Synthetic Biology Approaches for Reconstructing Transcriptional Networks in Eucalyptus and Re-Engineering Woody Biomass

Lignocellulosic biomass derived from fast-growing Eucalyptus trees may serve as a renewable feedstock for future biomaterials and bioenergy needs. Understanding and re-engineering the transcriptional networks governing wood formation in this hardwood, especially secondary cell wall (SCW) formation, may enable strategic improvement of wood property traits using synthetic biology approaches. We have designed and produced a three-part synthetic transcription factor and promoter panel comprising 286 constructs that serve the purpose of high-throughput SCW-associated protein-DNA interaction network reconstruction and future transcriptional network re-wiring attempts. Part A comprises 48 transcription factors cloned into the pIX-HALO vector, allowing for SP6 promoter-mediated in vitro transcription and translation of these proteins for DNA Affinity Purification sequencing (DAP-seq). Target genes identified from low-coverage DAP-seq data of six E. grandis R2R3-MYB transcription factors were significantly enriched for membership of SCW-related co-expression modules and candidate genes in Eucalyptus, suggesting functional relevance of these genome-wide in vitro DNA-binding data. Part B includes 173 SCW-associated transcription factor coding sequences that have been domesticated by elimination of illegal Type IIS restriction sites and tailored to the Phytobrick DNA assembly standard. Assembled using ligation-independent cloning in pCR8/GW/TOPO, the panel is also GATEWAY-compatible. Part C includes AarI-flanked and GATEWAY-compatible PCR-amplified promoter sequences of some 65 SCW-associated genes, partially domesticated by mutation of illegal restriction sites through substitution with known single nucleotide polymorphisms to preserve promoter function. Promoter-transcription factor-terminator assemblies with the LOOP method have been conducted, and panels B and C are compatible with GATEWAY-mediated enhanced yeast one-hybrid screening for targeted protein-DNA interaction detection. Finally, we have adopted Eucalyptus hairy root transformation as a simplified reverse genetics screening platform to alleviate the limitations of whole-plant Eucalyptus transformation and regeneration. This will allow for the study of SCW-related transcription factor and promoter function, and potentially the testing of SCW-related synthetic gene circuits, with increased efficiency.