Jiangnan University

Bloomage Biotechnology Corporation Limited

Zhen Kang

Jian Chen

Guocheng Du

Peng Jin

Yang Wayne Wang

Jianghua Li

Litao Hu

Zhengxiong Zhou

Jialian Li

Tianmeng Zhang

Wenwen Ding

The invention discloses a recombinantfor efficient synthesis of highly pure hyaluronic acid and oligosaccharides thereof, belonging to the technical field of bioengineering. The recombinantconstructed in the present invention can produce hyaluronic acid with a yield up to 40 g/L, and a crude product purity of 95%. Addition of exogenous hyaluronic acid hydrolase and optimization of the fermentation conditions results in hyaluronic acid oligosaccharides with specific molecular weight, and can further improve the yield of hyaluronic acid to 72 g/L. The invention lays a solid foundation for the efficient synthesis of highly pure hyaluronic acid by microorganisms, and the constructed recombinantis suitable for industrial production and application.

High-efficiency synthesis and high-purity hyaluronic acid, and recombinantfor oligosaccharide thereof

The present invention relates to a method for chondroitin sulfate biosynthesis, belongs to the field of pharmaceuticals. CS was biosynthesized by sulfating the chondroitin with C4ST or C6ST in Tris-HCl buffer assisted with 3'-phosphoadenosine 5&#x2032;-phophosulfate (PAPS). C4ST and C46ST came from bioengineeredor. Chondroitin came from bioengineered168.

Method for do novo biosynthesis of chondroitin sulfate

The present invention provides a method for scarless in vitro DNA assembly using thermostable exonucleases and ligase, which relates to the field of genetic engineering. The present invention provides a fast method for assembling DNA subfragments with homologous ends, which employs thermostable polymerases and ligase in a thermal cycle of denaturation, annealing, digestion and ligation. After denaturation, DNA subfragments are assembled together via annealing of the homologous end sequences, the unpaired single-stranded overhangs are digested by polymerases, and the resulting nicked gaps are sealed by a ligase. Using this method, 2-6 DNA subfragments were successfully assembled within two hours. This method can be used in conventional DNA recombination and be adapted to high throughput assembly operations. In addition, combinatorial mutations can be easily introduced into the assembled sequence by use of primers with mutated bases. It is particularly suitable for making enzyme and synthetic pathways mutation libraries with high diversity, which can be used in directed evolution to screen for enzymes and synthetic pathways with desirable properties.

In vitro method for fast scarless DNA assembly using thermostable exonucleases and ligase

The present invention relates to the field of biotechnology engineering. It provides a method of constructing a recombinantthat can produce specific-molecular-weight hyaluronic acids. By integranted expression of hasA fromand overexpression of genes of HA synthetic pathway, tuaD, glmU and glmS, high yield HA production was achieved in the recombinant strain. Additionally, introduction and functional expression of the leech hyaluronidase in the recombinant strain substantially increased the yield of HA to 19.38 g&#xb7;L. Moreover, HAs with a broad range of molecular weights (10Da to 10MDa) were efficiently produced by controlling the expression level of hyaluronidase using RBS mutants with different translational strengths. The method of the present invention can be used to produce low molecular weight HAs at large scale in industrial applications.

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The present invention provides a novel leech HAase and a method of producing low-molecular-weight HA oligosaccharides using the leech HAase. This invention successfully cloned the first leech HAase gene and provides a method for high-level expression of the leech HAase gene. By controlling the incubation condition, different HA oligosaccharides, particularly HA4, HA6, HA8 and HA10, can be selectively generated using the leech HAase. The large-scale expression of the leech HAase and the enzymatic production of specific HA oligosaccharides are not only useful for the cosmetic, healthcare and the medical industries but also can be a great help to polysaccharides chemical synthesis and cancer research.

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Wuxi, China

Zhen Kang