Engineering and application of LacI mutants with stringent expressions

Abstract

Optimal 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 P_lac /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).

FIGURE 1

LacI engineering. (A) GFP expression after 12‐h cultivation of Escherichia coli BLGR strain expressing LacI wild‐type (WT) or the selected LacI mutants (M1‐M7) from plasmid pLac7, in the presence (+) and absence (−) of 5 mM IPTG. (B) The IPTG dose–response curves of GFP expression in strain BLGR, which expresses wild‐type LacI (WT), M7 mutant (M7) or LacI with single‐mutation N246S (N246S) or I79T (I79T). (C) Basal expression levels (leaky expression) and the final inducing folds (calculated as the ratio of maximal induced fluorescence to the leaky expression) in strain BLGR expressing the indicated LacI regulators. (D) The modelled IPTG binding pocket of mutant M7, compared with wild‐type LacI (WT) (PDB ID: 2P9H) (Daber et al., 2007). (E) Comparison of GFP expression levels in strain BLGR containing pLac7‐M7 or pLac7 derivative with tandem lacOs, pLacO2 or pLacO3. Error bars indicate SD of three independent replicates. All the cells were allowed to grow under inducing condition at 37°C for 12 h before the measurements.

FIGURE 2

The binding affinities of purified 6×His‐tagged LacI wild‐type (A) and M7 mutant (B) for tac promoter DNA were measured by SPR. Different concentrations of purified wild‐type LacI (50, 100, 200, 400, 800 nM) or M7 (0.3125, 0.625, 1.25, 2.5, 5, 10 nM) were used for determination.

FIGURE 3

Feasibility of the M7 mutant‐based biosensor used for transgalactosylation activity screening. (A) Scheme of screening for improved transgalactosylation activity using biosensor. Inducing effects of IPTG and/or lactose on (B) wild‐type LacI and (C) M7 mutant. (D) GFP fluorescence of strain BLGR harbouring plasmids plac7‐M7 and pBADLacS‐R supplemented with inducer l‐arabinose and substrate lactose at the indicated concentrations. The cells were cultured at 37°C for 12 h.

FIGURE 4

LacS engineering aided by the M7 mutant‐based biosensor. (A) GFP fluorescence and the GOS production (by crude enzyme) of the selected mutants from two rounds of random mutagenesis. A dose of 1 μM arabinose (inducer) and 5 mM lactose (substrate) was supplemented, and the cells were cultured for 12 h at 37°C. The strain expressing wild‐type LacS (WT) was included as reference. Fluorescence in the presence (+) or absence (−) of substrate lactose was determined. Comparison between GOS production (B) and specific activities (C) of purified wild‐type LacS (WT) and mutant S5‐11 at 37 and 70°C. The reaction was incubated at indicated temperatures for 12 h, supplemented with 0.438 M lactose. One unit (U) of enzyme activity was defined as the amount of enzyme required to produce 1 μmol GOS from lactose per hour in (C). Error bars indicate SD of three independent replicates.