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CUT&RUN identifies centromeric DNA regions of Rhodotorula toruloides IFO0880.

J Carl Schultz1, Mingfeng Cao1, Andrea Mejia1

  • 1Department of Chemical and Biomolecular Engineering, U.S. Department of Energy Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

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|December 13, 2021
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Summary

Researchers identified centromeric DNA regions in Rhodotorula toruloides using CUT&RUN, a key step for developing stable episomal plasmids for bioproduction. This finding aids future efforts in yeast genetic engineering.

Keywords:
Rhodotorula toruloidesCUT&RUNCse4centromere

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Synthetic Biology

Background:

  • Rhodotorula toruloides is a promising yeast for producing lipids, fatty acids, and terpenoids.
  • Stable episomal plasmid maintenance is crucial for efficient bioproduction but lacks established centromere and autonomously replicating sequence (ARS) elements in R. toruloides.

Purpose of the Study:

  • To identify centromeric DNA sequences in Rhodotorula toruloides IFO0880.
  • To lay the groundwork for developing stable episomal plasmids for enhanced bioproduction in R. toruloides.

Main Methods:

  • Utilized cleavage under targets and release using nuclease (CUT&RUN) to map DNA-protein interactions.
  • Focused on identifying regions associated with the centromeric histone H3 protein Cse4.
  • Analyzed sequence characteristics of identified putative centromeres.

Main Results:

  • Identified fifteen putative centromeric regions in R. toruloides IFO0880, varying from 8 to 19 kb.
  • Found that these centromeric sequences have low GC content, are transcriptionally silent, and contain minimal repetitive or transposon-related sequences.
  • Tested four regions for autonomously replicating sequence (ARS) activity, which was not detected.

Conclusions:

  • The identified centromeric DNA sequences provide essential components for future development of stable episomal plasmids in R. toruloides.
  • These findings advance the genetic engineering toolkit for R. toruloides, supporting its use in industrial biotechnology.
  • Further research is needed to identify functional ARS elements in conjunction with these centromeric sequences.