Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in <i>E. coli</i>

Casas, Meritxell Galindo; Stargardt, Patrick; Mairhofer, Juergen; Wiltschi, Birgit

doi:10.1021/acssynbio.0c00298

Cited by 20 publications

(22 citation statements)

References 93 publications

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“…Previous work has demonstrated the feasibility of using SPSs as alternatives to DPSs in both Escherichia coli and mammalian cells. − In prior studies in E. coli, SPSs facilitated the evolution of aaRSs and tRNAs for enhanced ncAA incorporation. ,, In mammalian cells, SPSs lead to improved transfection or transduction efficiencies and more stable expression of ncAA-encoding gene(s) of interest. ,, Although these reports clearly demonstrated the utility of SPSs, we are not aware of direct, side-by-side comparisons of SPS and DPS performances.…”

Section: Introductionmentioning

confidence: 99%

Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast

2021

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Genetic code expansion is a powerful approach for advancing critical fields such as biological therapeutic discovery. However, the machinery for genetically encoding noncanonical amino acids (ncAAs) is only available in limited plasmid formats, constraining potential applications. In extreme cases, the introduction of two separate plasmids, one containing an orthogonal translation system (OTS) to facilitate ncAA incorporation and a second for expressing a ncAA-containing protein of interest, is not possible due to a lack of the available selection markers. One strategy to circumvent this challenge is to express the OTS and protein of interest from a single vector. For what we believe is the first time in yeast, we describe here several sets of single plasmid systems (SPSs) for performing genetic code manipulation and compare the ncAA incorporation capabilities of these plasmids against the capabilities of previously described dual plasmid systems (DPSs). For both dual fluorescent protein reporters and yeast display reporters tested with multiple OTSs and ncAAs, measured ncAA incorporation efficiencies with SPSs were determined to be equal to efficiencies determined with DPSs. Click chemistry on yeast cells displaying ncAA-containing proteins was also shown to be feasible in both formats, although differences in reactivity between formats suggest the need for caution when using such approaches. Additionally, we investigated whether these reporters would support the separation of yeast strains known to exhibit distinct ncAA incorporation efficiencies. Model sorts conducted with mixtures of two strains transformed with the same SPS or DPS both led to the enrichment of a strain known to support a higher efficiency ncAA incorporation, suggesting that these reporters will be suitable for conducting screens for strains exhibiting enhanced ncAA incorporation efficiencies. Overall, our results confirm that SPSs are well behaved in yeast and provide a convenient alternative to DPSs. SPSs are expected to be invaluable for conducting high-throughput investigations of the effects of genetic or genomic changes on ncAA incorporation efficiency and, more fundamentally, the eukaryotic translation apparatus.

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Section: Introductionmentioning

confidence: 99%

Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast

2021

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“…Previous work has demonstrated the feasibility of using SPSs as alternatives to DPSs in both E. coli and mammalian cells. [15][16][17][18] SPSs are slightly less flexible than DPSs and larger plasmid sizes may impact transformation efficiency, but they provide access to genetic code expansion that is otherwise unattainable using DPSs. Additionally, with SPSs the burden of maintaining two separate plasmids is eliminated along with the risk of losing one of the two plasmids during cell propagation.…”

Section: Introductionmentioning

confidence: 99%

Broadening the toolkit for quantitatively evaluating noncanonical amino acid incorporation in yeast

Stieglitz

Potts

Deventer

2021

Preprint

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Genetic code expansion is a powerful approach for advancing critical fields such as biological therapeutic discovery. However, the machinery for genetically encoding noncanonical amino acids (ncAAs) is only available in limited plasmid formats, constraining potential applications. In extreme cases, the introduction of two separate plasmids, one containing an orthogonal translation system (OTS) to facilitate ncAA incorporation and a second for expressing a ncAA-containing protein of interest, is not possible due to lack of convenient selection markers. One strategy to circumvent this challenge is to express the OTS and protein of interest from a single vector. For what we believe is the first time in yeast, we describe here several sets of single plasmid systems (SPSs) for performing genetic code manipulation and compare the ncAA incorporation capabilities of these plasmids against the capabilities of previously described dual plasmid systems (DPSs). For both dual fluorescent protein reporters and yeast display reporters tested with multiple OTSs and ncAAs, measured ncAA incorporation efficiencies with SPSs were determined to be equal to or improved relative to efficiencies determined with DPSs. Click chemistry on yeast cells displaying ncAA-containing proteins was also shown to be feasible in both formats, although differences in reactivity between formats suggest the need for caution when using such approaches. Additionally, we investigated whether these reporters would support separation of yeast strains known to exhibit distinct ncAA incorporation efficiencies. Model sorts conducted with mixtures of two strains transformed with the same SPS or DPS led to enrichment of a strain known to support higher efficiency ncAA incorporation, suggesting that these reporters will be suitable for conducting screens for strains exhibiting enhanced ncAA incorporation efficiencies. Overall, our results confirm that SPSs are well-behaved in yeast and provide a convenient alternative to DPSs. In particular, SPSs are expected to be invaluable for conducting high-throughput investigations of the effects of genetic or genomic changes on ncAA incorporation efficiency and, more fundamentally, the eukaryotic translation apparatus.Abstract Figure

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“…The yield of the tsPurple-Cou was 34.4% of the corresponding tsPurple-WT protein in unmodified chassis cells, as observed previously. 32,33 The phenomenon was most likely due to the premature termination of the tsPurple-Cou expression at the UAG codon by the release factor 1 (RF1), which could result in the accumulation of truncated protein and reduced production of the full-length protein. 5 Furthermore, the orthogonal tRNA synthetase/tRNA pair could reduce the yield of POI 34 through recognizing all of the amber codons in unmodified chassis cells, leading to the waste of translation machinery in the genome-wide infertile readthrough protein.…”

Section: Resultsmentioning

confidence: 99%

Simplified Construction of Engineered Bacillus subtilis Host for Improved Expression of Proteins Harboring Noncanonical Amino Acids

Zou

Tian

et al. 2023

ACS Synth. Biol.

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The UAG-based genetic code expansion (GCE) enables site-specific incorporation of noncanonical amino acids (ncAAs) harboring novel chemical functionalities in specific target proteins. However, most GCE studies were done in several whole-genome engineered chassis cells whose hundreds of UAG stop codons were systematically edited to UAA to avoid readthrough in protein synthesis in the presence of GCE. The huge workload of removing all UAG limited the application of GCE in other microbial cell factories (MCF) such as Bacillus subtilis, which has 607 genes ended with UAG among its 4245 coding genes. Although the 257 essential genes count only 6.1% of the genes in B. subtilis, they transcribe 12.2% of the mRNAs and express 52.1% of the proteins under the exponential phase. Here, we engineered a strain named Bs-22 in which all 22 engineerable UAG stop codons in essential genes were edited to UAA via CRISPR/Cas9-mediated multiple-site engineering to minimize the negative effect of GCE on the expression of essential genes. Besides the process of constructing GCE-compatible B. subtilis was systematically optimized. Compared with wild-type B. subtilis (Bs-WT), the fluorescence signal of the eGFP expression could enhance 2.25-fold in Bs-22, and the production of protein tsPurple containing l-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) was increased 2.31-fold in Bs-22. We verified that all purified tsPurple proteins from Bs-22 contained Cou, indicating the excellent fidelity of the strategy. This proof-of-concept study reported efficient overexpression of ncAA-rich proteins in MCF with minimized engineering, shedding new light on solving the trade-off between efficiency and workload.

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Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli

Cited by 20 publications

References 93 publications

Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast

Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast

Broadening the toolkit for quantitatively evaluating noncanonical amino acid incorporation in yeast

Simplified Construction of Engineered Bacillus subtilis Host for Improved Expression of Proteins Harboring Noncanonical Amino Acids

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