Difference between revisions of "Part:BBa K5136066"

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LMT

Biology

LMT

Lytic murein transglycosylase (LMT) is an enzyme that is able to degrade murein, a component of bacterial cell walls. There are two kinds of LMTs existing in E.coli: the membrane-binding one and the soluble one. The LMT signal peptide (named LMT in our parts) is from the LMT homolog, which can facilitate the secretion of the fused protein out of E.coli.

Building on the XMU-China 2022 (OMEGA) and 2023 (NAIADS) projects, we have further refined our research on signal peptides. In this year's study, we designed and compared the effects of eight different signal peptides, finding that the LMT signal peptide demonstrated superior performance in promoting protein excretion.

GGG linker

(GGGGS)₃ is commonly used in protein engineering because of its flexibility and resistance to proteases. Therefore, we selected (GGGGS)₃ flexible linker (1) as a short peptide to connect LMT and T7 lysozyme 119G in our autolytic system.

T7 lysozyme 119G

T7 lysozyme is a small molecular weight protein in bacteriophage T7, primarily functioning to degrade the cell wall of host bacteria during phage infection, facilitating the injection of phage DNA or the release of newly formed phage particles. In molecular biology research, it is widely used for the efficient lysis of Escherichia coli cells (2, 3). Moreover, it has been reported that higher levels of lysozyme provided by plasmids pLysE or pLysH can reduce the full induction activity of T7 RNA polymerase, allowing induced cells to continue growing indefinitely while producing non-toxic target proteins (3). This feature not only highlights the excellence of T7 lysozyme in promoting cell lysis but also makes it extremely useful in preparing cell extracts for protein purification. Notably, T7 lysozyme 119G sequence was found in pLysS (4), and it differs from the T7 lysozyme 119V sequence selected from the UniProt database (5), with a variation at the 119th amino acid position.

SSrA

The SsrA is a small peptide tag used to mark proteins for protein degradation. When fused with the target protein, SsrA could guide it to specific proteases, such as the ClpXP and ClpAP complexes, for degradation (6).

Usage and Design

Performance comparison of multiple signal peptides

To characterize the secretion efficiency of LMT, the signal peptide was fused to the N-terminus of sfGFP via a flexible linker (GGGGS)₃. This fusion was expressed under the control of the T7 promoter on the pET-28a(+) vector in E. coli BL21(DE3).

Verifying the function of LMT-GGG Iinker-T7 Lysozyme 119G-SsrA mediated autolytic system

In our design, we aim to induce cell autolysis to release enzymes into the supernatant, simplifying the complex protein purification process. By utilizing the dual-pathway signal peptide LMT, we direct T7 lysozyme to the peptidoglycan layer, enhancing cell lysis. Additionally, the SsrA tag is fused to the C-terminus of T7 lysozyme to ensure the degradation of any leaked T7 lysozyme, minimizing system cytotoxicity and ensuring the proper accumulation of the target enzyme in the correct location (7).

This composite part we constructed aims to express the LMT-T7 lysozyme-SsrA mediated autolytic system (LLSA), which includes T7 lysozyme 119G, under the control of an L-arabinose inducible promoter. To validate the efficiency of the LLSA system, we used sfGFP as a reporter.

Characterization

Performance comparison of multiple signal peptides

Agarose Gel Electrophoresis (AGE)

When constructing this circuit of composite part BBa_K5136066, colony PCR and gene sequencing were used to verify that the construction was correct. Target bands (1083 bp) can be observed at the position between 1000 and 2000 bp (Figure 3).

Comparative Tests of Performance of Multiple Signal Peptides

The LMT sequence has been identified as a signal peptide capable of directing recombinant proteins out of bacterial cells (XMU-China 2021). Given the variety of engineered signal peptides and corresponding translocation systems developed for the secretory production of heterologous proteins in E. coli(8), we sought to compare the performance of the LMT sequence against other commonly used signal peptides.

Specifically, several signal peptides or translocation systems associated with the Type II Secretion System (T2SS)—including both the Sec-dependent system and the TwinArginine Translocation (TAT) system—as well as the Type I Secretion System (T1SS), were selected and fused to the reporter protein sfGFP. The sequences of the various signal peptides are provided in Table 1. Most of the secretion systems utilized in this comparative analysis contain critical N-terminal regions necessary for recognition and function within the translocation machinery (9), which resulted in N-terminal fusions with sfGFP. However, the HlyABD secretion system, which belongs to T1SS, requires the C-terminal sequence of HlyA, as well as the heterologous expression of HlyB and HlyD as assistants (10, 11). Therefore, a C-terminal fusion of sfGFP with HlyA was constructed. All signal peptide-sfGFP fusions were expressed under the control of the T7 promoter on the pET-28a(+) vector in E. coli BL21(DE3) for parallel testing. Following induction, we monitored the fluorescence intensity of the culture, the supernatant after centrifugation, and the OD600 of each group over time. Secretion efficiency was calculated at each sampling point as the ratio of fluorescence intensity of supernatant to culture.

Our results revealed that both OsmY- and HlyA-mediated secretion, with the assistance of HlyB and HlyD, exhibited higher secretion efficiencies than any other signal peptides, including the newly discovered LMT sequence, which ranked third. YebF also performed well in the comparison (Figure 4A). Most of the other signal peptides exhibited secretion efficiencies below 2.5%. These findings demonstrate the variability in secretion efficiencies among different signal peptides, despite the long-term usage of some of them (8).

Interestingly, certain groups showed lower calculated secretion efficiencies than the sfGFP control group (which lacked a signal peptide, No SP). This may be due to the rapid growth and high levels of cytoplasmic sfGFP expression driven by the strong T7 promoter in E. coli BL21(DE3) (12). The normalized fluorescence intensity of the sfGFP-only (No SP) culture was much higher than that of sfGFP fusions with signal peptides, suggesting that even minor leakage due to cell growth or lysis contributed significantly to the fluorescence intensity in the supernatant (Figure 1B). In the case of the LMT signal peptide, despite producing only half the total sfGFP as the no-signal-peptide group (No SP), the fluorescence intensity in the supernatant was comparable to that of the control group and significantly higher than that of any other signal peptide after 10 hours of induction. This highlights the LMT sequence′s efficiency in exporting recombinant proteins out of the bacterial cells.

While OsmY- and HlyA-mediated secretion showed the highest secretion efficiencies, they also impaired cell growth (reflected by the lowest OD600 values) and reduced the normalized fluorescence intensity in the culture (Figure 4B). This indicates that overexpression of these systems places a metabolic burden on the host cells, possibly explaining the larger error bars associated with their calculated secretion efficiencies (Figure 4A). Additionally, the normalized fluorescence intensities in all groups with signal peptides were reduced (Figure 4B), potentially due to adverse effects on sfGFP folding caused by the fusion with signal peptides (13).

Classic signal peptides such as PelB and TorA, which direct recombinant proteins to the periplasmic space, exhibited relatively low efficiency for extracellular translocation (Figure 4A) (14). Similarly, OmpA, another well-known signal peptide, showed poor efficiency for recombinant protein secretion, unlike YebF and OsmY, which also mediate extracellular expression (15). Notably, the artificial intelligence-generated signal peptide AIgen, which functions efficiently in Bacillus subtilis, performed poorly in exporting recombinant proteins from E. coli (Figure 4A) (16). However, the OD600 values for the AIgen group suggested no significant growth burden (Figure 4B).

In summary, our self-discovered LMT signal peptide showed a slight growth defect but achieved considerable secretion efficiency for the extracellular expression of recombinant proteins, making it a competitive alternative compared to other commonly used signal peptides in E. coli.

Verifying the function of LMT-GGG Iinker-T7 Lysozyme 119G-SsrA mediated autolytic system

Agarose gel electrophoresis (AGE)

The composite part BBa_K5136223 constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into E. coli DH10β. The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (2278 bp) can be observed at the position around 3000 bp. (Figure 5).

sfGFP Release Efficiency Determination

After co-transforming BBa_I0500-BBa_B0034-LMT-GGG linker-T7 lysozyme 119G-SsrA-BBa_B0015_pSB1C3 and sfGFP_pET-28a(+) into E. coli BL21 (DE3), the cultures were grown overnight in LB medium containing corresponding antibiotics. The cultures were diluted and grown to OD600 0.6-0.8, followed by the addition of 0.5 mM IPTG to induce sfGFP expression at 18°C. After 10 hours, 0.25% L-arabinose was added to activate the autolytic system. The total fluorescence intensity was measured after 16 hours of expression of the induced autolysis system, and after centrifugation, the fluorescence intensity of the supernatant was measured too. The ratio of the fluorescence intensity of the culture and supernatant was used to assess the lysis efficiency of LLSA system.

We have proved that some CYP199A4 mutants showed stronger deinking, and LMT showed a good secretion effect. So, we try to verify the deinking efficiency of CYP199A4 mutants secreted to the supernatant by the LMT. The engineered bacteria were cultured at 25°C, and the supernatant culture was taken at 12 h, 18 h, 24 h, and 36 h, respectively, using SDS-PAGE to demonstrate that the fusion protein could be successfully secreted into the supernatant. Gray scale value analysis was performed on the bands, proving that the concentration of LMT-CYP199A4 T253E in the culture supernatant gradually increased with time (Figure 7A). At the same time, the supernatant from the culture in 36 hours was used for the pulp deinking experiment (see SOP for more details), and the results are shown in Figure 7B. As shown in the picture from the microscope, LMT-CYP199A4 T253E in the supernatant showed a perfect deinking effect. The above results showed that LMT signal peptide could secrete CYP199A4 T253E to the extracellular environment continuously, which further exhibits the perfect performance in removing the ink from the pulp.

The effect of different promoters on the function of LMT signal peptides

Construction of gene circuit

We constructed circuits driven by promoters of different strengths (J23100, J23103, J23104, J23106, J23110, J23114), each containing RiboJ-B0034-LMT-linker-sfgfp-B0010. The composite part J23104-RiboJ-B0034-LMT-linker-sfgfp-B0010 (BBa_K5136202 ) was used as an example, and the rest results of colony PCR are detailed in BBa_K5136200,BBa_K5136201,BBa_K5136203,BBa_K5136204,BBa_K5136205.

Characterization of secretion performance

By measuring the fluorescence intensity in the supernatant of each circuit, we can quantitatively analyze the secretion of the LMT signal peptide under different promoter strengths and thus evaluate its performance (Figure 9).

We find that this follows index kinetics, which is consistent with the T7-mediated secretion. As shown in Figure 10A, secretion efficiency is affected by many factors, but not positively related to promoter strength. The R square (R²) indicated that the fitting effect was better, and J23104 (0.9786) was the highest (Figure 3B).

To ensure that these results reflect actual secretion rather than differences in cell growth, we measured fluorescence intensity data in 30 h of culture (Figure 11A) and supernatant (Figure 11B) and calculated the secretion efficiency (supernatant/culture) (Figure 11C).

Interestingly, when we compared the secretion efficiency at 30 hours, we found that high secretion efficiency does not mean high protein content in supernatant and culture. Although the secretion efficiency of the weak promoter and the strong promoter were both higher, the weak promoter could fully realize extracellular secretion due to its low efficiency and less protein production by the exosystem, but the amount of protein in supernatant was less. For strong promoters, although the amount of protein secreted is large, the total fluorescence is low, indicating that the metabolic burden is heavy, which affects the survival rate. If the detection continues, very few bacteria will survive. Also, we use a multi-scale model to deconstruct the dynamic secretion process of signal peptides accurately. The experimental results aligned well with the model's predictions (see Model for more details). By trading off, BBa_K5136202 with promoter J23104 has the most appropriate secretion efficiency.

Reference

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2. J. Yun, J. Park, N. Park, S. Kang, S. Ryu, Development of a novel vector system for programmed cell lysis in escherichia coli. J Microbiol Biotechnol 17, 1162-1168 (2007).

3. F. W. Studier, Use of bacteriophage t7 lysozyme to improve an inducible t7 expression system. J Mol Biol 219, 37-44 (1991).

4. https://www.snapgene.com/plasmids/pet_and_duet_vectors_(novagen)/pLysS.

5. https://www.uniprot.org/uniprotkb/P00806/entry.

6. Q. Chai, Z. Wang, S. R. Webb, R. E. Dutch, Y. Wei, The ssra-tag facilitated degradation of an integral membrane protein. Biochemistry 55, 2301-2304 (2016).

7. F. Zhang et al., Development of a bacterial fhud-lysozyme-ssra mediated autolytic (flsa) system for effective release of intracellular products. ACS Synth Biol 12, 196-202 (2023).

8. R. Freudl, Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems. Microb. Cell Fact. 17, 52 (2018).

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10. B. D. Tzschaschel, C. A. Guzmán, K. N. Timmis, V. D. Lorenzo, An Escherichia coli Hemolysin Transport System-based Vector for the Export of Polypeptides: Export of Shiga-like Toxin IIeb Subunit by Salmonella typhimurium aroA. Nat. Biotechnol. 14, 765–769 (1996).

11. L. A. Fernández, I. Sola, L. Enjuanes, V. De Lorenzo, Specific Secretion of Active Single-chain Fv Antibodies into the Supernatants of Escherichia coli Cultures by Use of the Hemolysin System. Appl. Environ. Microbiol. 66, 5024–5029 (2000).

12. S.-I. Tan, I.-S. Ng, New Insight into Plasmid-driven T7 RNA Polymerase in Escherichia coli and Use as a Genetic Amplifier for a Biosensor. ACS Synth. Biol. 9, 613–622 (2020).

13. B. J. Feilmeier, G. Iseminger, D. Schroeder, H. Webber, G. J. Phillips, Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli. J. Bacteriol. 182, 4068–4076 (2000).

14. F. J. M. Mergulhão, D. K. Summers, G. A. Monteiro, Recombinant Protein Secretion in Escherichia coli. Biotechnol. Adv. 23, 177–202 (2005).

15. J. T. Boock, D. Waraho-Zhmayev, D. Mizrachi, M. P. DeLisa, “Beyond the Cytoplasm of Escherichia coli: Localizing Recombinant Proteins Where You Want Them” in Insoluble Proteins, E. García-Fruitós, Ed. (Springer New York, New York, NY, 2015; http://link.springer.com/10.1007/978-1-4939-2205-5_5)vol. 1258 of Methods in Molecular Biology, pp. 79–97.

16. Z. Wu, K. K. Yang, M. J. Liszka, A. Lee, A. Batzilla, D. Wernick, D. P. Weiner, F. H. Arnold, Signal Peptides Generated by Attention-based Neural Networks. ACS Synth. Biol. 9, 2154–2161 (2020)

Sequence and Features