Part:BBa_K4015001
Lcp1VH2
The Lcp1VH2 is a strain of latex clearing protein extracted from Gordonia polyisoprenivorans VH2, a species of Gordoniaceae, which is of interest because of its ability to damage natural rubber. Latex clearing protein was first described by Rose (Rose et al., 2005) and the synthesis of Lcp1VH2 into e.coli was first described by Hiessl et. al (Hiessl et al., 2014). The Lcp1VH2 belongs to oxygenate, which can catalyse the extracellular cleavage of poly (cis-1,4-isoprene) through adding two oxygen molecules to the chemical bond where the polyisoprene is attached(Ilcu et al., 2017).
Usage and Biology
In previous studies, we have discovered that lcp1VH2 was usually inserted into the pET23a plasmid and subsequently transferred into the C41 strain for expression. We tried that approach, but the bands of lcp1VH2 were indistinguishable and could not tell whether it was expressed in E. coli C41.
In order to obtain a viable expression system, we tested four expression protocols using existing strains on hand (E.coli C41 and E.coli BL21) with vectors (pet23a and pet28a): E.coli C41 pET23a:: Lcp1VH2, E.coli C41 pET28a:: Lcp1VH2, E. coli BL21 pET23a:: Lcp1VH2, E. coli BL21 pET28a:: Lcp1VH2 and E. coli BL21 pET28a:: cp1VH. coli BL21 pET23a:: Lcp1VH2 and E. coli BL21 pET28a:: Lcp1VH2. as shown in Fig1, we can see that E. coli BL21 pET23a:: Lcp1VH2 is the best system for Lcp1VH2 expression: compared to the control, we can see a clear induction band and the Lcp1VH2 expression and water solubility were excellent.
Fig 1. Optimization of the expression system of Lcp1VH2. SDS-PAGE of crude extracts(C) and soluble fractions(S) . A. Expression of pET23a::Lcp1VH2 and pET28a::Lcp1VH2 in E. coli C41: E. coli C41(1) as control, pET23a:: Lcp1VH2 (2)and pET28a:: Lcp1VH2 (3). B. pET23a:: Lcp1VH2 and expression of pET28a:: Lcp1VH2 in E. coli BL21: E. coli BL21(4) as control, pET28a:: Lcp1VH2 (5) and pET23a:: Lcp1VH2 (6).
Improvement
The team Brasil-USP designed the part LcpK30 (Part: BBa_K1819001) in 2015, which is a 42kDa enzyme extracted from actinomycete Streptomyces sp. strain K30 that can catalyze rubber cleavage at the cis double bonds to multiple products ranging from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid.
In this year, we improved the pre-existing Lcp(LcpK30) by adding new characterization data for it and designing a new Lcp type- Lcp1VH2 ,which is a rubber oxygenase extracted by a Gram positive rubber degrading bacteria named Gordonia Polyisoprenivorans VH2.
Lcp1VH2 has performed better expression and enzyme activity compared to LcpK30.
Better expression
In order to compare LcpK30 and Lcp1VH2, we tested two proteins using E.coli BL21 with plasmid pET23a:: Lcp1VH2 (C.2 & S.2 presented on figure 1) and pET23a:: LcpK30 (C.3 & S.3 presented on figure 1). From the SDS-Page result in fig.1, Lcp1VH2 expressed a clear and distinct band at 42 kDa, while the band trace of LcpK30 at the same position was obviously much weaker in comparison, indicating that its expression was much inferior to that of Lcp1VH2.
Fig. 1 Expression of Lcp1VH2 and LcpK30 when inserted into pET23a plasmid.SDS-PAGE of crude extracts(C) and soluble fractions(S) of E. coli BL21(1) as control, Lcp1VH2 (2) and fusion proteins NusA-Lcp1VH2 (3).
Better enzyme activity
The process for Lcp to degrade rubber requires oxygen consumption. It utilizes the process of β oxidation to break down bonds within polyisoprene. During β oxidation, Lcp adds two oxygen molecules in between the chemical bonds of polyisoprene. The oxygen consumption rate in the sample tube represents the enzyme activity of Lcp1VH2. As shown from the oxygen dissolving results below, the initial dissolved oxygen in the sample is about 8.5 mg/l. After 6 hours, the dissolved oxygen in the sample tube (Supernatant containing Lcp1VH2) creates a downward slope , The value dropped to approximately 0mg/l eventually. In the sample tube containing LcpK30, oxygen was consumed at a slower rate, and after 9.5 hours, oxygen was roughly depleted. However, the dissolved oxygen in the control tube(Supernatant only of BL21) slowly drops to 6mg/l after 24 hours. This indicates that the LCP1VH2 protein has a higher enzymatic activity.
Fig 2. Dissolved oxygen experiment to verify the activity of Lcp1VH2 and LcpK30
Citation:
1. Altenhoff, A. L., Thierbach, S., & Steinbüchel, A. (2020). High yield production of the latex clearing protein from Gordonia polyisoprenivorans VH2 in fed batch fermentations using a recombinant strain of Escherichia coli. Journal of Biotechnology, 309, 92-99.
2. Andler, R., Heger, F., Andreeßen, C., & Steinbüchel, A. (2019). Enhancing the synthesis of latex clearing protein by different cultivation strategies. Journal of Biotechnology, 297, 32-40.
3. Ilcu, L., Röther, W., Birke, J., Brausemann, A., Einsle, O., & Jendrossek, D. (2017). Structural and Functional Analysis of Latex Clearing Protein (Lcp) Provides Insight into the Enzymatic Cleavage of Rubber. Scientific reports, 7(1), 6179. https://doi.org/10.1038/s41598-017-05268-2
4. Hiessl, S., Böse, D., Oetermann, S., Eggers, J., Pietruszka, J., Steinbüchel, A., 2014. Latex clearing protein-an oxygenase cleaving poly(cis-1,4-isoprene) rubber at the cis double bonds. Appl. Environ. Microbiol. 80, 5231–5240.
5. Rose, K., Steinbüchel, A., 2005. Biodegradation of natural rubber and related compounds:
recent insights into a hardly understood catabolic capability of microorganisms. Appl.
Environ. Microbiol. 71, 2803–2812.
6. Rose, K., Tenberge, K.B., Steinbüchel, A., 2005. Identification and characterization of
genes from Streptomyces sp. strain K30 responsible for clear zone formation on natural rubber latex and poly(cis-1,4-isoprene) rubber degradation. Biomacromolecules 6, 180–188.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 508
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 127
Illegal NgoMIV site found at 485 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 112
Illegal SapI.rc site found at 100
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