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Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers
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Codon-tRNA Coadaptation Bias for Identifying Strong Native Promoters in Komagataella phaffii.

Louise La Barbera Kastberg1, Mykhaylo S Petrov2, Tomas Strucko1

  • 1Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads Building 223, 2800 Kgs. Lyngby, Denmark.

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|February 21, 2024
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Summary

We identified strong native promoters in Komagataella phaffii (formerly Pichia pastoris) using a computational method based on codon bias. This expands the toolkit for engineering microbial production strains in bioprocesses.

Keywords:
Komagataella phaffiiTranslational selectionnumber of effective codonspromotersstrain engineeringtRNA adaptation index

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Area of Science:

  • Microbiology
  • Synthetic Biology
  • Biotechnology

Background:

  • Promoters are essential for microbial strain engineering in bioprocesses.
  • A limited set of characterized promoters hinders data-driven engineering of Komagataella phaffii (formerly Pichia pastoris).

Purpose of the Study:

  • To develop an in silico method for identifying strong, condition-independent native promoters in K. phaffii.
  • To expand the promoter toolbox for K. phaffii strain engineering.

Main Methods:

  • Utilized tRNA-codon coadaptation analysis of K. phaffii coding sequences.
  • Employed two scores: number of effective codons and tRNA adaptation index.
  • Validated computationally identified promoters using fluorescent reporter assays.

Main Results:

  • Identified promising strong promoter candidates based on codon bias.
  • Experimentally characterized 50 promoters, revealing a 76-fold expression range in glucose medium.
  • Reported several novel promoters suitable for K. phaffii engineering.

Conclusions:

  • The study presents a novel in silico approach for de novo promoter discovery in microbial genomes.
  • The identified promoters enhance the molecular toolbox for K. phaffii engineering.
  • This method facilitates the development of improved microbial production strains.