Repurposing ribosomes for synthetic biology

Abstract

The translation system is the cell’s factory for protein biosynthesis, stitching together hundreds to thousands of smaller units of amino acids into proteins, which are required for the structure, function, and regulation of living systems. The extraordinary synthetic capability of this system, which includes the ribosome and its associated factors required for polymerization, has driven extensive efforts to harness it for societal needs in areas as diverse as energy, materials, and medicine. An example is recombinant protein production that has impacted the lives of patients through the synthesis of biopharmaceuticals such as insulin. In nature, however, only limited sets of monomers are utilized, thereby resulting in limited sets of biopolymers (i.e., proteins). Expanding nature’s repertoire of ribosomal monomers could yield new classes of enzymes, therapeutics, materials, and chemicals with diverse genetically encoded chemistry. Here, we discuss recent progress towards engineering ribosomes both in vivo and in vitro. These fundamental and technical breakthroughs open doors for advanced applications in biotechnology and synthetic biology.

Toward an orthogonal central dogma

“…20-22 Creating orthogonal ribosomes in which both the large and small ribosomal subunits are selectively directed to an orthogonal message, and do not cross-assemble with endogenous ribosomal subunits, might expand the sequence space that can be explored for large subunit evolution, and potentially the scope of monomers that can be accommodated by ribosomal translation. 23 Progress towards these goals may be accelerated by the observation that the ribosomal RNA of the large subunit can be circularly permuted, 24 which has enabled the creation of functional ribosomes in which the subunits are covalently linked through RNA. 25,26…”

Incorporation of Modified Amino Acids by Engineered Elongation Factors with Expanded Substrate Capabilities

“…7,14 Components that have previously been somewhat tolerant of ncAA incorporation, like EF-Tu, are beginning to come to the forefront as obstacles that must be addressed to achieve these challenging goals. 11,12 These concurrent efforts illustrating the urgent need for comprehensive and creative strategies to expand the genetic code support the argument for novel approaches to engineer EF-Tu.…”

A Bacterial Machinery for Surface Displayed Enzymes

“…In this regard, there have been studies to immobilize enzymes and protein on solid supports to provide a longer lifetime and better durability for them [21]. In a whole cell system these optimizations can be achieved through engineering of the cellular genetic regulation systems [22]. In our study we have formed a system where the timely control of the enzyme secretion was achieved through an inducible system.…”

“…This cell is suitable for the production of the proteins under the control of the LacI repression along with pLac promoter system. [22] As descried in materials and methods section the cell were grown and induced to initiate the engineered autotransporter protein expression. Following the expression and folding of the protein the cell membrane of the protein was expected to be displayed on the cell surface.…”