Synthetic Prokaryotic Genome Codon Compression for Viable Cells
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Solution Overview
Problem
Existing methods for genome-wide synonymous codon compression in prokaryotic genomes have not been able to produce viable organisms with significantly reduced sense codon usage, and there is a need for improved methods to produce synthetic genomes with minimal sense codons.
Innovation Solution
A method combining recombination-mediated genetic engineering (REXER and GENESIS) with directed conjugation is used to efficiently replace large portions of a prokaryotic genome, allowing for genome-wide synonymous codon compression, reducing sense codons to 5 or fewer occurrences, and replacing stop codons like TAG with TAA, while using defined recoding and refactoring schemes to handle non-tolerated positions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If genome-wide synonymous codon compression is attempted to reduce sense codon usage, then the number of sense codons is reduced, but the viability of the organism is compromised
Solution Approach 1:
The genome is divided into multiple synthetic DNA fragments that are assembled iteratively. The replacement process is segmented into multiple rounds, each handling specific genomic regions, allowing systematic recoding while maintaining organism viability through controlled progression
Solution Approach 2:
Synthetic DNA fragments are designed and prepared in advance with the desired codon replacements before being introduced into the organism. The recoding scheme is planned preliminarily to ensure essential genes retain necessary codons while non-essential regions undergo compression
Solution Approach 3:
A synthetic intermediate genome serves as a mediator between the original genome and the final compressed genome. This intermediate synthetic DNA contains the recoded sequences that are gradually integrated through multiple rounds of recombination-mediated engineering
2Manufacturing precision
If site-directed mutagenesis is used to replace sense codons, then individual codons can be replaced, but the process is inefficient for genome-wide compression
Solution Approach 1:
Multiple codon replacement operations are merged into a single synthetic DNA fragment that contains all desired recodings. This combined approach allows simultaneous replacement of numerous codons across different genes in one integration event, dramatically increasing productivity while maintaining precision
Solution Approach 2:
The approach transitions from single-point mutagenesis (one dimension) to genome-wide recoding (multiple dimensions). By working with entire synthetic DNA fragments containing multiple recoded codons simultaneously, the method expands from incremental changes to comprehensive genome engineering
3Quantity of substance
If large portions of the genome are replaced with synthetic DNA, then more codons can be compressed, but the complexity of the engineering process increases
Solution Approach 1:
The complex genome replacement process is segmented into manageable rounds, each handling specific synthetic DNA fragments. This segmentation breaks down the overall complexity into discrete, controllable steps that can be systematically executed and verified
Solution Approach 2:
The genome engineering is performed through periodic rounds of recombination-mediated replacement. Each round follows a standardized protocol for introducing synthetic DNA, selecting successful integrants, and preparing for the next round, creating a repetitive yet progressive engineering cycle
Data Source
AI summary
The current invention provides a synthetic prokaryotic genome comprising 5 or fewer occurrences of one or more sense codons; and/or a synthetic prokaryotic genome derived from a parent genome, wherein the synthetic prokaryotic genome comprises less than 10%, 5%, 2%, 1%, 0.5%, 0.1% of the occurrences of one or more sense codons, relative to the parent genome; and/or a synthetic prokaryotic genome comprising 100 or more, 200 or more, or 1000 or more genes with no occurrences of one or more sense codons.