Thermophilic Genetic Tool Development for Engineering and Functional Genomics in Clostridium thermocellum
Authors:
Carrie A. Eckert1,3* ([email protected]), Yasemin Kaygusuz1,3, Nandhini Ashok1,3, Mitchell Long2,3, Elise Phillips1,3, Bill Alexander1,3, Adam Guss1,3, Gerald Tuskan1,3
Institutions:
1Oak Ridge National Laboratory; 2University of Tennessee–Knoxville; 3Center for Bioenergy Innovation, Oak Ridge National Laboratory
URLs:
Goals
The Center for Bioenergy Innovation (CBI) vision is to accelerate domestication of bioenergy-relevant, non-model plants and microbes to enable high-impact innovations along the bioenergy and bioproduct supply chain while focusing on sustainable aviation fuels (SAF). CBI has four overarching innovation targets: (1) develop sustainable, process-advantaged biomass feedstocks; (2) refine consolidated bioprocessing with cotreatment to create fermentation intermediates; (3) advance lignin valorization for bio-based products and aviation fuel feedstocks; and (4) improve catalytic upgrading for SAF blendstocks certification.
Abstract
Domestication and genetic engineering of non-model plants and microbes for optimal bioconversion performance is essential to achieve efficient and sustainable production of fuels and chemicals from lignocellulose. Clostridium thermocellum is an anaerobic thermophile capable of directly degrading lignocellulose to fermentable sugars―which eliminated the additional step of chemical or enzymatic deconstruction of lignocellulosic biomass and mediating cost-effective consolidated bioprocessing (CBP) by also fermenting the sugars into intermediates (e.g., ethanol). While C. thermocellum has powerful capabilities in biomass solubilization, further strain improvement is needed for yield , titer, and selectivity, Available genetic engineering tools for C. thermocellum were limited by inefficient conventional homologous recombination; however, recent work introduced two alternative, efficient, and timesaving CRISPR-Cas genome editing systems utilizing the native Type I-B from C. thermocellum and the heterologous Type II CRISPR system from Geobacillus stearothermophilus, with Lambda Red exo/beta homologs from Acidithiobacillus caldus for RecA-independent homologous recombination (Marcano-Velazquez et al 2019). In addition to continued efforts to improve transformation efficiency through plasmid methylation platforms, researchers are developing genetic tools towards high throughput genome scale studies to enable gene-to-trait identification. Traditional homologous recombination in C. thermocellum is challenging and time-consuming, especially for chromosomal insertion of heterologous genes. To enable faster and easier heterologous gene insertion in C. thermocellum, researchers developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) technique using the recombinase from Geobacillus sp. Y412MC61 (Walker et al 2020). Using the tSAGE method to integrate the tested genetic elements, researchers tested 15 homologous and 32 heterologous promoters, four inducible promoters, five riboswitches, 10 fluorescent reporter genes, and a library of gene cassettes with variable distances between the ribosome binding site and the start codon of the promoter. The tSAGE method allowed researchers to quickly screen and identify the genetic elements that will improve strain engineering for CBP of lignocellulose into sustainable fuels and chemicals. tSAGE will also be key to efficiently screen modified metabolic pathways in C. thermocellum.
The CRISPR-Cas genome editing tools are useful for permanent genetic engineering such as deletions and insertions. To enable temporary perturbations in gene expression, researchers repurposed the native Type I-B CRISPR system and generated a catalytically dead CRISPR system capable of CRISPR-interference (CRISPRi) and gene knockdown by deleting the Cas3 nuclease. To achieve reversibility and temporal control in gene knockdowns, the team identified a xylose-inducible promoter and a 2-aminopurine-inducible riboswitch (Yang et al 2014) that function efficiently in a dose-dependent manner in C. thermocellum. Researchers are currently working on generating a C. thermocellum strain capable of inducible CRISPRi by integrating the inducible gene expression systems into its genome to drive the expression of the Cas3-deleted CRISPRi system. The reversibility and temporal control of the inducible CRISPRi system will allow rapid and high-throughput genome-wide screens for genotype-phenotype discovery and enable identification of new gene function in C. thermocellum. The inducible gene expression systems identified in this work will also be useful to control gene expression for pathway engineering and optimizing CBP of lignocellulose into ethanol in C. thermocellum.
References
Marcano-Velazquez, J. G., et al. 2019. “Developing Riboswitch-Mediated Gene Regulatory Controls in Thermophilic Bacteria,” ACS Synthetic Biology 8(4), 633–40.
Walker, J. E., et al. 2020. “Development of Both Type I–B and Type II CRISPR/Cas Genome Editing Systems in the Cellulolytic Bacterium Clostridium thermocellum,” Metabolic Engineering Communications 10, e00116.
Yang, L., et al. 2014. “Permanent Genetic Memory with >1-Byte Capacity,” Nature Methods 11(12), 1261–66.
Funding Information
Funding was provided by the Center for Bioenergy Innovation (CBI) led by Oak Ridge National Laboratory. CBI is funded as a U.S. DOE Bioenergy Research Center supported by the BER Program in the DOE Office of Science under FWP ERKP886. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. DOE under contract no. DE-AC05-00OR22725.