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Engineering and application of LacI mutants with stringent expressions.

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Engineered LacI protein (M7 mutant) significantly reduces lac promoter leakiness and broadens induction range. This breakthrough enables improved biosensors and enhanced galacto-oligosaccharides (GOS) production.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biotechnology

Background:

  • Transcriptional regulatory circuits require stringent control for optimal function, with minimal expression in the absence of inducers and a wide dynamic range upon induction.
  • The lac operon's Plac promoter in Escherichia coli exhibits leakiness due to the moderate binding affinity of the LacI repressor protein.
  • Engineering regulatory proteins can overcome limitations in natural systems, improving control and expanding applications.

Purpose of the Study:

  • To engineer the LacI repressor protein to enhance its regulatory properties, specifically reducing promoter leakiness and increasing the induction dynamic range.
  • To investigate the individual contributions of specific mutations to the improved regulatory characteristics of the engineered LacI.
  • To utilize the enhanced LacI mutant in the development of a novel biosensor for high-value sugar production.

Main Methods:

  • Site-directed mutagenesis was employed to create LacI variants, including the M7 mutant (I79T and N246S).
  • Surface plasmon resonance (SPR) was used to quantify the binding affinity of wild-type and mutant LacI to the operator sequence.
  • A biosensor was constructed utilizing the M7 mutant, followed by directed evolution to select for improved galactosidase activity.

Main Results:

  • The M7 LacI mutant demonstrated a 95% reduction in lac promoter leakiness and a significantly broader induction dynamic range compared to wild-type LacI.
  • SPR studies confirmed that the M7 mutant exhibits tighter binding to the operator sequence.
  • The developed biosensor facilitated the selection of mutant galactosidases with a seven-fold increase in specific activity for transgalactosylation.

Conclusions:

  • The engineered M7 LacI mutant provides a superior transcriptional control system with reduced leakiness and enhanced dynamic range.
  • The improved LacI regulatory protein is a valuable tool for constructing more efficient biosensors.
  • This advancement directly enables enhanced biosynthesis of valuable galacto-oligosaccharides (GOS).