In Silico Analysis and Development of the Secretory Expression of D-Psicose-3-Epimerase in Escherichia coli

Abstract

D-psicose-3-epimerase (DPEase), a key enzyme for D-psicose production, has been successfully expressed in Escherichia coli with high yield. However, intracellular expression results in high downstream processing costs and greater risk of lipopolysaccharide (LPS) contamination during cell disruption. The secretory expression of DPEase could minimize the number of purification steps and prevent LPS contamination, but achieving the secretion expression of DPEase in E. coli is challenging and has not been reported due to certain limitations. This study addresses these challenges by enhancing the secretion of DPEase in E. coli through computational predictions and structural analyses. Signal peptide prediction identified PelB as the most effective signal peptide for DPEase localization and enhanced solubility. Supplementary strategies included the addition of 0.1% (v/v) Triton X-100 to promote protein secretion, resulting in higher extracellular DPEase (0.5 unit/mL). Low-temperature expression (20 °C) mitigated the formation of inclusion bodies, thus enhancing DPEase solubility. Our findings highlight the pivotal role of signal peptide selection in modulating DPEase solubility and activity, offering valuable insights for protein expression and secretion studies, especially for rare sugar production. Ongoing exploration of alternative signal peptides and refinement of secretion strategies promise further enhancement in enzyme secretion efficiency and process safety, paving the way for broader applications in biotechnology.

Keywords: D-psicose-3-epimerase; protein expression; protein secretion; psicose; signal peptide.

Figure 1

Plasmid construction map for this study. The synthesized DPEase genes were cloned into pET28a with and without OmpA and PelB signal peptides at the 5′ NcoI and 3′ XhoI sites. The expression of all constructs was under the control of the T7 promoter.

Figure 2

SDS-PAGE analysis of recombinant DPEase in the E. coli Psicose strain (A,D), the OmpA_Psicose strain (B,E), and the PelB_Psicose strain (C,F) cultured in LB medium without (A–C) and with (D–F) 0.1% (v/v) Triton X-100. The samples from various compartments were analyzed. (Lane M: protein marker; lane 1: fermentation broth fraction; lane 2: periplasm fraction; lane 3: cell lysate fraction and lane 4: cell debris).

Figure 3

Effect of temperature on DPEase expression. (A) SDS-PAGE analysis of recombinant DPEase in the E. coli Psicose strain cultured at 20 °C (lane 1), 25 °C (lane 2), 30 °C (lane 3), and 37 °C (lane 4). SDS-PAGE analysis of DPEase expression at 20 °C of the Psicose strain (B), the OmpA_Psicose strain (C), and the PelB_Psicose strain (D) with 0.1% (v/v) Triton X-100. The samples from various compartments were analyzed. (lane M: protein marker; lane 1, 2: fermentation broth fraction; lane 3, 4: periplasm fraction; lane 5, 6: cell lysate fraction and lane 7, 8: cell debris). Blue arrows represent the DPEase position.