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Related Experiment Videos

Centromeric DNA Facilitates Nonconventional Yeast Genetic Engineering.

Mingfeng Cao1, Meirong Gao1, Carmen Lorena Lopez-Garcia1

  • 1Department of Chemical and Biological Engineering, ‡NSF Engineering Research Center for Biorenewable Chemicals (CBiRC), §Genome Informatics Facility, Office of Biotechnology, ∥Interdepartmental Microbiology Program, and ⊥The Ames Laboratory, Iowa State University , 4140 Biorenewables Research Laboratory, Ames, Iowa 50011, United States.

ACS Synthetic Biology
|April 11, 2017
PubMed
Summary

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This summary is machine-generated.

Researchers identified centromeric DNA in Scheffersomyces stipitis, enabling stable episomal plasmids for enhanced biomanufacturing. This breakthrough improves gene editing and compound production in nonconventional yeasts.

Area of Science:

  • Biotechnology
  • Synthetic Biology
  • Microbial Engineering

Background:

  • Nonconventional yeasts like Scheffersomyces stipitis offer unique metabolic capabilities for biorenewables.
  • S. stipitis shows promise for producing high-value compounds via the shikimate pathway.
  • Current limitations in S. stipitis include a lack of stable episomal expression and precise genome-editing tools.

Purpose of the Study:

  • To identify and characterize centromeric DNA in S. stipitis for stable extra-chromosomal DNA segregation.
  • To develop a stable episomal expression platform for S. stipitis.
  • To enhance genome editing efficiency and compound production in S. stipitis.

Main Methods:

  • Identification of centromeric DNA sequences in S. stipitis.
Keywords:
CEN epigeneticityCRISPR/Cas9Scheffersomyces stipitiscentromeresepisomal plasmidsnonconventional yeasts

Related Experiment Videos

  • Construction and testing of stable episomal plasmids utilizing the identified centromeric DNA.
  • Evaluation of gene expression stability and compound titer.
  • Assessment of gene knockout efficiency using CRISPR-Cas9 on the stable plasmid.

    • Main Results:

    • Successfully pinpointed centromeric DNA as a partitioning element for stable DNA segregation.
    • Developed a stable minichromosome-like expression platform.
    • Achieved homogeneous gene expression and a 3-fold increase in commercial compound titer.
    • Dramatically increased gene knockout efficiency from <1% to >80%.

    Conclusions:

    • The identified centromeric sequence is crucial for stabilizing episomal plasmids in S. stipitis.
    • Establishment of a stable expression platform is key for functional modification of nonconventional yeasts.
    • This work expands the potential of S. stipitis as a microbial biomanufacturing platform.