Synthetic genome
Inventors
FREDENS, Julius • Wang, Kaihang • DE LA TORRE, Daniel • FUNKE, Louise F. H. • Robertson, Wesley E. • Chin, Jason W.
Assignees
United Kingdom Research and Innovation • Scarab Genomics LLC
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Publication Number
US-12385035-B2
Publication Date
2025-08-12
Expiration Date
Abstract
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.
Core Innovation
The invention relates to synthetic E. coli genomes that implement genome-wide synonymous codon compression for one or two serine codons, with very low frequency of the serine codons while maintaining viability. The synthetic E. coli genome contains 5 or fewer occurrences of one or two serine codons, or 10% or less of the occurrences relative to a parent E. coli genome.
The genome lacks a functional gene encoding an endogenous cognate tRNA for at least one of the serine codons, and each serine codon is recoded such that the endogenous cognate tRNA for the serine codon is dispensable. The recoded design includes synonymous codon replacement and handling of target codons such as TCG, TCA, and TAG, including replacement with sense and/or stop codons such as TAA.
The document also describes genome refactoring for overlapping ORFs, including synthetic inserts to preserve protein-coding and regulatory context in overlapping regions, head-to-tail and tail-to-tail overlaps, duplicated overlaps, and in-frame TAA stop codons plus insertions to resolve problematic overlap/refactoring situations. A fully recoded genome is constructed by conjugation-based section assembly, and the resulting synthetic strain is evaluated for viability and functional/phenotypic consequences associated with reduced serine codon usage and endogenous cognate tRNA dispensability.
Claims Coverage
The independent claims cover two related inventive concepts: a viable synthetic E. coli genome with highly constrained serine codon occurrences and dispensable endogenous cognate tRNA, and a viable synthetic E. coli genome derived from a parent genome that reduces serine codon occurrences relative to the parent while removing functional endogenous cognate tRNA and recoding the serine codons. Across the independent claims, there are three core inventive elements: constrained occurrence of one or two serine codons, removal of functional endogenous cognate tRNA genes for those serine codons, and viability of an E. coli comprising the synthetic genome.
Highly constrained serine codon occurrences with dispensable endogenous cognate tRNA
A synthetic E. coli genome comprises 5 or fewer occurrences of one or two serine codons; the genome lacks a functional gene encoding an endogenous cognate tRNA for at least one of the serine codons; and each serine codon has been recoded such that the endogenous cognate tRNA is dispensable, wherein an E. coli comprising the synthetic E. coli genome is viable.
Relative reduction of serine codon occurrences derived from a parent genome
A synthetic E. coli genome derived from a parent E. coli genome comprises 10% or less of the occurrences of one or two serine codons relative to the parent; the synthetic genome lacks a functional gene encoding an endogenous cognate tRNA for at least one of the serine codons; and each of the serine codons has been recoded such that the endogenous cognate tRNA is dispensable, wherein an E. coli comprising the synthetic E. coli genome is viable.
The claim coverage centers on viability-preserving synthetic E. coli genomes that reduce occurrences of one or two serine codons to a low absolute threshold or to 10% or less relative to a parent genome, remove functional endogenous cognate tRNA genes for at least one of those serine codons, and recode the serine codons so the corresponding endogenous cognate tRNA is dispensable.
Stated Advantages
Achieves viability of an E. coli comprising the synthetic E. coli genome.
Enables a viable E. coli comprising the synthetic E. coli genome under the stated serine-codon and endogenous tRNA gene constraints.
Documented Applications
Production of polypeptides containing one or more non-proteinogenic amino acids by culturing an E. coli host cell.
Culturing an E. coli host cell to produce polypeptides containing one or more non-proteinogenic amino acids.
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