Nanoscale surface engineered living cells with extended substrate spectrum

Abstract

We report on cell surface engineering of living microorganisms by using Layer-by-Layer (LbL) technology to extend the substrate spectrum. The yeast Arxula adeninivorans LS3 (Arxula) was employed as a model organism and biological template. By using LbL technology, Arxula cells were encapsulated by polyelectrolyte and enzyme layers. The biological activity of the Arxula was retained after the encapsulation process. The polymeric capsule surrounding the Arxula provides a stable interface for surface engineering of living cells. LbL of polyelectrolytes followed by an enzyme layer of lactate oxidase were assembled. The outer enzyme layer provides an additional biological function for Arxula to convert the unfavourable substrate lactate into the favourable substrate pyruvate, thus extending the substrate spectrum of the organism. Moreover, capsule stability and enzyme conjugate stability of the surface engineered Arxula were studied.

Truly Nonionic Polymer Shells for the Encapsulation of Living Cells

“…[85] The absence of such cofactors in dead cells leads to no conversion and no fluorescence can be detected. [86] Figure 7 shows viability of cells encapsulated with truly nonionic LbL shells in comparison with the same cells encapsulated but with cationic PEI pre-layer as a control series explored in previous study with different PVPON molecular weight. [61] In this study, different molecular weights of PVPON (360 or 1 300 kDa) and different numbers of TA/PVPON bilayers (two or four) have been explored.…”

Layer‐by‐Layer Nano‐Encapsulation of Microbes: Controlled Cell Surface Modification and Investigation of Substrate Uptake in Bacteria

“…[16,17] Furthermore, different yeast species have been encapsulated, and for Arxula adeninivorans LS3, LbL nano self-assembly demonstrated a spectrum of substrate utilization. [18,19] These studies show the promising prospects for the application of LbL nano self-assembly on questions in microbiology because the method allows construction of cell surfaces with a defined architecture on living and metabolically active microorganisms, while the complete shell characterizes cell colloidal stability, and the outermost layer characterizes the surface charge and a specific hydrophilicity or hydrophobicity. [20] Furthermore, defining the incorporation (uptake) and defined release of different molecules, like proteins or nanoparticles in the shells, are possible for eukaryotes and prokaryotes.…”

“…[17] Therefore, it was the aim of our investigations to demonstrate substrate uptake for bacteria, similar to experiments done with the eukaryotic A. adeninivorans LS3. [19] As a model organism with a versatile sulfur metabolism, the gram-negative, phototrophic purple sulfur bacterium Allochromatium vinosum was chosen.…”

“…In contrast to Mak et al [19] who assembled an outermost enzyme layer to the cells that provided usage of an unfavorable metabolic substrate, we used two different electrostatic LbL nano self-assembly approaches to investigate controlled substrate uptake for metabolism in A. vinosum. Specifically, we measured changes in the z-potential of the cell surface with very thin shells of three and less layers and evaluated the effect of building a physical barrier around the cell with shells of four and more layers.…”

Biomedical Applications of Layer‐by‐Layer Self‐Assembly for Cell Encapsulation: Current Status and Future Perspectives

“…Such result shed light on the feasibility of manipulating the function of single cells by LbL cell encapsulation. From then on, several researches focused on the encapsulation of yeast and bacterial spore by LbL assembly and observe the viability and metabolic activity changes of the encapsulated cells . For example, Diaspro et al reported the encapsulation of yeast ( S. cerevisiae ) with PAH and PSS and observed that microbial cells maintained their normal metabolic activities and viability after encapsulation .…”