Periplasmic and extracellular production of cellulase from recombinant Escherichia coli cells

Abstract

BACKGROUND Secretory production of recombinant enzymes in E. coli has significant advantages in bioprocess development, such as simplicity of purification, preventing proteolytic degradation, and providing greater access to substrates such as cellulose that opens the path to consolidated bioprocessing. RESULTS In this study, an endoglucanase (Cel5A) from Fusarium graminearum was investigated for extracellular production in E. coli as a fusion to the YebF carrier protein, and compared with periplasmic production enabled by fusing the DsbA signal peptide to the N‐terminus of Cel5A. Yields of extracellular YebF‐Cel5A (175 mg L−1) were nearly 2‐fold higher than yields of periplasmic Cel5A. Periplasmic but not extracellular production of Cel5A increased ∼1.5‐fold following the addition of 0.4 mmol L−1 MgSO4 to the growth medium. While most of the divalent cations had an inhibitory effect on the periplasmic Cel5A activity, all cations tested enhanced the extracellular endoglucanase activity, with Co2+ having the highest activation (4.3‐fold increase). Moreover, YebF‐Cel5A had 20% higher residual activity than Cel5A following 1 h heat treatment. CONCLUSION The differences demonstrated for periplasmic and extracellular production of cellulases in E. coli provide the basis of bioprocess design parameters, paving the way to consolidated bioprocessing. © 2016 Society of Chemical Industry

Genetically Engineered Proteins to Improve Biomass Conversion: New Advances and Challenges for Tailoring Biocatalysts

“…Even though this section focused on eukaryotic models, it is worth noting that several approaches were also taken in prokaryotic hosts to pursue a more efficient protein secretion and display, given that purification of such enzymes is often troublesome due to the typical formation of inclusion bodies in these systems [126]. Works by Yildirim et al, in which a recombinant endoglucanase was efficiently engineered for periplasmic and extracellular targeting by a native secretion signal of E. coli [138], and Huang et al, who showed the advantages of using protease-deficient strains of B. subtilis for cell-surface display of heterologous endoglucanase [139], gave insights for overcoming current limitations of prokaryotic systems.…”

Enhanced heterologous expression of Trichoderma reesei Cel5A/Cel6A in Pichia pastoris with extracellular co‐expression of Vitreoscilla hemoglobin

“…To date, the heterologous expression of Cel5A and Cel6A enzymes in different hosts under control of various promoters has been widely reported . Mellitzer et al .…”

“…8 To date, the heterologous expression of Cel5A and Cel6A enzymes in different hosts under control of various promoters has been widely reported. [9][10][11][12] Mellitzer et al 11 heterologously expressed codon optimized T. reesei Cel6A gene in Pichia pastoris under the AOX1 promoter variant, maximizing the CMCase activity of 9.05 U mL -1 . Akbarzadeh et al 12 successfully expressed the codon optimized Trichoderma reesei Cel5A in Pichia pastoris and constructed the recombinant host capable of producing a maximum CMCase activity of 2 358 U mL -1 with 1% methanol induction for 72 h. Researchers have argued that the Pichia pastoris host appears a desirable candidate for the expression.…”