Engineered Host Cells for Unnatural Nucleic Acid Production
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Solution Overview
Problem
The limited chemical and physical diversity of the natural genetic alphabet restricts the ability to sequence-specifically synthesize or amplify oligonucleotides, limiting the information that can be stored in cells and the creation of novel gene expression products.
Innovation Solution
The use of engineered host cells with modified transposition-associated proteins, modified DNA repair proteins, and a modified nucleoside triphosphate transporter to increase the production of nucleic acid molecules containing unnatural nucleotides, thereby expanding the genetic alphabet and enhancing gene expression capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If natural nucleotides (A, C, G, T) are used for DNA synthesis, then the genetic alphabet maintains its natural simplicity and compatibility with existing cellular machinery, but the chemical and physical diversity is limited, restricting information storage capacity and novel gene expression products
Solution Approach 1:
The patent segments the genetic alphabet into natural nucleotides (A, C, G, T) and unnatural nucleotides (dNaM, dTPT3), allowing each segment to serve specific functions. The unnatural nucleotides are introduced as distinct segments that expand information capacity without completely replacing the natural system, thereby increasing adaptability while managing complexity through modular integration
Solution Approach 2:
The patent introduces intermediary components including modified polymerases (e.g., PolB with specific mutations), modified nucleoside triphosphate transporters (e.g., PtNTT2), and DNA repair proteins that mediate between the unnatural nucleotides and the natural cellular machinery. These intermediaries enable the unnatural nucleotides to be incorporated and maintained without requiring complete redesign of the genetic system
Solution Approach 3:
The patent changes key parameters of the cellular system by introducing unnatural nucleotides with distinct chemical properties (dNaM with amino group, dTPT3 with thio and propargyl groups). These parameter changes in nucleotide chemistry enable expanded information storage while the accompanying modifications in polymerase and transporter parameters maintain system compatibility
2Adaptability or versatility
If unnatural nucleotides are incorporated into DNA, then the information storage capacity and gene expression diversity are expanded, but the retention and stability of unnatural base pairs in newly synthesized DNA strands decrease
Solution Approach 1:
The patent employs preliminary action by pre-modifying the cellular machinery before unnatural nucleotide incorporation. Specifically, modified polymerases are expressed in advance to recognize and incorporate unnatural nucleotides, and modified nucleoside triphosphate transporters are prepared to import unnatural nucleoside triphosphates into the cell, ensuring stable incorporation and retention before synthesis occurs
Solution Approach 2:
The patent implements feedback mechanisms through modified DNA repair proteins (e.g., MutS, MutL) that monitor DNA synthesis and correct errors involving unnatural nucleotides. This feedback loop ensures high fidelity replication of unnatural base pairs by detecting mismatches and facilitating corrective repair, thereby maintaining retention stability
Solution Approach 3:
The patent creates composite DNA structures containing both natural nucleotides (A, C, G, T) and unnatural nucleotides (dNaM, dTPT3) in the same genome. This composite approach allows the system to leverage the stability of natural base pairs while incorporating the information capacity of unnatural pairs, achieving both stability and versatility
3Productivity
If modified transposition-associated proteins and DNA repair proteins are used to increase unnatural nucleotide production, then the production efficiency is improved, but the system complexity and difficulty of engineering increase
Solution Approach 1:
The patent employs universal components that perform multiple functions. For example, the modified polymerase PolB serves both as a replicative polymerase for genome maintenance and as a mechanism for incorporating unnatural nucleotides during replication. The modified nucleoside triphosphate transporter PtNTT2 universally imports both natural and unnatural nucleoside triphosphates, reducing the need for separate specialized components and simplifying the overall system
Solution Approach 2:
The patent implements self-service mechanisms where the modified DNA repair proteins (MutS, MutL, RecA) automatically detect and correct errors involving unnatural nucleotides during normal DNA replication and repair processes. The system self-regulates unnatural nucleotide incorporation through intrinsic proofreading and repair mechanisms, eliminating the need for external control systems and reducing engineering complexity
Data Source
AI summary
Disclosed herein are methods, cells, engineered microorganisms, and kits for increasing the production of polypeptides comprising one or more unnatural amino acids. Further provided are cells, engineered microorganisms, and kits for increasing the retention of unnatural nucleic acids encoding the unnatural amino acids in an engineered cell, or semi-synthetic organism.