Engineering and application of LacI mutants with stringent expressions

Author:

Wu Jieyuan12,Liang Chaoning1,Li Yufei12,Zeng Yueting3,Sun Xu4,Jiang Peixia1,Chen Wei1,Xiong Dandan12,Jin Jian‐Ming4,Tang Shuang‐Yan1ORCID

Affiliation:

1. CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources Institute of Microbiology, Chinese Academy of Sciences Beijing China

2. University of Chinese Academy of Sciences Beijing China

3. School of Life Sciences Hebei University Baoding China

4. Beijing Key Laboratory of Plant Resources Research and Development Beijing Technology and Business University Beijing China

Abstract

AbstractOptimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the Plac/LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild‐type LacI. An in‐depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild‐type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high‐value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven‐fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto‐oligosaccharides (GOS).

Publisher

Wiley

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