Engineering Lactococcus lactis for the scaffold protein-mediated surface display of recombinant enzymes

摘要

Multi-enzyme complexes are responsible for the synthesis of a number of biochemical compounds and the degradation of complex polymers. An example of the latter is the degradation of cellulose by enzyme complexes termed “cellulosomes” which are produced by several bacteria of the class Clostridia. The basic structure of a cellulosome comprises a central scaffold protein, which associates with a multitude of cellulases via cohesin-dockerin interactions. The advent of cellulose utilization as feedstock for producing biofuels has garnered much interest towards designing custom-tailored recombinant cellulosomes and expressing them in microbes of interest. The metabolic diversity among bacteria also make this approach an appealing strategy for bestowing cellulolytic capabilities upon organisms which produce non-biofuel commodity chemicals such as lactic acid, succinic acid, acetone, amino acids, food additives and carotenoids. In addition, the display of recombinant multi-enzyme complexes in bacteria can yield novel insights into the mechanisms and parameters affecting their secretion, assembly and function. The industrially relevant lactic acid bacterium, Lactococcus lactis, is a model organism for the secretion and display of recombinant proteins, and the numerous biological techniques available for its manipulation make this organism particularly appealing for such a task. In this thesis, I present my work describing the incremental steps taken towards the surface display of custom-tailored multi-enzyme complexes on the surface of L. lactis. Chapter 1 describes the proof of concept for this project, including the choice of promoters, secretion signal peptide, and reporter enzymes. It also discusses the major bottlenecks observed based on the organism’s physiology. Chapter 2 describes the engineering of scaffold chimeras with cohesins of different specificity and the display of two enzymes on such scaffolds. I also investigated the catalytic profiles of the resulting complexes when enzymes were simultaneously or sequentially bound to the displayed scaffold. Finally, chapter 3 describes the optimization of the type 2 dockerin-cohesin interaction by the inclusion of the CipA “X” module, as well as the engineering of enzyme complexes with novel architectures by use of secondary “adapter” scaffolds and the subsequent assembly of multi-scaffold complexes. Also investigated is the potential of using a dual-plasmid system for the full in vivo assembly of such complexes without the exogenous addition of components.