Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization, yield and activity

Abstract

Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest.

Periplasmic and extracellular production of cellulase from recombinant Escherichia coli cells

“…A recent study showed that heterologous expression of Bacillus maltogenic amylase was obtained at the highest yields with a DsbA signal peptide, which leads to the SRP pathway, compared with PelB (50‐fold higher), FhuD, MalE, OmpA, TorA, MdoD and YcdO signal peptides . In this study, the extracellular YebF‐Cel5A (0.30 IU mL −1 , 175 mg L −1 ) was obtained with about 1.7‐fold higher yields compared with the periplasmic Cel5A (0.18 IU mL −1 , 100 mg L −1 ) (Fig.…”

“…The choice of the signal sequence for efficient secretion should be individually tested for each protein. 24 The signal sequence is cleaved by a signal peptidase during transport of proteins out of the cytoplasm, to yield the mature protein product. 25 The major mechanism driving the post-translational translocation of unfolded proteins in E. coli is the sec-dependent type-II secretion (Sec) pathway, however, since the Sec pathway requires the use of chaperone proteins to aid translocation, many heterologous proteins when expressed in E. coli with Sec signal peptides cannot be exported due to lack of recognition by the host chaperones.…”

“…The choice of the signal sequence for efficient secretion should be individually tested for each protein . The signal sequence is cleaved by a signal peptidase during transport of proteins out of the cytoplasm, to yield the mature protein product .…”

Establishment of a functional system for recombinant production of secreted proteins at 50 °C in the thermophilic Bacillus methanolicus

“…It has been demonstrated that fusion of signal peptides to different proteins can result in hampering of secretion and thermodynamic destabilization, reduced activity and increased tendency to aggregate of the target protein. The in-depth inspection of interactions between signal peptides fused to different proteins suggests that for each signal peptide-protein fusion, the effect on protein thermodynamic stability is unpredictable and dependent on both components of the structure and their interplay [ 61 – 63 ]. This leads to a need for screening large signal peptide libraries in order to identify optimal combinations of signal peptides and proteins when establishing secretion of heterologous proteins [ 61 , 64 , 65 ].…”

Efficient production of extracellular pullulanase in Bacillus subtilis ATCC6051 using the host strain construction and promoter optimization expression system

“…Recombinant expression is an important method to facilitate the production of target proteins. Many genetic strategies have been developed to improve the production of recombinant proteins, such as the use of protease-deficient host strains to prevent degradation [ 5 ], the deletion of extracellular protein genes to reduce secretion stress [ 6 ], and the optimization of promoters and signal peptides [ 7 , 8 ]. Specifically, B. subtilis strains were engineered to serve as extracellular-protease-deficient strains for the overproduction of heterologous proteins such as B. subtilis WB600 [ 9 ], B. subtilis WB700 [ 10 ], and B. subtilis WB800 [ 11 ] (Table 1 ).…”