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Distance-dependent translational coupling and interference in Lactococcus lactis.

M van de Guchte1, J Kok, G Venema

  • 1Department of Genetics, University of Groningen, Haren, The Netherlands.

Molecular & General Genetics : MGG
|May 1, 1991
PubMed
Summary

Researchers enhanced heterologous gene expression in Lactococcus lactis by optimizing translational coupling. Varying the proximity of stop and start codons in fused open reading frames (ORFs) significantly boosted lacZ gene expression.

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Area of Science:

  • Molecular Biology
  • Microbial Genetics
  • Biotechnology

Background:

  • Heterologous gene expression in Lactococcus lactis is crucial for industrial applications.
  • Enhancing protein production in L. lactis remains a key challenge.
  • Translational coupling is a known mechanism for co-regulating gene expression.

Purpose of the Study:

  • To investigate the potential of translational coupling for increasing heterologous gene expression levels in Lactococcus lactis.
  • To determine the optimal arrangement of upstream and downstream open reading frames (ORFs) for maximal gene expression.

Main Methods:

  • Constructing transcriptional fusions between a lactococcal ORF and the Escherichia coli lacZ gene.
  • Introducing a Shine-Dalgarno (SD) sequence at the ORF boundary.

Related Experiment Videos

  • Systematically varying the spacing between the stop codon of the upstream ORF and the start codon of the lacZ gene on plasmids.
  • Quantifying lacZ gene expression levels under different codon arrangements.

    • Main Results:

    • Gene expression of lacZ increased incrementally as the stop and start codons of adjacent ORFs were brought closer.
    • A transition from translational interference to translational coupling was observed with decreasing codon proximity.
    • Optimal lacZ expression was achieved when the stop and start codons partially overlapped.

    Conclusions:

    • Translational coupling is an effective strategy for enhancing heterologous gene expression in Lactococcus lactis.
    • Fine-tuning the positioning of stop and start codons, particularly partial overlap, maximizes gene expression.
    • This approach offers a valuable tool for metabolic engineering and protein production in L. lactis.