Modular functionalization of engineered polyhydroxyalkanoate scaffolds : a thesis presented in partial fulfilment of the requirements for degree Doctor of Philosophy in Microbiology at Massey University, New Zealand

Abstract

Microbial polyhydroxyalkanoates (PHAs) are spherical polyesters that are naturally synthesized in vivo by a variety of microorganisms as carbon and energy reserves under imbalanced nutrition environments. Notably, PHA particles can be functionalized by the genetic modification of surface-exposed PHA-associated proteins, e.g. PHA synthase (PhaC), and this approach has led to multiple successful proof-of-concept demonstrations for bio-technology applications. However, current recombinant methods to functionalize PHAs require a certain biological complexity, such as simultaneous polyester and protein synthesis within a single cell. The less defined nature of this technology means limited control over particle morphology and surface functionalization. Seeking to overcome these limitations, the work presented in this thesis is to introduce the concept of modularity to the PHA particle technology, by merging the PHA particle technology with Tag/Catcher protein ligation systems. The Catcher domain can rapidly form a covalent bond with its pairing short peptide tag in a site-specific manner, without the need of additional reagents nor enzymes at broad ligation conditions. The SpyTag/SpyCatcher pair was merged recombinantly with PHA particle technology, where the resulting SpyCatcher-coated PHA particles were able to immobilize various SpyTagged proteins in vitro in a tunable manner and remained functional. This thesis further demonstrates several functionalization processes to streamline this modular strategy by assessing the possibility of whether non-purified SpyTagged proteins could ligate with the PHA particles in complex environments. The results demonstrated that SpyCatcher-coated PHA particles could be functionalized adequately using two of the proposed methods. To further expand the design space of this generic modular platform towards programmable multi-functionalization, various bimodular PHA particles utilizing alternative Tag/Catcher pairs (e.g. SdyTag/SdyCatcher and SnoopTag/Snoop-Catcher pairs) were designed and studied. One of the constructs resulted in the simultaneous multi-functionalization of plain PHA particles in one-step with two differently tagged proteins in in vivo and ex vivo reaction conditions. This work presents the modular design of PHA scaffolds and several streamlined manufacturing processes to the production of task-specific designer PHAs. Introducing the concept of modularity to the PHA particle technology enabled better control of particle uniformity, reproducibility, and immobilized protein density while remaining functional. These concepts should be broadly applicable to the design and manufacture of advanced functional materials for industrial applications.