Glycosylation Circuit Enables Improved Catalytic Properties for Recombinant Alkaline Phosphatase.

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins. Here, we engineered a Campylobacter jejuni N-linked glycosylation machinery by overexpressing one of the core glycosylation-related enzymes, PgIB, to increase the glycosylation rate. It has been previously shown that by utilizing N-linked glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. We utilized N-linked glycosylation using an engineered glycosylation pathway to glycosylate a model enzyme, the alkaline phosphatase (ALP) enzyme in Escherichia coli. We have investigated the effects of glycosylation on enzyme properties. Considering the glycosylation mechanism is highly dependent on accessibility of the glycosylation tag, ALP constructs carrying the glycosylation tag at different locations of the gene have been constructed, and glycosylation rates have been calculated. Our results showed that, upon glycosylation, ALP features in terms of thermostability, proteolytic stability, tolerance to suboptimal pH, and denaturing conditions are dramatically improved. The results indicated that the N-linked glycosylation mechanism can be employed for protein manipulation for industrial applications.

Engineering of biofilms with a glycosylation circuit for biomaterial applications.

Glycosylation is a crucial post-translational modification for a wide range of functionalities. Adhesive protein-based biomaterials in nature rely on heavily glycosylated proteins such as spider silk and mussel adhesive proteins. Engineering protein-based biomaterials genetically enables desired functions and characteristics. Additionally, utilization of glycosylation for biomaterial engineering can expand possibilities by including saccharides to the inventory of building blocks. Here, de novo glycosylation of Bacillus subtilis amyloid-like biofilm protein TasA using a Campylobacter jejuni glycosylation circuit is proposed to be a novel biomaterial engineering method for increasing adhesiveness of TasA fibrils. A C. jejuni glycosylation motif is genetically incorporated to tasA gene and expressed in Escherichia coli containing the C. jejuni pgl protein glycosylation pathway. Glycosylated TasA fibrils indicate enhanced adsorption on the gold surface without disruption of fibril formation. Our findings suggest that N-linked glycosylation can be a promising tool for engineering protein-based biomaterials specifically regarding adhesion.

Synthetic Genetic Circuits for Self-Actuated Cellular Nanomaterial Fabrication Devices.

Genetically controlled synthetic biosystems are being developed to create nanoscale materials. These biosystems are modeled on the natural ability of living cells to synthesize materials: many organisms have dedicated proteins that synthesize a wide range of hard tissues and solid materials, such as nanomagnets and biosilica. We designed an autonomous living material synthesizing system consisting of engineered cells with genetic circuits that synthesize nanomaterials. The circuits encode a nanomaterial precursor-sensing module (sensor) coupled with a materials synthesis module. The sensor detects the presence of cadmium, gold, or iron ions, and this detection triggers the synthesis of the related nanomaterial-nucleating extracellular matrix. We demonstrate that when engineered cells sense the availability of a precursor ion, they express the corresponding extracellular matrix to form the nanomaterials. This proof-of-concept study shows that endowing cells with synthetic genetic circuits enables nanomaterial synthesis and has the potential to be extended to the synthesis of a variety of nanomaterials and biomaterials using a green approach.