Dual-Layer Engineering of Heparin 6-O-Sulfotransferase: PROSS-Guided Design Meets Synonymous Rare-Codon Replacement

Heparin has been the most important drug for treating thrombotic disorders for more than 60 years. However, the traditional production of heparin involves the slaughter of animals. Therefore, there is a demand for the animal-free production of heparin, such as enzymatic synthesis based on the heparin biosynthetic pathway. To achieve this, robust 6-O-sulfotransferases (6OSTs) are required to produce the 6-O-sulfation pattern in heparin, which is crucial for the biological activity. However, most native 6OSTs are derived from animal tissues and exhibit poor recombinant expression, low catalytic efficiency, and insufficient stability in E. coli. To overcome these limitations, we systematically established a two-tier engineering framework that integrated structure-guided protein repair and optimization of translation. Cross-species screening identified Oryzias melastigma 6OST-1 as an engineering-competent template. First, we constructed the variant 6OST-M10 through protein repair one-stop service-guided structural restoration combined with targeted reverse mutations. To address the long-standing challenge of low heterologous expression, we generated a synonymous rare-codon (SRC)-guided ultrahigh-throughput screening platform based on split-GFP complementation. This platform systematically tunes the translation kinetics for sulfotransferases. Ultimately, the 6OST-M10(SRC) variant was generated, which displayed an 8.375-fold increase in soluble expression and a 27-fold improvement in catalytic activity that reached 4400 IU/L under high-density fermentation. This dual-layer strategy couples structural stabilization with translational optimization to resolve the trade-offs among activity, stability, and recombinant expression that have previously limited bacterial production of animal-derived sulfotransferases and heparin synthesis in E. coli This framework provides a universal, scalable paradigm for protein engineering and enables the synthesis of bioengineered heparin and other glycosaminoglycan products without the involvement of animals.