Conventional genetic engineering often struggles with the complexity of glycosylation pathways, which involve dozens of coordinated enzymatic reactions. Achieving the desired glycotype requires more than just a gene knockout or knock-in; it demands the simultaneous regulation of multiple genomic loci to balance metabolic flux and eliminate competing pathways. CD BioGlyco provides the precision and scale required to rewire these intricate networks, ensuring that your host strain, whether it be Escherichia coli, Saccharomyces cerevisiae, or Chinese hamster ovary (CHO) cells, is perfectly tailored to your specific biomanufacturing goals.
Key Technologies
Multiplex Gene Editing Systems
Our cornerstone is the use of multiplexed gene editing systems, enabling the simultaneous modification of 5 to 10+ genes in a transformation event. This approach drastically reduces the time required for strain construction and minimizes the metabolic stress associated with repeated selection and curing cycles.
Modular DNA Assembly
We integrate advanced DNA assembly techniques with multiplex automated genome engineering (MAGE). This synergy allows for the rapid construction of large multigene circuits that are integrated into the host genome at pre-validated "safe harbor" sites. By combining modular assembly with high-throughput (HTP) screening, we generate and evaluate libraries of strain variants to identify the optimal configuration for glycan yield and purity.
Engineering Glycobiology with Multi-dimensional Genomic Precision
As a specialized subset of our broader chassis strain development service, our multi-gene editing offerings are designed to handle the unique challenges of glycobiology. This service primarily focuses on the systematic overhaul of host genomes to support heterologous glycosylation pathways. We provide solutions for:
Pathway Implantation
Integration of entire metabolic clusters (e.g., nucleotide-sugar biosynthetic pathways) into the chassis.
Host De-glycosylation
Knocking out endogenous glycosylation genes (e.g., OCH1 in yeast) to prevent "hyper-mannosylation" or other non-human glycan structures.
Byproduct Elimination
Deleting genes involved in competing metabolic pathways to redirect carbon and energy toward the target glycan or glycoprotein.
Protease Reduction
Simultaneously knocking out multiple protease genes to increase the stability and yield of secreted recombinant glycoproteins.
Whether you are looking to "humanize" a yeast strain for therapeutic protein production or optimize a bacterial chassis for milk oligosaccharide synthesis, CD BioGlyco provides the technical framework to turn your vision into a reality.
Workflow
1. Project Assessment and Pathway Design
Our experts use computational modeling to identify the essential genes, regulatory bottlenecks, and potential metabolic conflicts. We work closely with clients to define the final strain specifications, including the desired glycotype and production scale.
2. Guide RNA and Vector Synthesis
Utilizing proprietary algorithms, we design highly specific guide RNAs (sgRNAs) for multiplexed editing. For multi-gene insertions, we design large donor DNA fragments with optimized codon usage to match the host chassis's translational machinery.
3. Transformation and Genomic Integration
The host cells are transformed with the multi-gene editing machinery using delivery methods such as electroporation. We utilize "scarless" techniques, ensuring that no antibiotic resistance markers or foreign DNA sequences remain in the final strain.
4. High-Throughput Phenotypic Screening
We employ automated systems to screen thousands of colonies. This step involves identifying candidates with the correct genomic modifications and verifying their performance using micro-scale fermentation and rapid glyco-profiling techniques.
5. Multi-omics Validation and Genotyping
We perform whole genome sequencing (WGS) to confirm the precision of the edits and ensure the absence of unintended off-target mutations. Transcriptomic and metabolomic analyses are conducted to verify that the rewired pathways are functioning as intended.
6. Strain Stability and Scale-up Assessment
The optimized chassis undergoes rigorous stability testing across multiple generations. We provide initial scale-up data to demonstrate that the engineered traits remain robust during industrial fermentation processes.
Publication Data
DoI: 10.1093/nar/gkz669
Journal: Nucleic acids research
IF: 13.1
Published: 2019
Results: This study demonstrates that catalytically inactive DNA-PKcs (via K3753R mutation) or transient inhibition by small molecule M3814 enhances homology-directed repair (HDR) efficiency for precise genome editing (PGE) in human cells (hiPSCs, HEK293, K562). By blocking non-homologous end joining (NHEJ) without reducing ATM levels, HDR frequencies increased 3.3-fold on average (up to 81%) across 14 genes, while NHEJ decreased 8.5-fold. This approach enabled simultaneous PGE of up to five genes, with ~33% of single-cell colonies achieving homozygous edits for three targets and 6% for four targets. Critically, DNA-PKcs-inactive cells maintained genomic stability, with normal karyotypes and low mutation rates. M3814 matched the mutation's HDR-enhancing effect (18% to 81% in K562 cells) with moderate toxicity. These findings address the key limitation of NHEJ dominance over HDR, enabling efficient multiplexed precise edits for research and potential therapeutic applications.
Fig.1 Multiplexed precise genome editing using catalytically inactive DNA-PKcs. (Riesenberg, et al., 2019)
Applications
Therapeutic Glycoprotein Production
Engineering CHO or yeast chassis to produce human-like glycosylation patterns on monoclonal antibodies and enzymes, reducing immunogenicity and enhancing the efficacy of biologics.
Human Milk Oligosaccharides (HMOs) Synthesis
Modifying E. coli or Bacillus subtilis to express multiple glycosyltransferases and sugar transporters for the sustainable, large-scale production of high-purity HMOs.
Metabolic Flux Optimization
Rewiring the central carbon metabolism of industrial strains to maximize the availability of nucleotide-sugar precursors like UDP-GlcNAc or CMP-sialic acid significantly boosts glycan titers.
Synthetic Glycan Libraries
Creating sets of engineered strains that express varied glyco-enzymes, allowing researchers to generate diverse glycan libraries for high-throughput functional screening and drug discovery.
Advantages
Unparalleled Multiplexing Capability
We allow for the simultaneous editing of numerous genes, cutting development timelines from years to months while maintaining high cell viability and genomic integrity.
Glyco-centric Expertise
We possess a deep understanding of the unique biochemical constraints of glycosylation, ensuring that pathway designs are biologically feasible and productive.
High Targeting Accuracy
We utilize advanced bioinformatic tools and "nuclease-plus" technologies to achieve near-zero off-target rates, which is essential for developing safe and stable industrial production strains.
Scarless and Marker-free Editing
Our proprietary systems ensure that final strains are free of selection markers, making them ideal for pharmaceutical applications and simplifying the submission process.
Frequently Asked Questions
Customer Review
"The multi-gene editing team was instrumental in our project to humanize Pichia pastoris. They successfully knocked out four endogenous genes and integrated a human glycosylation cluster."
– W.J., Biopharmaceutical Research Institute
"CD BioGlyco's multi-gene editing service allowed us to overexpress several key transporters while silencing competing pathways simultaneously. The result was a 5-fold increase in our target glycoform."
– Q.T., Biotech Company
"CD BioGlyco delivered exactly what they promised—a marker-free, stable E. coli strain optimized for HMO production. Their analytical validation was particularly impressive."
CD BioGlyco offers a sophisticated and reliable multi-gene editing service that empowers researchers to overcome the complexities of chassis development in glycobiology. Our commitment to quality, transparency, and scientific excellence ensures that your project is in the best hands, from initial design to final validation. Please feel free to contact us for detailed information on our services or to request a formal quotation.
Reference
Riesenberg, S.; et al. Simultaneous precise editing of multiple genes in human cells. Nucleic acids research. 2019, 47(19): e116. (Open Access)
For research use only. Not intended for any clinical use.
Fig.1 Multiplexed precise genome editing using catalytically inactive DNA-PKcs. (Riesenberg, et al., 2019)
