Abstract
Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. However, an imbalance between cell growth and bioproduction often reduces production efficiency. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. Here, GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. We demonstrate GCE-CGBBE in genome-recoded Escherichia coli Δ321AM by precisely balancing glycolysis and N-acetylglucosamine production, resulting in a 4.54-fold increase in titer. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Moreover, the development of ncAA-dependent essential gene expression regulation shows efficient biocontainment of engineered B. subtilis to avoid unintended proliferation in nature.
Highlights
Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds
Recent progress in genome engineering and writing has enabled the construction of genetically recoded Escherichia coli, including E. coli with all amber stop codons replaced by ochre stop codons (TAA), or with a 61-codon genome (serine codons (TCG and TCA) and amber stop codons were replaced by their synonyms AGC, AGT, and TAA, respectively), which provides a favorable chassis cell for applying GCEbased orthogonal translation systems by reassigning free codons to ncAAs30,31
The orthogonal translation system for non-canonical amino acids (ncAAs) incorporation is composed of mutant aminoacyl-tRNA synthetase (aaRS)/tRNA pairs for translating noncanonical codons, which has been well established in E. coli[35]
Summary
Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Metabolic connections between cell growth and target product biosynthesis increase the complexity of balancing cellular metabolic pathways. By inserting amber stop codons into genes that are important for controlling cell growth, such as genes involved in central carbon metabolic pathways, should be regulated by ncAA abundances. Further titration of ncAAs to determine optimum concentrations to precisely control cell growth rates can potentially be used for balancing cell growth and bioproduction (Fig. 1a, c)
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