A versatile silk-based coacervate as protocell model towards structural and functional protocell evolution

Abstract

Molecularly crowded coacervate microdroplets derived from associative liquid-liquid phase separation, are often utilized as protocell models but the absence of a physical membrane limits their application. Up till now, numerous auxiliary surface-active or complexation agents have been employed to stabilize the coacervate droplets by irreversibly producing an exterior membrane on the surface while minimal attention was focused on the endogenous pathway for reversible membranization. Besides, with the developed structure of coacervate vesicle resulting from the continuous coacervate membranization, almost no more effort has been given to achieve a further inner structural/functional complexation of it for high-ordered cellular aspects. Thus, in this thesis, we are aiming to solve these problems.
In the first section, we introduce a dynamic alginate/silk coacervate system capable of reversible structural reconfiguration from membrane-less coacervate droplets to semipermeable coacervate vesicles by triggering the self-organization of amphiphilic silk-based polymers for membranization in the absence of auxiliary agents. The endogenous membranization and reconfiguration of coacervate can be reversibly controlled by employing the antagonistic GOx/urease for pH manipulation such that the protocell reconfigurations are coupled to dynamic sequestration/extrusion of water-soluble cargoes.
In the second section, by involving the endosymbiotic theory, the produced silk-based coacervate vesicle was utilized as the host for housing guest protocells (proteinosomes), to produce a proteinosome-in-coacervate vesicle (Prot.-in-PCV) structure by triggering the coacervate vesicle reconfiguration into membrane-less coacervate droplets to capture the guest entities and thereby produce a proteinosome-in-coacervate (Prot.-in-C) entity, and then reversing the coacervate reconfiguration to produce a Prot.-in-PCV protocell. The results indicate that the host/guest entities of the produced Prot.-in-C/PCV protocell are capable of synergetic and symbiotic behaviors for enhanced tolerance to environmental pressures (e.g., TCEP reduction, salt-triggered disassociation) and polysaccharide uptake/digestion to power the structural evolution of symbiont and fluorescence signal output.
In the third section, considering the versatility and biocompatibility of silk fibroin, the yeast cells were harnessed as the living guests incorporated by the dynamic silk-based coacervate to produce an endosymbiotic Yeast-in-C/PCV protocell capable of living behaviors, like synergetic respiration, structural protocell evolution, intracellular guest division. Taken all, our results open new perspectives in the design of coacervate-based protocells capable of evolution by endogenous membranization and reconfiguration, as well incorporation of guest entities for endosymbiotic behaviors. This can possibly offer a new route for protocell construction and complexation by following the endosymbiotic theory.

Date of Award	27 Sept 2022
Original language	English
Awarding Institution	University of Bristol
Supervisor	Stephen Mann (Supervisor) & Nick C Norman (Supervisor)