The ability to genetically modify microorganisms is foundational to performing rational strain engineering, but most organisms are not transformable when first isolated. We have deep expertise in genetic tool development and strain engineering in non-model fungi and bacteria, including work with extremophilic organisms that thrive at elevated temperature and extreme pH, including both aerobes and strict anaerobes.

We can develop the full suite of tools, methodologies, and protocols necessary to use new organisms to enable efficient biomanufacturing. These capabilities include everything from transformation protocols, validated genetic part libraries, efficient genome modification tools (e.g. CRISPR, phage integrase, etc.) for rapid, targeted gene deletion or gene regulation, transposon mutant and overexpression libraries to allow high throughput phenotype screening or selections, and plate based transformation and screening protocols amenable to automation.

Bacterial Genetic Tools 

We have capabilities to “start from scratch” in the development of bacterial hosts for biomanufacturing. These efforts encompass everything from the initial host onboarding to advanced high-throughput genome engineering. 

Transformation

Transformation is an essential first step for metabolic engineering, and we have expertise in developing transformation methods for bacterial strains. We can evade bacterial host defenses against foreign DNA via targeted DNA methylation to protect DNA from native restriction systems. We have extensive collections of plasmids that replicate in phylogenetically diverse hosts, which can be introduced into organisms via electroporation or conjugation.  

Genetic Parts

Effective rational engineering requires an expanded set of parts to control gene expression. We have large collections of genetic parts for phylogenetically diverse bacteria. This includes selectable and counter-selectable markers, plasmid origins of replication, constitutive and regulated promoters, terminators, riboregulators, biosensors, and aerobic and anaerobic reporter genes. These parts can be rapidly prototyped and optimized in diverse new organisms.

Engineering Tools

We have demonstrated and optimized a number of tools required for efficient genome engineering to enable production and rapid optimization of bioproducts in bacteria. This includes methodologies for targeted and random introduction of heterologous DNA and targeted genome editing using tools such as homologous recombination, recombineering, and CRISPR that enable gene deletion, decreased expression, and overexpression of native and heterologous genes. These tools are connected with other Agile BioFoundry capabilities for analysis of transcripts, proteins, and metabolites which are used to verify the genetic modifications function as desired. We have also developed orthogonal DNA integration tools that use site-specific recombinases, which can enable multiplexed DNA insertion into the chromosome for combinatorial pathway evaluation in the organism of choice.

High-throughput Methods

We develop and adapt high throughput methods for bacterial strain engineering and evaluation. Automation can be applied to bacterial genetic transformation to increase throughput. Transposon mutagenesis and CRISPRi can be applied for genome-scale analysis of genotype-phenotype relationships involved in substrate utilization, product formation, and robustness. Site-specific recombination enables simple, rapid, and high throughput insertion of DNA into the chromosome for rapid evaluation of heterologous DNA at single copy instead of from plasmids. Biosensors combined with fluorescence-activated cell sorting (FACS), especially in combination with other genome-scale methods, allows rapid screening of large libraries of strains and constructs.

Fungal Genetic Tools

The Agile BioFoundry has the capabilities to “start from scratch” in the development of fungal hosts for biomanufacturing, including both filamentous fungi and yeasts. These efforts encompass everything from the initial host onboarding to advanced high-throughput genome engineering. We have successfully implemented or developed capabilities for multiple yeast and filamentous fungi including: Aspergillus niger, A. pseudoterreus, A. oryzae, Rhodosporidium toruloides, Lipomyces starkeyi, Yarrowia lipolytica, Pichia kudriavzevii, etc.

Below is a list of some of these capabilities:

Transformation

Transformation is an essential part of biomanufacturing and we have expertise in development and application of electroporation, chemical and Agrobacterium-based transformation methods. Development may also include preparation and stabilization of protoplasts for filamentous fungi and optimizing the various parameters for each transformation method for optimal efficiency. 

Genetic Parts

Effective rational engineering requires an expanded set of parts to control gene expression. We have developed methods for omics-enabled identification and validation of promoters and terminators to control expression of native and heterologous genes and is developing a set of universal fungal promoters to expedite engineering of non-model fungi. We also have an extensive library of selectable and counter-selectable markers (antibiotic resistance and auxotrophies) to enable marker and marker-free engineering.

Engineering Tools

We have demonstrated and optimized a number of tools required for efficient genome engineering to enable rapid optimization of fungal strains for producing bioproducts and biofuel precursors. This includes methodologies for targeted and random introduction of heterologous DNA and targeted genome editing using tools such as CRISPR that enable reduction and overexpression of native and heterologous genes. These tools are connected with the Agile BioFoundry Test capabilities for analysis of transcripts, proteins, and metabolites which are used to verify the genetic modifications function as desired and identify additional gene targets for improving the efficiency and productivity of the desired product in the host.

High-throughput Methods

We have expertise in modifying bench-scale protocols for transformation and strain screening to a plate-based format that is compatible with automation platforms to reduce the time and resources required for strain building and functional validation. Examples include, plate-based chemical transformation of yeast, PCR-based methods for rapid validation of genetic modifications and assessment of heterologous gene copy number, plate-based HPLC analysis of products and metabolites, etc.