Genetic Tools

Genetic Tools

Another critical factor for the future success and economic viability of algal biofuels is the development of co-products, which are non-fuel products that add value to the algae biomass. In order to enable co-product production and empower algae crop protection, it is necessary to develop genetic tools in genetic engineering and synthetic biology. CAB-Comm aimed to develop these genetic tools for not only the collaborating labs and corporate partners, but also for the greater academic and industry communities.

Objectives

  1. Develop additional selectable markers and crop protection tools for green algae and diatoms
  2. Develop new methods to control gene expression and cell viability in green algae
  3. Develop cyanobacterial genetic tools
  4. Developing genetic tools and co-products for brown algae
  5. Develop methods for the rapid generation and expression of high affinity nanobodies to promote crop protection, facilitate harvesting, and express high-value co-products
  6. Create a model and analysis of benefits of algae co-products

Accomplishments and Discoveries

rainbow algae

Genetically modifed "rainbow" green algae with different fluorescent proteins in different cellular structures

rainbow algae

Increased triacylglycerol (precursor to biodiesel) accumulation in metabolically engineered diatom (1A6) vs. natural wild type (WT) shown by fluorescent dye (BODPIY) and fatty acid profiles. 

Perhaps our most important successes were achieved in developing genetic tools for cyanobacteria, green algae, and diatoms, as these tools have enabled the entire algal research community to be more productive.

For cyanobacteria, CAB-Comm researchers created a web application called CYANO-VECTOR for building in silico design of genetic tools. They then created and tested over 70 genetic parts and devices including origins of replication, improved broad-host-range plasmids, homology regions for chromosome engineering, antibiotic markers, and functional devices for gene expression. As a proof of concept, these same researchers engineered the production of polyunsaturated fatty acids into several different cyanobacterial species. 

For green algae, researchers developed and validated a set of eight fluorescent protein and enzymatic reporters for the use in the alga Chlamydomonas reinhardtii, expressed camelid VHH domains in C. reinhardtii chloroplast, which function as antitoxins against the botulinum neurotoxin and demonstrated that the antitoxin was stable inside the gastrointestinal tract of mice, and developed a recombination detection system for C. reinhardtii nuclear genome and significantly increased homologous recombination efficiency.

In diatoms (brown algae), scientists developed new promoters, new antibiotic resistance selectable markers, markers that enables selection without GMO classification, RNAi and antisense approaches for gene regulation, and an array of fluorescent proteins and targeting vectors for sending proteins to multiple cellular locations. 

In terms of improving the energy return on investment (EROI) and reducing life cycle greenhouse gas (GHG) emissions during algae biofuel production, it was determined that anaerobic digestion of biomass would perform best under a number of scenarios and assumptions, however, hydrothermal gasification showed the potential to be better. Researchers also showed that algal oil production systems are likely to achieve greater environmental performance by maximizing energy and nutrient recovery and recycling within the production system rather than using residual biomass for external products like animal feed.

Publications