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

Synthetic Biology Tool Development for Precision Engineering of Oilseed Crops

Authors:

Michael J. Smanski1* (smanski@umn.edu), J. Armando Casas-Mollano1, Jonathan Cors1, Tingyuan Xiao2, Alisha Lnu2, Timothy P Durrett2* (tdurrett@ksu.edu), Edgar Cahoon3

Institutions:

1University of Minnesota–Twin Cities; 2Kansas State University–Manhattan; 3University of Nebraska–Lincoln

URLs:

Goals

  • Establish safe-harbor landing pads for predictable transgene
  • Characterize seed-specific promoters with a range of expression
  • Define the impact of compositional genetic context effects on transgene expression with respect to alternative chromosomal loci for gene integration.
  • Develop and use Programmable Transcriptional Activators (PTAs) to regulate expression of multiple (endogenous) genes for seed oil manipulation.
  • Targeted gene replacement at the native genomic locus in pennycress.

Abstract

The random nature of Agrobacterium-mediated transgene insertion into plant genomes affects expression strength, resulting in unpredictable product accumulation and the need to characterize many independent transgenic lines. This greatly limits throughput of different gene combinations to efficiently explore the expression space needed for effective multi-gene pathway metabolic engineering. To overcome this limitation, researchers are creating a suite of tools for reliable engineering of multi-gene systems to provide predictable control of the level of transgene expression in camelina and pennycress seeds. Specifically, this team will (1) generate camelina and pennycress lines with safe harbor landing pads; (2) develop a new set of seed specific promoters with a range of expression strengths; and (3) leverage sequence-programmable transcription activators (PTAs).

  1. Safe harbor landing pads: Researchers are developing camelina and pennycress lines with safe harbor landing pads that will use site-specific recombinase systems to enable the targeted insertion of transgenic cargos into predetermined genomic loci, allowing better control of transgene expression strength and reducing the number of independent transgenic lines to be characterized. Using a protoplast expression system, this group screened five recombinase systems for their effectiveness in pennycress. Two serine recombinases, Bxb1 and phiC31, possessed the highest recombination rate. Landing pads using the recombination sites of these two recombinases have been generated and transformed into both pennycress and camelina.
  2. Novel seed specific promoters: The relative expression levels of genes involved in primary or secondary metabolism can dramatically affect product profile and titer. To augment the suite of very strong seed specific promoters (e.g., napin, glycinin) currently used for oil seed engineering, the researchers have mined existing gene expression databases to identify nine seed specific genes expressed at levels 1x, 0.1x and 0.01x relative to the strong seed specific promoters in these species (e.g., napin orthologs). Promoter and terminator sequences have been cloned for these genes to be compatible with the GoldenBraid cloning system and used to express two different lipid biosynthetic Transgenic lines have been generated for all promoter/gene combinations and T2 seeds analyzed for the accumulation of target lipid molecules.
  3. Sequence-programmable transcription activators (PTAs): Catalytically deactivated Cas9 (dCas9 or ‘dead’ Cas9) can be used as an RNA-guided DNA binding domain to deliver transcriptional activation or repression domains to a promoter of interest. These give unprecedented ability to control and fine-tune the expression level of multiple endogenous genes in parallel. Researchers have recently improved the performance of dCas9-based PTAs for plant applications by altering the mechanism by which activation domains are recruited to the dCas9 (Casas-Mollano et al. 2023) and by swapping out transcriptional activation domains derived from human viruses for those evolved in plant cells (Zinselmeier et al. 2022). Researchers now have transgenic lines of pennycress and camelina that stably express the components of MoonTag PTAs and are in position to begin leveraging these tools for seed oil engineering.

Image

Precision Engineering Oilseed Crops

Figure 1. Tool development for rational genome engineering. (a) Strategic design for detecting site-specific genome integration via serine recombinases. (b) Protoplast-derived characterization of serine integrase efficiency. (c) Seed fluorescence measurements confirms visual binning of transgenic events.

References

Casas-Mollano, J. A., et al. 2023. “Efficient Gene Activation in Plants by the MoonTag Programmable Transcriptional Activator ,” NucleicAcids Research 51(13), 7083–93. DOI:10.1093/nar/gkad458.

Zinselmeier, M. H., et al. 2022. “Optimized dCas9 Programmable Transcription Activators for Plants,” bioRxiv. DOI:10.1101/2022.06.10.495638.

Funding Information

This research was supported by the DOE Office of Science, BER program, grant no. DE-SC0023142.