Recombinant Cell Biosynthesis of pN-Phe for Vaccine Immunogenicity
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Solution Overview
Problem
Current methods for biosynthesizing para-nitro-L-phenylalanine (pN-Phe) within live cells are non-existent, limiting its application in enhancing live vaccines and immunotherapies due to the inability to incorporate this immunostimulatory compound into proteins within recombinant cells.
Innovation Solution
Engineering recombinant cells to produce pN-Phe from native metabolites like chorismate through expression of heterologous enzymes such as PapA, PapB, PapC, N-monooxygenase, and aminotransferase, allowing for the conversion of these metabolites into pN-Phe without external supplementation, and incorporating pN-Phe into target polypeptides using an aminoacyl-tRNA synthetase and transfer RNA pair.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If recombinant cells are engineered to biosynthesize pN-Phe from native metabolites, then the ability to incorporate pN-Phe into proteins within live cells is improved, but the device complexity increases due to the need to express multiple heterologous enzymes
Solution Approach 1:
The biosynthesis pathway is divided into multiple enzymatic steps, with each step catalyzed by a separate heterologous enzyme (PapA, PapB, PapC, N-monooxygenase, and aminotransferase). This segmentation allows the complex transformation from chorismate to pN-Phe to be achieved through a series of manageable reactions, each performed by a specialized enzyme from a different organism.
Solution Approach 2:
The patent introduces intermediate metabolites (para-amino-phenylpyruvate and para-nitro-phenylpyruvate) that serve as bridges between the starting material (chorismate) and the final product (pN-Phe). These intermediaries enable the stepwise transformation and allow each heterologous enzyme to act on a specific intermediate rather than requiring a single complex enzyme.
2Productivity
If pN-Phe is produced within recombinant cells without exogenous supplementation, then the productivity of pN-Phe production is improved, but the loss of substance increases due to the consumption of native metabolites
Solution Approach 1:
The patent utilizes native metabolites (chorismate, para-amino-phenylpyruvate, or para-nitro-phenylpyruvate) that are already present in the cell's metabolic pathway as starting materials. By performing the biosynthesis steps using these pre-existing metabolites rather than requiring external supplementation, the system achieves self-sufficiency and improves productivity while minimizing the need for additional resource input.
3Manufacturing precision
If heterologous enzymes are expressed to convert native metabolites to pN-Phe, then the manufacturing precision of pN-Phe biosynthesis is improved, but the ease of manufacture decreases due to the complexity of introducing and maintaining multiple heterologous genes
Solution Approach 1:
The patent identifies and utilizes native metabolites that serve multiple functions: they are both endogenous to the host cell's metabolism and serve as substrates for the heterologous biosynthesis pathway. This universality allows the same cellular machinery to support both normal metabolism and the engineered pN-Phe production, simplifying the manufacturing process while maintaining precision.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the production of pN-Phe within recombinant cells, enhancing the immunogenicity of target polypeptides by at least 50% without exposing the cells to exogenous pN-Phe, facilitating improved immune responses for vaccine development and immunotherapy.
Implementation Method 1
The recombinant cell expresses the one or more heterologous enzymes and a native metabolite. As a result, the native metabolite is converted to the pN-Phe in the recombinant cell.
Implementation Method 2
incorporating pN-Phe into target polypeptides using an aminoacyl-tRNA synthetase and transfer RNA pair
Data Source
AI summary
The present invention provides a recombinant cell for producing para-nitro-L-phenylalanine (pN-Phe). The recombinant cell comprises heterologous genes encoding heterologous enzymes. The recombinant cell expresses the heterologous enzymes and contains a native metabolite. The native metabolite is converted to the pN-Phe in the recombinant cell. The biosynthesized pN-Phe may be incorporated into a target polypeptide in the recombinant cell without requiring exposure of the recombinant cell to exogenous pN-Phe. A cell culture comprising the recombinant cell is also provided. Further provided is a method of producing pN-Phe by a recombinant cell comprising heterologous genes encoding heterologous enzymes. The method comprises expressing a native metabolite by the recombinant cell, expressing the heterologous enzymes, and converting the native metabolite to the pN-Phe in the recombinant cell. The method may further comprise incorporating the pN-Phe into the target polypeptide in the recombinant cell.