Part:BBa_K4949006

RPA1511

RPA1511 is a carboxyl esterase originally identified in Rhodopseudomonas palustris. RPA1511 has been shown to degrade a variety of polymers, including PLA, with an optimal temperature range of 50-60℃ (Hajighasemi et al. 2016). Our team modified the RPA1511 genetic sequence to include the lpp-OmpA anchor (BBa_K2302003) in order to characterize the surface-display mechanism.

Reference
Hajighasemi, M., Nocek, B. P., Tchigvintsev, A., Brown, G., Flick, R., Xu, X., Cui, H., Hai, T., Joachimiak, A., Golyshin, P. N., Savchenko, A., Edwards, E. A., & Yakunin, A. F. (2016). Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases. Biomacromolecules, 17(6), 2027–2039. https://doi.org/10.1021/acs.biomac.6b00223

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 279
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 105
  Illegal NgoMIV site found at 114
  Illegal NgoMIV site found at 340
  Illegal NgoMIV site found at 514
  Illegal NgoMIV site found at 574
  Illegal AgeI site found at 139
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 817
  

Whole-cell catalysis is performed by mixing cells expressing Lpp-OmpA-Enzyme constructs with the substrate (either NPO or pNOB) in PBS (0.137 M NaCl, 2.7 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, pH 7.4). Cell suspensions used in whole cell-catalysis experiments are normalized to have OD600 of 1.0 in 1 mL (i.e. all samples contain ~109 cells/mL). Control cases are prepared by diluting substrates in PBS. These reactions are then incubated at room temperature. After a set interval (every 5 minutes), 100 µL of the mixture is aliquoted into a clean tube, centrifuge to pellet all cells, and then 2 µL sample is measured using a nanodrop. Absorbance at 410 nm was measured to detect the presence of 4-nitrophenyl, a product of esterase activity. When the absorbance is above 1, samples are diluted until the absorbance is below 1 for measurement and then, calculate the final value, taking the dilution factor into account.The hypothesis is if cells are decorated with enzymes on the outer membrane, there would be a high absorbance of 410 nm.

Figure 1. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3) + RPA1511(ΔLpp), bacteria with a Lpp deficient plasmid, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD₆₀₀=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 2. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3) + RPA1511(ΔLpp), bacteria with a Lpp deficient plasmid, were washed and resuspended in PBS with 100µM pNOB with a cell density normalized to OD₆₀₀=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 1 and 2 support a functional Lpp-OmpA-RPA1511 surface display mechanism. The absorbance at 410 nm increases for treatment reactions with cells containing enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This indicates that our whole cell catalysis approach works. The absorbance at 410 nm for treatment cases containing cells with Lpp-OmpA-RPA1511 increases faster than cases containing cells with Enzymes. These observations suggest two conclusions:

1) Cells with Lpp-OmpA-RPA1511 have enzymes anchoring on the outer membrane and thus can readily access substrates, which enhance catalysis rate and result in a higher rate of increase in absorbance at 410 nm.

2) Cells with Enzymes without anchors can also catalyze cleavage of substrate, unlike our expectation. This suggests that substrates can cross cell membranes to reach enzymes. As cells are pellet before measurement, it also suggests that the product can then cross the membrane again and diffuse into the solution. This hypothesis is further supported by the general higher rate of increase of absorbance at 410 nm when pNOB is used instead of NPO. pNOB has a shorter carbon chain (4) in comparison to NPO (8) and thus, is expected to cross the membrane more readily.

Figure 3. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression, E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3)ΔLpp +RPA1511, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD₆₀₀=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant was measured at 410 nm.

Figure 4. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression, E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3)ΔLpp + RPA1511, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM pNOB with a cell density normalized to OD₆₀₀=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

BL21(DE3) Δlpp are E. coli strains that were genetically modified to remove their endogenous genes with Lpp signal. By removing endogenous genes with an Lpp signal, there will be less competition with our Lpp-OmpA-Est119 for secretion machinery and thus, more enzymes will be exported and decorated on the outer membrane. Figure 3 and 4 show that in all cases for Lpp-OmpA-RPA1511, the absorbance at 410 nm increases for treatment reactions with cells decorated with enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This is an indication that enzymes, which are anchored on the outer cell membrane, catalyze the cleavage of substrate into 4-nitrophenol. The rate of increase in absorbance at 410 nm is higher for pNOB substrate than NPO substrate in both enzymes. Furthermore, there is no significant difference between two different cell lines (BL21(DE3) and BL21(DE3) ΔLpp). This indicates the competition between lpp-OmpA-RPA1511 and endogenous genes with Lpp secretion signal for secretion machinery in cells is likely to be not the rate-determining step.