Contribution

Overview

As we embarked on our project, we read about the research and contributions of our predecessors. Their insights and experiences sparked our creativity and guided our design, significantly advancing our work. We aim to enrich the knowledge base for future iGEM participants and hope our contributions inspire other teams, fostering a vibrant and diverse iGEM community.

Contribution from XMU-China 2025

The LMT sequence, a signal peptide discovered from Vibrio natriegens (Please see the Results of 2021 XMU-China), is capable of directing recombinant proteins out of both V. natriegens and E. coli bacterial cells. This part (BBa_K3739010) was first registered in 2021 (2021 XMU-China) and was used to construct expression systems and helped prove the function of some signal peptides in V. natriegens. The effect of different promoters on the function of LMT signal peptides was tested in 2024 (2024 XMU-China).

This year, for the contribution of this part and for further improving the secretion (Sec) performance of LMT signal peptide, we supplemented some knowledge about Sec pathway-dependent secretion process and identified the sequence features of LMT signal peptide, which are closely related to the interactions with elements in the Sec pathway. Based on these, we then conducted targeted mutations in LMT sequence to investigate the influence of specific residues on secretion of recombinant proteins.

1. Basic of Sec secretion pathway and sequence features of LMT signal peptide

Figure 1 Sec pathway-dependent secretion process and the corresponding sequence structures of signal peptides. (A) Secretion of recombinant proteins through Sec pathway. (B) The typical sequence features of signal peptide can be identified in the sequence of LMT.

The secretion of most proteins is primarily achieved through the General Secretion pathway (Figure 1A) (1) . The typical sequence structure of signal peptides that guide secretion via this pathway is illustrated in Figure 1B. In this pathway, proteins first bind to SecB via the positively charged N-region of their signal peptides while SecB inhibits premature folding of the protein. Subsequently, SecA recognizes the hydrophobic H-region of the signal peptide and translocates the protein to the cell membrane, in which the SecYEG translocon acts as a channel, guiding the protein to the periplasmic space. After the signal peptidase (SPase) recognizes and cleaves at the cleavage site (AXA) in the C-region of signal peptides, the protein then correctly folds in the periplasm (2).

Figure 2 Probe the key secA and secB genes in E. coli DH5α via colony PCR. DNA gel electrophoresis of colony PCR products to probe secA (A) and secB (B). Target bands, 2706 bp and 468 bp, can be observed at the position around 3000 bp and 500 bp, respectively. Specific primers were designed according to the genome sequence of DH5α (GCF_002899475.1).

The LMT signal peptide we discovered has the typical sequence features of signal peptides, which can be divided into 3 regions described above as well (Figure 1B). Thus, we turned to investigate the influence of some specific residues in this signal peptide on protein (here, sfGFP) secretion. We first confirmed the presence of the key genes secA and secB of the Sec pathway in the chassis E. coli DH5α via colony PCR (Figure 2), which set the foundation of downstream works and characterizations. Two specific variants of the N-terminal positively charged region (presumed to be the SecB binding region) and the C-terminal AXA cleavage site (SPase recognition site) were then constructed, namely LMT Δ2-4 and LMT A19V, respectively (Figure 3A). Gibson Assembly was performed to construct the expression module (BBa_25G6VM6I and BBa_25CWGZSX, respectively) of these variants at pSB1C3 vector and positive transformants were selected and confirmed by colony PCR (Figure 3B) and sequencing.

Figure 3 Construction of the expression modules of LMT Δ2-4 and A19V. (A)Sequence alignments of LMT and two variants. Mutated sites were colored in black. (B) DNA gel electrophoresis of the colony PCR products of expression modules of LMT Δ2-4 and A19V fused with sfgfp at pSB1C3 in DH5α. Target bands, 1383 bp and 1374 bp, can be observed at the position between 1500 bp and 1000 bp, respectively.

After incubation, the fluorescence intensity of the culture and the supernatant after centrifugation as well as the OD₆₀₀ of each group were monitored as time progressed. Our results showed that the fluorescence intensity of the two variants, no matter for the culture or supernatant, was much lower than that of the wild-type (WT) LMT group (Figure 4B and 4C), although no significant growth defect due to the modifications was observed (Figure 4A). Both the reduction of positive charges in N-region (LMT Δ2-4) and mutation on SPase cleavage site (A19V) are “lethal” to the function of LMT signal peptide, which highlights the importance of critical interactions between transportation machinery of host and signal peptide during secretion process. In addition, the results in Figure 4 have given a lesson about engineering signal peptide in bacteria that the mutations of some residues are intolerable, which might even disturb the normal folding of cargo proteins.

Figure 4 Characterization of secretion performance of LMT and two variants. The OD₆₀₀ (A), fluorescence intensity of culture (B) and supernatant (C) was measured as time progressed. Relative fluorescence units (RFU) was defined as the fluorescence of bacterial culture subtracted the background fluorescence of growth media.

2. Targeted mutations and screening of high-performance variants

Based on the identified features of LMT sequence, and inspired by the high-throughput experiments and analysis by Gresso et al. (3) , we turned to engineer the sequence of LMT through targeted mutations on H-region and C-region and tried to screen variant(s) of high-performance on secretion. After verifying the secretion pathway mediated by the LMT sequence and its functional domain partition, we performed precise point mutations and structural optimizations with the goal of obtaining LMT signal peptide variants with better secretion performance.

Figure 5 Targeted mutations in H-region of LMT signal peptide. (A) Sequence alignments of LMT and 4 variants. Mutated sites were colored in black. (B) DNA gel electrophoresis of the colony PCR products of expression modules of LMT variants fused with sfgfp at pSB1C3 in DH5α. Target bands (1383 bp) can be observed at the position between 2000 bp and 1000 bp. (C) Each variant's fluorescence intensity of supernatant of 10 h was normalized to LMT WT, for the first characterization. (D) Fluorescence intensity of supernatant of M13L variant with LMT WT was measured as time progressed, for the second characterization.

It was reported that increasing the hydrophobicity of the H-region core can enhance secretion efficiency (1-3). Thus, we designed 4 LMT variants: G12L, M13L, C14L, and A15L (Figure 5A) and constructed corresponding expression modules (Figure 5B) using the same method mentioned above. For the first characterization, two variants, M13L and C14L showed higher fluorescence intensity of the supernatant than LMT (Figure 5C). Given the poor reproducibility observed in C14L group, we evaluated M13L only for the second time of characterization with LMT as control. However, as time progressed, the M13L variant presented a little bit lower fluorescence intensity of supernatant than the wild-type (Figure 5D), inconsistent with the previous observation. Taken together, the strategy of increasing the hydrophobicity of the H-region core might not be feasible and robust for the case of LMT signal peptide.

Figure 6 Construction of the expression modules of the variants increasing the number of glutamine residues in the C-region. (A) Sequence alignments of LMT and four variants. Mutated sites were colored in black. (B) DNA gel electrophoresis of the colony PCR products of four variants' expression modules fused with sfgfp at pSB1C3 in DH5α. Target bands (1383 bp) can be observed at the position between 1500 bp and 1000 bp.

Demonstrated through high-throughput experiments, the number of glutamine residues (Q) in the C-region of most signal peptides is positively correlated with secretion efficiency (3). Therefore, we designed to increase the number of glutamine residues in the C-region of LMT and constructed four C-terminal point mutants (Figure 6), named LMT A15Q, LMT S16Q, LMT A17Q, and LMT F18Q. In characterization, none of the variants impaired the cell growth (Figure 7A). However, even though some variants presented higher culture fluorescence intensity than the wild-type LMT (Figure 7B), all variants showed much lower fluorescence intensity of supernatant than LMT (Figure 7C), which means no improvements of these variants in secretion performance. Especially, A17Q mutation disrupts the AXA cleavage site like A19V variant, thus resulting in the same “abortive” phenomenon of fluorescence intensity.

Figure 7 Characterization of secretion performance of LMT and the variants increasing the number of glutamine residues in the C-region. The OD₆₀₀ (A), fluorescence intensity of culture (B) and supernatant (C) was measured as time progressed.

With the failure experience of increasing the number of glutamine residues in the C-region, we then turned to focus on the key factor for SPase cleavage efficiency-the composition of residues in C-region (3). This time, we designed to increase the number of alanine residues in LMT and constructed three C-terminal point mutants, named LMT M13A, LMT C14A, and LMT F18A (Figure 8A and 8B) . The LMT C14A presented outstanding performance on protein secretion as time progressed (Figure 8C). The results of characterization for the second time showed that this variant has a better secretion performance when compared to the wild-type LMT signal peptide (Figure 8D), which indicates that more recombinant proteins can be transported outside the bacterial cell due to the C14A mutation.

Figure 8 Increase the number of alanine residues in LMT. (A) Sequence alignments of LMT and 3 variants. Mutated sites were colored in black. (B) DNA gel electrophoresis of the colony PCR products of expression modules of LMT variants fused with sfgfp at pSB1C3 in DH5α. Target bands (1383 bp) can be observed at the position between 1500 bp and 1000 bp. (C) Fluorescence intensity of the supernatant was measured as time progressed. (D) Fluorescence intensity (at 10 h) of culture and supernatant of C14A and LMT WT were compared, for the second time of characterization. p-value: 0.0008 (***) and 0.0098 (**).

Summary

For the contribution, we supplemented the knowledge about Sec pathway-dependent secretion process and identified the sequence features of LMT signal peptide, which are closely related to the interactions with elements in the Sec pathway. We then conducted several rounds of targeted mutations for the purpose of screening higher-performance variant(s) that can be used to secrete more recombinant proteins produced by the microbial cell factories. Finally, we obtained an outstanding variant LMT C14A, which has been further applied in secreting chitosanase for food preservation (see Proof of Concept for details) . The new information and data have been added to the page of previous part BBa_K3739010 and we hope that the knowledge and engineering process about LMT signal peptide will IGNITE other teams even the SynBio community, since the protein secretion is a promising approach for real-world applications.

References

1. Kaushik, S., He, H. & Dalbey, R. E. Bacterial Signal Peptides- Navigating the Journey of Proteins. Front. Physiol. 13(2022).
2. Owji, H., Nezafat, N., Negahdaripour, M., Hajiebrahimi, A. & Ghasemi, Y. A comprehensive review of signal peptides: Structure, roles, and applications. Euro. J. Cell Biol. 97, 422-441 (2018).
3. S. Grasso, Signal Peptide Efficiency: From High-Throughput Data to Prediction and Explanation. ACS Synth. Biol. 12, 390-404 (2023).