Non-canonical lipoproteins with programmable assembly and architecture and method of making non-canonical lipoproteins
Inventors
Assignees
Publication Number
US-11884941-B2
Publication Date
2024-01-30
Expiration Date
2041-11-18
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Abstract
Artificial lipoproteins bearing non-canonical post-translational modifications that are synthesized by leveraging substrate promiscuity of an acyltransferase. The non-canonical functionality of these lipoprotein results in a distinctive hysteretic assembly that is absent from the canonical lipoproteins and is used to prepare hybrid multiblock materials with precise and programmable patterns of amphiphilicity due to the unique assembly and function of the non-canonical post-translational modifications.
Core Innovation
The invention provides a method for synthesizing non-naturally occurring lipoproteins using the substrate promiscuity of post-translational modification (PTM) machinery. By co-expressing a lipid modification enzyme, such as N-myristoyl transferase, together with an intrinsically disordered peptide-polymer fused to a peptide substrate, and supplementing the expression media with a non-natural fatty acid, artificial lipoproteins bearing non-canonical post-translational modifications are produced. These lipoproteins are uniquely characterized by their ability to form distinctive assemblies and functionalities that are absent from their canonical counterparts.
The problem addressed by the invention is the evolutionary and functional constraint on the repertoire of lipid modifications in biological systems, where conventional genetic engineering methods only allow incorporation of amino acid-like constituents, limiting the chemical and functional diversity of proteins. Previous methods for producing non-natural lipidated proteins are technically challenging, time-consuming, and expensive, and biosynthetic routes have been hindered by the narrow substrate scope of lipidation enzymes.
The core innovation harnesses the ability of N-myristoyltransferase to accept a variety of artificial fatty acids, such as 12-azidododecanoic acid, to generate lipoproteins with non-canonical PTMs. These artificial lipoproteins can have functional groups suitable for further chemical derivatization, and display unique, programmable phase behaviors and assembly morphologies, including stimuli-responsive and hysteretic transitions not achievable with canonical lipoproteins. This allows for the synthesis of hybrid multiblock materials with precise patterns of amphiphilicity and opens new design spaces for the development of protein-based nanomaterials.
Claims Coverage
There is one independent claim, which defines the main inventive features surrounding the method for synthesizing non-naturally occurring lipoproteins via coexpression in a host organism.
Method for synthesizing non-naturally occurring lipoprotein using coexpression and non-natural fatty acid substrate
The inventive method includes: 1. Modifying a host organism to express Saccharomyces cerevisiae N-myristoyl transferase. 2. Modifying the host organism to express an intrinsically disordered peptide polymer fused to a peptide substrate specific to the N-myristoyl transferase. 3. Coexpressing both the N-myristoyl transferase and the peptide-polymer fusion in the host organism, while providing an expression medium supplemented with a non-natural fatty acid of the formula R—COOH, with R being a linear chain of no more than 15 atoms of C, N, O, or S. 4. Enabling the host organism to carry out post-translational modification of the peptide-polymer by attaching the non-natural fatty acid, thereby forming a non-naturally occurring lipoprotein.
The independent claim focuses on an integrated biosynthetic method, leveraging the promiscuity of a specific acyltransferase (N-myristoyl transferase) for non-natural lipid attachment to recombinant peptide polymers, resulting in non-canonical lipoproteins.
Stated Advantages
The invention enables the production of non-naturally occurring lipoproteins with distinctive assembly behaviors and functionalities absent from canonical lipoproteins.
It provides access to a wider chemical and functional design space, overcoming the constraints of natural lipidation machinery and genetic engineering limitations.
The method allows for programmable and precise patterns of amphiphilicity, facilitating synthesis of hybrid multiblock materials with unique assembly and functions.
The approach is biosynthetic and can use readily available tools and organisms, circumventing technically challenging, time-consuming, and expensive chemoenzymatic methods.
Documented Applications
Programming morphological transitions from nanoparticles to fibers for applications such as simultaneous release of encapsulated cargo and providing scaffolds for cell adhesion and growth.
Using non-canonical lipids as chemical handles for structural elaboration and synthesis of hybrid materials with precise and unique amphiphilic patterns.
Programming assemblies into complex 2D and 3D morphologies to form materials with unique optical and mechanical properties.
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