Cell-free protein synthesis platforms derived from clostridia extracts
Inventors
Jewett, Michael Christopher • Kruger-Gericke, Antje • Mueller, Alexander Paul • Koepke, Michael
Assignees
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Publication Number
US-12157908-B2
Publication Date
2024-12-03
Expiration Date
Abstract
Disclosed are compositions, methods, and kits for performing cell-free RNA transcription and/or cell-free protein synthesis (CFPS). The disclosed compositions, methods, and kits include or utilize components prepared from a species of Clostridia such as cellular extracts from Clostridium autoethanogenum.
Core Innovation
The document discloses a cell-free protein synthesis platform that performs in vitro transcription of mRNA and in vitro translation of polypeptides using a cellular extract prepared from a cell culture of a species of Clostridia. The platform is based on engineering the Clostridia species to express an upregulated gene product that functions as a positive effector for CFPS, where the positive effector is identified as the Clostridia homolog of E. coli ackA.
A key aspect of the disclosed innovation is that the platform is used in an aerobic CFPS context, with motivation and optimization results addressing oxygen availability effects. The extract preparation and CFPS reaction conditions are tuned, including sensitivity of extract preparation to lysis energy and the choice between runoff versus dialysis, along with an unusually high Mg(Glu)2 requirement and temperature tuning around 30°C.
The document further discloses that the DNA template design affects performance, including use of linear DNA with phosphorothioate-modified ends. Performance is demonstrated using luciferase reporter readouts under batch and semi-continuous CFPS formats, and the platform is also used to prototype clostridial genetic parts using native promoters and 5′UTRs, to assess endogenous promoter activity, and to express full-length enzymes and multi-enzyme metabolic pathway components.
Claims Coverage
The independent claims center on a CFPS platform that uses an extract from engineered Clostridia expressing a specified positive effector, the Clostridia homolog of E. coli ackA. The main coverage includes one explicitly independent claim, with dependent claim refinements adding specific reaction-ingredient constraints, engineered components, and a genetic parts characterization and testing use case.
Engineered Clostridia extract with upregulated CFPS positive effector
A cell-free protein synthesis platform comprising a cellular extract prepared from a cell culture of a species of Clostridia, wherein the species is engineered to express an upregulated gene product that is a positive effector for CFPS, and wherein the positive effector for CFPS is the Clostridia homolog of E. coli ackA.
Defined energy source with PEP concentration range
The platform further includes an energy source comprising phosphoenol pyruvate (PEP) at a concentration greater than about 30 mM but less than about 100 mM, together with translation and transcription templates and a DNA dependent RNA polymerase.
Magnesium concentration window
The platform further includes magnesium at a concentration above about 1 mM and below about 60 mM.
Potassium concentration window
The platform further includes potassium (K+) at a concentration greater than about 10 mM and less than about 500 mM.
Engineered expression of T7 RNA polymerase in the Clostridia source
The platform is configured with an engineered Clostridia species that expresses T7 RNA polymerase.
Genetic parts identification and characterization method
A method identifies and characterizes genetic parts of Clostridia and assesses Clostridia gene expression used for transcription and/or translation by creating a test library of Clostridia genetic parts or variant gene sequences and testing the genetic parts and/or an alternative codon expressed on the platform.
Across the independent and dependent claim set provided, the core coverage is a Clostridia-extract CFPS platform where an upregulated CFPS positive effector is the Clostridia homolog of E. coli ackA, with refinements directed to defined reaction-ingredient constraints, optional T7 RNA polymerase engineering in the Clostridia source, and a method for genetic parts identification and characterization using the platform.
Stated Advantages
Supports CFPS for in vitro transcription of mRNA and in vitro translation of polypeptides using a Clostridia-derived cellular extract.
Enables aerobic CFPS operation as described by oxygen-availability motivation and optimization results.
Provides improved luciferase performance with described higher outputs in semi-continuous operation compared with batch operation.
Documented Applications
Prototyping clostridial genetic parts using native promoters and 5′UTRs, including assessing endogenous promoter activity.
Expression of full-length enzymes and prototyping metabolic pathways using multi-enzyme expression.
Metabolomics-based evaluation of oxygen-independent pathway activity, described with metabolite profiling consistent with glycolysis/gluconeogenesis and carbon fixation.
Characterization and testing of genetic parts by creating a test library of Clostridia genetic parts or variant gene sequences and testing the parts and/or an alternative codon on the claimed platform.
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