et al. 2021. “Characterizing the Portability of Phage-Encoded Homologous Recombination Proteins,” Nature Chemical Biology 17, 394–402.
Goodchild-Michelman, I. M., et al. 2023. “Light and Carbon: Synthetic Biology Toward New Cyanobacteria-Based Living Biomaterials,” Materials Today Bio 19, 100583. DOI:10.1016/j.mtbio.2023.100583.
Gray, A. N., et al. 2015. “High-Throughput Bacterial Functional Genomics in the Sequencing Era,” Current Opinion in Microbiology 27, 86–95.
Lajoie, M. J., et al. 2013. “Genomically Recoded Organisms Expand Biological Functions,” Science 342, 357–60.
Mandell, D. J., et al. 2015. “Biocontainment of Genetically Modified Organisms by Synthetic Protein Design,” Nature 518, 55–60.
Mills, L. A., et al. 2022. “Development of a Biotechnology Platform for the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901,” Biomolecules 12(7), 872.
Schubert, M. G., et al. 2021. “High-Throughput Functional Variant Screens via In Vivo Production of Single-Stranded DNA,” Proceedings of the National Academy of Sciences of the United States of America 118(18), e2018181118.
Wannier, T. M., et al. 2020. “Improved Bacterial Recombineering by Parallelized Protein Discovery,” Proceedings of the National Academy of Sciences of the United States of America 117, 13689–98.
Wannier, T. M., et al. 2021. “Recombineering and MAGE,” Nature Reviews Methods Primers 1, 1–24.
Włodarczyk, A., et al. 2020. “Newly Discovered Synechococcus sp. PCC 11901 Is a Robust Cyanobacterial Strain for High Biomass Production,” Communications Biology 3, 215.
Funding Information
This project has been funded by DOE grant DE-FG02-02ER63445. Dr. Church is a founder of companies in which he has related financial interests: EnEvolv (Ginkgo Bioworks); and 64x Bio. For a complete list of Dr. Church’s financial interests, see arep.med.harvard.edu/gmc/tech.html.