The genetic engineering of Escherichia coli for the synthesis of dermatan sulfate epimerase

Abstract

Glycosaminoglycans (GAG) are a fast-growing industry. Currently they are primarily extracted from animals, which presents several limitations and risk. For this reason, several animal free manufacturing methods have been developed as a way to support the increasing GAG demand. Microbial biosynthesis has shown to be a promising alternative to GAG production. Though this has been successful for the production of some GAGs, like heparin and chondroitin sulfate, other GAGs like dermatan sulfate (DS) are yet to be synthesized by microbial organisms. This is primarily because a human expressed protein, dermatan sulfate epimerase (DS-Epi), catalyzes the synthesis of dermatan sulfate. This study is the first attempt at expressing human dermatan sulfate epimerase in E. coli. Several homologous mutants were generated in this study, with the intention of developing a more soluble and stable version of DS-Epi that can be expressed in E. coli. To express DS-Epi in E. coli, these generated mutant variants were cloned into a vector to form recombinant plasmids that transformed competent E. coli cells. In this study the dermatan sulfate epimerase gene was not successfully expressed in E. coli. Though the majority of the gene was successfully cloned and fostered bacterial growth, unexpected mutations in the desired gene led to its inexpression. Overall, this study showed that the DS-epi gene could be successfully cloned and be used in a recombinant plasmid to transform E. coli. Future research can expand on the work found in this study to successfully express dermatan sulfate epimerase in E. coli and allow for microbial synthesis of dermatan sulfate.