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Directed evolution study unveiling key sequence factors that affect translation efficiency in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
20548 43180 2013 6 PDF Available
Title
Directed evolution study unveiling key sequence factors that affect translation efficiency in Escherichia coli
Abstract

Synonymous mutations in protein coding genes significantly impact translation efficiency. We synthesized a pair of genes encoding green fluorescent protein that were separated by 160 synonymous mutations to investigate key factors that affect translation efficiency. One sequence was optimized for Escherichia coli (GFPEco) and the other for Bacillus subtilis (GFPBsu). When the genes were expressed in E. coli, GFPEco fluoresced 12-fold stronger than GFPBsu, confirming the suboptimal nature of the GFPBsu gene. We then employed directed evolution to improve the expression of GFPBsu. Random mutagenesis and DNA shuffling was used to generate mutant libraries, which were screened for fluorescence. A variant showing 6-fold fluorescence enhancement was identified, which contained a single mutation (G10A) in a rare codon for Gly-4. However, the substitution generated another type of rare codon, AGA, for Arg, suggesting that the improvement was caused by a factor other than the rare codon. We next applied saturation mutagenesis to Gly-4. The darkest variant contained a GGG codon (GFPBsu-G) for Gly-4. Taking the location of the mutation into account, we hypothesized that destabilization of the mRNA secondary structure around the initiation codon improved the expression. We then randomized the nucleotide triplet in 5′-untranslated region (5′UTR) of GFPBsu, which is complementary to the Gly-4 codon. A variant showing 6-fold fluorescence enhancement was identified, which exhibited a destabilized secondary structure. When this 5′UTR sequence was combined with GFPBsu-G, 22-fold fluorescent improvement was achieved. Collectively, the stability of the mRNA secondary structure around the initiation codon predominantly affected the translation efficiency.

Keywords
Codon usage; Directed evolution; Green fluorescence protein; mRNA folding; Synonymous mutations; Translation efficiency
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Directed evolution study unveiling key sequence factors that affect translation efficiency in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 116, Issue 5, November 2013, Pages 540–545
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us