Engineering microorganisms for biosensor development and bioelectricity generation

Abstract

The advent of recombinant DNA technology, as well as newly-emerging fields including synthetic biology and metabolic engineering, has brought great potential for genetically modified microorganisms to serve useful purposes. However, to date, most of the efforts have been devoted into studies using a few model host microorganisms, such as Escherichia coli and Saccharomyces cerevisiae, whereas much is left unexplored for the majority of other microorganisms that makes promising alternative hosts. While the conventional hosts exhibit certain strengths in their feasibility to be genetically modified and the availability of tools to be employed, it is highly desirable for researchers to look beyond the technical perspectives and into alternative hosts where we could find ways to complement the current systems. In this thesis, both a conventional microorganism host, S. cerevisiae and a newly-arising host of biotechnological significance, photosynthetic Rhodobacter sphaeroides were engineered for novel applications in biosensor development and bioelectricity generation. The thesis study presents two examples where the unique metabolic capacities of microbes can be leveraged to address major social issues, e.g., healthcare and energy. In the first part, an ultrasensitive immunoassay, YSD-CCI has been developed using engineered S. cerevisiae that can display immunogenic antigens on the surface and express intracellular fluorescent proteins simultaneously. The amount of analytes, namely antibodies, could be determined by counting the number of fluorescent yeast cells bound with corresponding antibody, realizing high sensitivity and multiplex detection capability of the platform. In the second part of the study focusing on genetic engineering of a nonconventional host R. sphaeroides, different strategies were investigated to improve the electrogenic activities of R. sphaeroides in a solar-powered microbial fuel cell. By manipulating endogenous electron fluxes through genetic engineering approaches, significant increase in the current generation was observed. In order to facilitate the development of R. sphaeroides as an alternative expression host, a vector based tRNA supplementation system was ultimately developed for R. sphaeroides to overcome its codon bias issues in heterologous expression. Via co-expressing rare tRNA genes on a multi-copy plasmid, the expression levels of two exemplary rare-codon containing genes, ribU and mtrA, in R. sphaeroides were improved accordingly. It is anticipated that the tRNA supplementation system can be further extended to other species of Rhodobacter, and thus will facilitate the engineering of purple bacteria for interesting applications in microbial technology.

Date of Award	2015
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology