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Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers
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Strong negative correlation between codon usage bias and protein structural disorder impedes protein expression after

Kunshan Liu1, Yaqi Ouyang1, Ru Lin1

  • 1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.

Journal of Biotechnology
|November 11, 2021
PubMed
Summary

Codon optimization did not improve protein expression in P. pastoris and even decreased it for some genes. This strategy may negatively impact mRNA and protein stability, especially for disordered proteins.

Keywords:
Codon optimizationCodon usage biasPichia pastorisProtein expression

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Codon usage bias influences gene expression, affecting translation speed, protein folding, and transcription.
  • Pichia pastoris (P. pastoris) is a key expression system, but the role of codon usage bias in its protein expression remains understudied.
  • A negative correlation exists between codon usage bias and protein structural disorder in many P. pastoris genes, with unclear implications for expression.

Purpose of the Study:

  • To investigate the impact of codon optimization on protein expression in P. pastoris.
  • To determine if eliminating the negative correlation between codon usage bias and protein structural disorder affects gene expression.
  • To assess the effects of codon optimization on mRNA and protein stability and structure.

Main Methods:

  • Selected four P. pastoris genes with a strong negative correlation between codon usage bias and protein structural disorder.
  • Performed full-length codon optimization to eliminate this correlation.
  • Utilized protein and RNA assays, including trypsin sensitivity and in vitro/in vivo RNA degradation, to compare gene expression before and after optimization.

Main Results:

  • Codon optimization failed to increase protein expression levels and, in some cases, led to a decrease.
  • Protein structure was altered by codon optimization, as shown by trypsin sensitivity assays for specific genes (0616, 0788).
  • Codon optimization affected mRNA levels and stability, altering the mRNA degradation rates for genes 0616, 0788, and 0135.

Conclusions:

  • Codon usage bias is a significant regulator of gene expression, mRNA stability, and protein stability in P. pastoris.
  • Aggressive codon optimization may be detrimental for genes with a strong negative correlation between codon usage bias and protein structural disorder.
  • Compromised codon optimization strategies might be necessary for successful expression, and this approach could severely impact the conformation of disordered proteins.