Engineering Protein Secretion Platforms for Living Materials in Caulobacter Crescentus

Living organisms create biomaterials with exceptional mechanical properties. These biomaterials also respond to various environmental cues and can self-generate & self-heal [1]. These characteristics lead to the construction of the engineered counterparts: engineered living materials (ELMs). However, our ability to create these new biomaterials is largely limited by how well we can program living organisms to synthesize biomolecules at the right place and amount [1]. Here we programmed Caulobacter crescentus to secrete proteins via the Type I Secretion System (T1SS) and the Type II Secretion System (T2SS) to synthesize and functionalize biomaterials. First, we engineered the T1SS of C. crescentus to secrete an extracellular matrix protein composed of an elastin-like polypeptide (ELP) hydrogel fused to supercharged SpyCatcher [SC^(−)]. This biopolymer was secreted at levels of 60 mg/liter, an unprecedented level of biomaterial secretion by a native T1SS [3]. Given our success with unfolded protein secretion via the T1SS, we sought to develop our platform for folded proteins via the native T2SS. To accomplish this, we used a native lipoprotein (ElpS), substrate of T2SS [4], as a native carrier for functional folded proteins. Furthermore, to control and increase the secreted folded protein yields, we upregulated the gene expression of the T2SS apparatus via the pho regulon’s response to phosphate starvation. Taken together, this work develops protein design guidelines for T1SS & T2SS in C. crescentus and demonstrates the autonomous secretion and assembly of extracellular protein matrices to form environmentally responsive engineered living materials.

1. Gilbert and Ellis, (2018). Biological Engineered Living Materials: Growing Functional Materials with Genetically Programmable Properties. ACS Synth Biol.
2. Orozco-Hidalgo et al., (2021). Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials. mSystems.
3. Le Blastier et al., (2010). Phosphate starvation triggers production and secretion of an extracellular lipoprotein in Caulobacter crescentus. PLoS One.