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Dual-Layer Engineering of Heparin 6-O-Sulfotransferase: PROSS-Guided Design Meets Synonymous Rare-Codon Replacement.
Yu-Han Zhao1, Yi Li1, Xin-Yu Li1
1State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Shandong Basic Science Academic Special Zone/Research Center (Pharmacy), Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), School of Pharmaceutical Science, Cheeloo College of Medicine, Shandong University, Jinan 250012, China.
Researchers developed an animal-free method for producing heparin, a vital anticoagulant drug. They engineered a key enzyme, 6-O-sulfotransferase (6OST), for improved expression and activity in bacteria, enabling sustainable heparin synthesis.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Drug Development
Background:
- Heparin is a crucial anticoagulant drug, but traditional production relies on animal sources.
- Existing methods for producing heparin enzymatically are hindered by limitations of animal-derived enzymes, including poor expression and low activity in bacterial systems.
- Robust 6-O-sulfotransferases (6OSTs) are essential for synthesizing the specific sulfation pattern in heparin critical for its biological activity.
Purpose of the Study:
- To develop an animal-free production method for heparin.
- To engineer 6-O-sulfotransferases (6OSTs) for enhanced recombinant expression, stability, and catalytic efficiency in *E. coli*.
- To establish a scalable protein engineering framework for producing bioengineered glycosaminoglycans.
Main Methods:
- Implemented a two-tier engineering framework combining structure-guided protein repair and translation optimization.
- Identified *Oryzias melastigma* 6OST-1 as a suitable template for engineering.
- Developed a synonymous rare-codon (SRC)-guided ultrahigh-throughput screening platform using split-GFP complementation to optimize translation kinetics.
Main Results:
- Engineered a variant, 6OST-M10, through protein repair and targeted mutations.
- Achieved an 8.375-fold increase in soluble expression and a 27-fold improvement in catalytic activity for the engineered 6OST.
- The final variant, 6OST-M10(SRC), reached 4400 IU/L under high-density fermentation, overcoming previous limitations in bacterial production.
Conclusions:
- A dual-layer strategy successfully enhanced enzyme stability and translational efficiency, resolving trade-offs limiting bacterial production of sulfotransferases.
- This framework provides a universal and scalable approach for protein engineering.
- Enabled the synthesis of bioengineered heparin and other glycosaminoglycans without animal involvement, paving the way for sustainable production.
