Genetically Encoded Stimuli-Responsive Cytoprotective Hydrogel Capsules for Single Cells Provide Novel Genotype–Phenotype Linkage

Abstract

Modification of cell surfaces with synthetic polymers is a promising approach for regulating cellular behavior. Here we describe a genetically controlled strategy for selectively encapsulating single yeast cells in synthetic microniches comprised of cross-linked phenol-modified alginate and chitosan hydrogel capsules. Our system links inducible gene expression with enzyme-mediated hydrogel polymerization and provides a novel genotype-phenotype linkage whereby only cells carrying a requisite gene encoding a flavin adenine dinucleotide (FAD)-dependent oxidoreductase undergo autonomous enzyme-mediated surface polymerization resulting in formation of hydrogel capsules. The composition of the hydrogel capsules is highly tunable and the capsule sizes are pH-responsive, allowing for control of capsule porosity and shell diameters over a range of 15-80 µm. The hydrogel capsules prevent extracellular proteins from reaching the cell surface, thereby conferring cellular immunity to lytic enzyme cocktails and rendering the hydrogel capsules cytoprotective against osmotic shock. We demonstrate the utility of this genetically controlled artificial hydrogel-encapsulated cell phenotype by isolating and enriching uniform eukaryotic cell lineages from genetically heterogeneous cell mixtures at 95-100% efficiency. The encapsulated cells remained viable and were capable of dividing and breaking free from their hydrogel capsules, allowing further propagation of selected cells. Our bottom-up approach to cellular compartmentalization links inducible intracellular genetic components with an artificial extracellular matrix that resists enzymatic lysis and mediates communication with the surrounding environment through a size-tunable and permeable hydrogel capsule.

Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

“…To produce the macromonomers, we modified sodium alginate (M W = 70,000 g/mol) with tyramine and aminofluorescein (Figure b; Liu, Sakai, Kawa, & Taya, ; Sakai & Kawakami, ) using carbodiimide activation chemistry. 1 H‐NMR confirmed the presence of aromatic phenol and fluorescein groups in the resulting polymer (Vanella et al, ). The approach for directed evolution of GOx is presented in Figure c.…”

“…During its catalytic cycle, GOx utilizes molecular oxygen as an electron acceptor, oxidizing β‐ d ‐glucose to d ‐gluconolactone and producing hydrogen peroxide as a byproduct (Swoboda & Massey, ; Figure a). We developed the current system relying on GOx and HRP working in tandem as a bienzymatic initiation system for polymerization of phenolated fluorescent macromonomers (Sakai, Komatani, & Taya, ; Sakai et al, ; Shoda, Uyama, Kadokawa, Kimura, & Kobayashi, ; Sigg et al, ; Uyama, Kurioka, & Kobayashi, ; Vanella et al, ; Zavada, Battsengel, & Scott, ). To produce the macromonomers, we modified sodium alginate (M W = 70,000 g/mol) with tyramine and aminofluorescein (Figure b; Liu, Sakai, Kawa, & Taya, ; Sakai & Kawakami, ) using carbodiimide activation chemistry.…”

“…In previous work (Vanella et al, ), we validated the hydrogel encapsulation reaction as a cell screening tool by screening cell mixtures of diluted GOx positive cells in a background of pYD1 empty plasmid‐containing cells, and observing a direct correspondence of selected positive cell concentrations in FACS plots. Green fluorescent cells sorted in single‐cell mode from a reference mixture containing 10% GOx positive cells were recultured without any noticeable effect of the gel capsules on cell growth rate, and all were found to be positive for GOx when tested using ABTS top agar assay.…”

“…Alginate macromonomers grafted with phenol moieties and aminofluorescein was prepared through carbodiimide activation chemistry as previously reported (Sakai & Kawakami, ; Vanella et al, ). Sodium alginate (avg.…”

“…Directed evolution of accelerated or stabilized glucose oxidase (GOx) variants is of considerable interest for the food and textile industries, as well as for blood glucose detection (Wong, Wong, & Chen, ), deoxygenation of buffers for single‐molecule fluorescence applications (Aitken, Marshall, & Puglisi, ), and oxygen inhibitor removal for radical polymerization (Chapman, Gormley, Herpoldt, & Stevens, ). Recently we reported a new method for single‐cell encapsulation in hydrogel capsules that relied on a GOx and horseradish peroxidase (HRP) enzyme cascade to cross‐link phenol moieties grafted onto alginate and chitosan macromonomers (Vanella, Bazin, Ta, & Nash, ). The system we reported was made possible by GOx initiator enzymes that were synthesized by the cells from single‐copy plasmids carrying the GOx gene, and surface‐displayed on the outer cell wall.…”