Construction of a functional assembly of modular enzyme for direct biosynthesis of cephalosporins
Introduction: The worldwide sale of β-lactam antibiotics represents a more than US$20 billion of business per annum. The development of novel β-lactam antibiotics has been a hot issue in the past few decades. Nevertheless, most of them are synthesised via chemical means, whose processing put extensive burden to our environment. 7-Aminocephalosporanic acid (7-ACA) is a key cephem nucleus for a number of semi-synthetic cephalosporins. Conventional chemical synthesis of 7-ACA is environmentally-damaging, and we therefore aim to develop a “greener” approach to synthesise 7-ACA with potential for industrial applications.
Objectives: (1) To create a collection of yeast strains that express scaffoldin and modular enzymes; and (2) To evaluate the potential applications of these yeast strains for one-pot biosynthesis of 7-ACA.
Methods: We aim at creating a functional assembly of modular enzymes for direct 7-ACA production. The basis of the functional assembly is based on the creation of Saccharomyces cerevisiae strains that express DAAO, GL-7-ACA acylase, and catalase. S. cerevisiae strain EBY100 was used to express scaffoldin and S. cerevisiae strain BY4742 was used for protein expression. Microbial cohesins and dockerins were obtained by PCR and cloned in an in-frame fashion with Aga2. Genotypes were confirmed by genomic Southern hybridisation. Cell surface display of scaffoldin was determined by specific antibodies and viewed under fluorescent microscope.
Results: The cohesins and dockerins have been cloned and functionally expressed in S. cerevisiae strains. The three dockerins were assembled as a trifunctional scaffoldin. DAAO, GL-7-ACA acylase, and catalase have been expressed in yeast. Their enzymatic activities have been detected using an array of biochemical tests. Conclusion: We have constructed a collection of yeast strains that express scaffoldin and the three modular enzymes.
THEi 2018 Research Symposium
Tsang, W.,& Fong, W. (2018). Construction of a functional assembly of modular enzyme for direct biosynthesis of cephalosporins. THEi 2018 Research Symposium. Retrieved from https://repository.vtc.edu.hk/thei-fac-gen-ed-sp/240