Enhance Biochemical Production via Codon Recoding of Constructs in Different Expression Systems

通過對不同表達系統中的密碼子重編以增加生物化學產物的產量

Student thesis: Doctoral Thesis

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Award date22 Aug 2024

Abstract

Synonymous codon recoding enhances protein expression in heterogeneous sequences, but existing approaches inadequately address the systematic reduction of sequence diversity when combining synonymous codon reduction with nucleobase skews. In this thesis, we present a novel recoding strategy for mNeonGreen2 fluorescent protein, which optimizes adenine nucleobase usage across 12 target amino acids through successive recoding steps in different biological systems. In the first section, mNeonGreen2 fluorescent protein expression of recoded sequences to one codon per amino acid in tandem with adenine skew was tested in vivo. 22 different sequences with varying fluorescence levels in E. coli Marionette BL21, formed two clusters, with the upper cluster showing 2.3- to 11.7-fold higher fluorescence while maintaining comparable growth rates. Strong correlations were found between the minimum free energy of recoded sequences and mNeonGreen2 mRNA abundance, and between mRNA abundance and fluorescent expression. To further elucidate the transcriptional and translational mechanism of these sequences, an E.coli-based cell-free protein synthesis (CFPS) was developed. This in vitro approach increases flexibility by directly dissecting and manipulating the transcription/translation with rapid feedback through key components tuning. Our results showed that expression of mNeonGreen2 fluorescent protein is comparable to those from commercial kit at a fraction of the cost ($0.012/µL of reaction). Finally, this CFPS method was utilized to express 40 recoded sequences of mNeonGreen2. Fluorescent protein expression formed a continuous expression pattern for all 40 sequences, with the highest expression is 3.75-fold compared to the reference sequence. Furthermore, the relationship between T7 RNA polymerase and nucleobase concentration on transcription/translation rate of fluorescent was shown, suggesting that transcription and translation mechanisms are much more complex than suggested. The knowledge from these works improves the understanding of heterogeneous expression of specific recoded sequences in different biological systems.

    Research areas

  • synthetic biology, Gene expression, Cell free DNA, transcriptome, Synonymous codon recoding, mNeonGreen2, artificial codon reduction, nucleotide skews