Metabolic engineering and applied enzymology for the preparation of nutraceutical/ pharmaceutical chondroitin sulfate
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Authors
He, Wenqin
Issue Date
2017-05
Type
Electronic thesis
Thesis
Thesis
Language
ENG
Keywords
Chemical engineering
Alternative Title
Abstract
Chondroitin sulfate (CS) is an important glycosaminoglycan that is predominantly present in cartilage and the surfaces of many cells and extracellular matrices. It has been widely used in treating osteoarthritis due to its anti-inflammatory and chondroprotective properties. Given the importance of CS in medicine and as a dietary supplement, the traditional animal extraction method which risks virus and/or prion contamination should be replaced by a standardized bioengineering process. The general mechanism of synthesizing CS involves the enzymatic reaction of a membrane-associated sulfotransferase adding sulfo group onto the polymeric chondroitin backbone by adopting a sulfo donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The capsular polysaccharide of pathogenic Escherichia coli K4 strain shares a similar structure to the CS precursor, chondroitin. We first overexpressed the genes involved in the biosynthesis of K4 polysaccharide in a non-pathogenic production strain BL21Star™ (DE3).
This strain produced ~2.4g/l chondroitin in DO-STAT fed-batch fermentor. The next challenge is the functional expression of membrane-associated chondroitin sulfotransferase, which typically requires post modification of N-glycosylation site. The recent success of expressing Homo sapiens chondroitin 4-sulfotransferase in BL21Star™ (DE3) by applying several protein engineering strategies enables a high level of conversion of chondroitin to CS type-A in vitro. The next challenge was improving the availability of the sulfur donor PAPS. PAPS is traditionally generated by chemical synthesis, which results in low yield and high cost. To resolve this issue, a simple and efficient enzymatic scheme was utilized by single plasmid system consisting of three related genes expressed in E. coli BL21 Star™ (DE3). With all the efforts, the metabolic engineering approach of chondroitin synthesis, expression of C4ST-1 and enzymatic synthesis of PAPS, will open up a new regime in bioengineering microbial chondroitin sulfate.
This strain produced ~2.4g/l chondroitin in DO-STAT fed-batch fermentor. The next challenge is the functional expression of membrane-associated chondroitin sulfotransferase, which typically requires post modification of N-glycosylation site. The recent success of expressing Homo sapiens chondroitin 4-sulfotransferase in BL21Star™ (DE3) by applying several protein engineering strategies enables a high level of conversion of chondroitin to CS type-A in vitro. The next challenge was improving the availability of the sulfur donor PAPS. PAPS is traditionally generated by chemical synthesis, which results in low yield and high cost. To resolve this issue, a simple and efficient enzymatic scheme was utilized by single plasmid system consisting of three related genes expressed in E. coli BL21 Star™ (DE3). With all the efforts, the metabolic engineering approach of chondroitin synthesis, expression of C4ST-1 and enzymatic synthesis of PAPS, will open up a new regime in bioengineering microbial chondroitin sulfate.
Description
May 2017
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY