CD BioGlyco offers a specialized Chassis Strain Development Service designed to transform standard microbial or mammalian hosts into high-performance "glycofactories". Our approach integrates the principles of synthetic biology with deep expertise in glycometabolism. By re-engineering the internal cellular environment, including modifying sugar donor pools, fine-tuning glycosyltransferase (GT) expression, and eliminating competing metabolic pathways, we provide our clients with optimized chassis strains that exhibit superior growth kinetics and high-fidelity product output. Whether you are developing next-generation biologics or sustainable glyco-materials, our chassis development service serves as the robust foundation for your project's success.
Key Technologies
Precision Genome Editing
To achieve complex glycosylation, multiple genetic modifications are often required simultaneously. Our genome editing platform leverages advanced systems to perform concurrent knockouts of endogenous proteases or glycosidases and the site-specific integration of heterologous glyco-pathways. This technology allows us to "humanize" yeast or bacterial strains by swapping entire biosynthetic clusters in a single engineering cycle, reducing development timelines while maintaining genomic integrity.
Metabolic Flux Analysis (MFA) and Pathway Reconstruction
We utilize MFA to quantify the flow of carbon and energy through the host's metabolic network. By identifying bottlenecks in the synthesis of nucleotide-sugar donors, we rationally reconstruct pathways to redirect metabolic resources toward the target glycan. This ensures that the engineered chassis produces the desired glycomolecules without compromising cellular fitness or growth rate.
From Genetic Blueprint to Industrial Reality: Tailor-Made Chassis for the Glyco-Revolution
At CD BioGlyco, our service scope is designed to cover every aspect of chassis development within the hierarchy of synthetic glycobiology. Our service bridges the gap between basic research and commercial production. We offer development across various host systems, including Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, and mammalian cell lines like CHO and HEK293. By optimizing these organisms, we enable the efficient synthesis of N-linked and O-linked glycoproteins, human milk oligosaccharides (HMOs), and specialized glycolipids.
We provide specialized support for the following critical editing services:
Our multi-gene editing service enables the simultaneous integration or deletion of multiple genetic loci. For example, moving a complete human sialylation pathway into a yeast or bacterial host. We utilize advanced multi-gene editing and iterative integration strategies to ensure that all genes are expressed in the correct stoichiometric ratios, minimizing metabolic bottlenecks and preventing the accumulation of undesirable intermediate glycoforms.
Our gene family editing service targets the clusters with surgical precision. By knocking out entire families of interfering enzymes or replacing them with optimized variants, we "clean" the cellular background. This results in a simplified and homogenous glycan landscape, which is essential for the production of biopharmaceuticals.
Our promoter editing service involves the replacement of native promoters with a library of characterized constitutive or inducible promoters of varying strengths. This allows us to "dial in" the exact expression level of each enzyme in the pathway. By balancing the metabolic flux between host maintenance and glycan synthesis, we create a chassis that is both highly productive and physiologically robust.
Unintended genetic modifications (off-target effects) lead to phenotypic instability or unexpected metabolic shifts. Our OGE service provides rigorous quality assurance. We utilize high-fidelity nucleases and conduct comprehensive whole-genome sequencing (WGS) paired with advanced bioinformatic algorithms to detect and quantify any off-target events.
For long-term stability, heterologous glyco-pathways are integrated into "safe harbor" regions of the genome where they will not disrupt essential genes or be subjected to gene silencing. Our targeted genome editing service specializes in site-specific insertion. We have mapped the genomes of major chassis strains to identify optimal integration sites that support high levels of transcription.
To push the boundaries of glycobiology, we offer the genetic code expansion service. This allows for the site-specific incorporation of non-canonical amino acids (ncAAs) into the protein backbone of the product. These ncAAs serve as chemical handles for "click chemistry" or as specific attachment points for synthetic glycans.
Workflow
1. Rational Design and Host Selection
We select the most appropriate host organism based on the required post-translational modifications (PTMs) and the final application. Using computational modeling, we design the optimal genetic architecture for the new chassis.
2. Genetic Tool and Pathway Assembly
We construct the necessary genetic tools, including specialized plasmids and guide RNAs (gRNAs). Entire biosynthetic pathways, comprising codon-optimized glycosyltransferases and sugar-transporters, are assembled.
3. Multi-Locus Genome Engineering
Utilizing our platform, we implement the designed genetic changes. This involves knocking out competing metabolic pathways that drain sugar-nucleotide pools and integrating the heterologous glyco-machinery into the host genome to ensure long-term stability.
4. Genetic Stability and Off-Target Validation
Every engineered strain undergoes deep sequencing to confirm the precision of the edits. We perform OGE analysis to ensure no deleterious mutations have occurred, guaranteeing that the chassis remains robust across multiple generations.
5. Phenotypic Characterization and Flux Optimization
We use MFA to identify any remaining metabolic friction and perform "fine-tuning" of promoter strengths to balance the expression of pathway enzymes, maximizing the titer of the target glycan.
6. Scale-up Feasibility and Quality Assurance
We test the best-performing strains under simulated industrial fermentation conditions. We provide a report including glycan structural verification via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a stability certificate.
Publication Data
DoI: 10.3389/fbioe.2024.1378873
Journal: Frontiers in Bioengineering and Biotechnology
IF: 4.8
Published: 2024
Results: This study completes the genome sequence of Paenibacillus polymyxa DSM 365 (5,889,536 bp, 45.6% GC, 5,370 coding sequences) and identifies a novel non-ribosomal peptide synthase (NRPS) potentially involved in tridecaptin production. To develop it as a microbial chassis, two genome-reduced strains (GR1, GR2) were constructed via gene editing. GR1 lacks 178.5 kb (3.0% of genome), including biosynthetic gene clusters (BGCs) for exopolysaccharides and antibiotics, reducing metabolic competition. GR2 lacks 38.5 kb (0.6% of genome), removing insertion sequence ISPap1 to enhance genetic stability. Both strains maintained wild-type (WT) growth rates (μmax = 0.32 h⁻¹) and 2,3-butanediol titers (17-19 g/L) in bioreactor batch cultures. GR1 showed no exopolysaccharide production, while GR2 retained WT-level exopolysaccharide synthesis. These streamlined strains validate P. polymyxa DSM 365 as a promising chassis for biotechnological production of value-added compounds.
Fig.1 Circular genome map of P. polymyxa DSM 365. (Ravagnan, et al., 2024)
Applications
Biopharmaceutical Development
Our engineered chassis is used to produce therapeutic glycoproteins (antibodies) with customized human-like glycosylation patterns, reducing immunogenicity and enhancing half-life.
Glyco-Vaccine Production
We develop specialized strains for the bioconjugation of bacterial O-antigens to carrier proteins, enabling the rapid and cost-effective production of potent conjugate vaccines against infectious diseases.
Nutraceuticals Development
CD BioGlyco assists in creating microbial factories for the large-scale synthesis of HMOs, such as 2'-fucosyllactose, for use in infant formula and health supplement development.
Diagnostics and Research Tools
Our chassis serve as platforms for producing high-purity glycan standards, lectins, and labeled glyco-conjugates, supporting advanced research in glycomics and the development of sensitive diagnostic assays.
Advantages
Unrivaled Multi-Host Versatility
We possess established protocols and proprietary toolkits for a wide array of hosts, ranging from simple prokaryotes to complex mammalian systems, ensuring the best fit for your specific glycan.
Superior Editing Efficiency
Our multi-gene editing technology allows for the simultaneous modification of up to 10+ genetic loci, drastically shortening the R&D cycle compared to traditional sequential engineering methods.
Enhanced Strain Fitness
We prioritize the "health" of the chassis. Our MFA-guided approach ensures that the metabolic burden of glyco-engineering is minimized, resulting in robust strains that excel in high-density fermentation.
Modular "Glyco-Parts" Library
CD BioGlyco maintains an extensive collection of validated promoters, terminators, and glycosyltransferases, allowing for the rapid "plug-and-play" assembly of customized biosynthetic pathways.
Frequently Asked Questions
Customer Review
"The team at CD BioGlyco successfully engineered a Pichia chassis for us that eliminated high-mannose structures and delivered consistent human-type G0F glycans."
– W.S., Senior Scientist
"The multi-gene editing they performed on our E. coli strain resulted in a five-fold increase in titer compared to our internal prototypes. Highly recommended for complex metabolic engineering."
– A.W., Director of R&D
"The level of detail in the final validation report was impressive. Not only was the chassis stable, but the off-target analysis gave us the confidence."
CD BioGlyco is dedicated to empowering the scientific community with the most advanced tools in synthetic glycobiology. Our chassis strain development service represents a fusion of precision genome editing, metabolic expertise, and industrial-grade validation. Please feel free to contact us for detailed information on our services or to request a formal quotation.
Reference
Ravagnan, G.; et al. Genome reduction in Paenibacillus polymyxa DSM 365 for chassis development. Frontiers in Bioengineering and Biotechnology. 2024, 12: 1378873. (Open Access)
For research use only. Not intended for any clinical use.
Fig.1 Circular genome map of P. polymyxa DSM 365. (Ravagnan, et al., 2024)
