Part:BBa_K5325002

RPA1511_plaA

An extracellular polylactic acid (PLA) depolymerase taken from Rhodopseudomonas palustris. able to digest PLA into oligomers and lactic acid monomers.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 70
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 820
  

Usage and Biology

Poly lactic acid (PLA) depolymerases have been found naturally in several microorganisms and they are able to facilitate digestion of the biopolymer PLA, producing oligomer chains and lactate monomers. This makes the protein a promising option for the clean up of PLA waste in the environment, which is not quite as biodegradable as the plastic was advertised to be and is still capable of persisting and polluting natural habitats. With that in mind, our team designed this part as a candidate for a PLA depolymerase part, originating from Rhodopseudomonas palustris (R. palustris) to allow an engineered bacteria with the part to cleave PLA to its oligomers and monomers, which can then be metabolized by the bacteria.

PLA depolymerases can be categorized into 2 types: Type I (proteases) - specific to cleave poly L-lactic acid (PLLA) - and Type II (lipases/cutinases/esterases) - preferentially cleave poly D-lactic acid (PDLA), with BBa_K5325002 matching the description of Type II as the original protein is an esterase with a preference towards cleaving poly(D,L)-lactate over strictly PLLA or PDLA^[1,2]. While the mechanism of how PLA depolymerase binds to and hydrolyze PLA is still unknown, PLA depolymerase hydrolysis activity is known to increase as temperature approaches the hydrolysis temperature of PLA (>50 °C).

SDS-PAGE and Western Blot Results

Figure 1: Western Blot of cell lysate from S. oneidensis cell with each carrying the empty vector pRL814, BBa_K5325000 (ABO),BBa_K5325001 (Amy),BBa_K5325002 (RPA), and BBa_K5325003 (Pa). All cell lysate samples were treated with DTT to prevent the dimerization of the proteins of interest. Column with the empty vector was used as a negative control for the experiment.

S. oneidensis culture was diluted to OD_600nm=1, then the culture was incubated at 95°C and spun down before the resulting cell lysate was run through the SDS-PAGE gel. Collected SDS-PAGE gel was then visualized using Western Blot with anti-FLAG antibodies to highlight the proteins of interest in the gel. The Western blot result is shown in Figure 1, which showed the resulting protein band size of RPA at 25-30 kDa. While this is below the predicted size of 34 kDa from RPA1511, the resulting band was considered to be within the expected range for the part BBa_K5325002^[2].

Cell Lysate and Cell Supernatant HPLC Analysis

Figure 2: Concentration of lactate and acetate in supernatant culture of S. oneidensis expressing BBa_K5325003 (Pa), empty vector pRL814, BBa_K5325000 (ABO), BBa_K5325002 (RPA), and BBa_K5325001 (Amy). The molecules' concentrations were measured at mM.

Figure 3: Concentration of lactate and acetate in culture with S. oneidensis lysate containing BBa_K5325003(Pa), empty vector pRL814, BBa_K5325000(ABO), BBa_K5325002(RPA), and BBa_K5325001(Amy). The molecules' concentrations were measured at mM.

Five S. oneidensis strains with empty pRL814 vector, BBa_K5325000, BBa_K5325001, BBa_K5325002, and BBa_K5325003, respectively, were grown in LB broth cultures overnight before each culture had the supernatant and cells separated into different new LB broth media. All cells were lysed using the freeze-thaw method in which the cell cultures were subjected to cooling to -20 °C for 30 minutes before being heated to 37 °C for 3 times consecutively. Supernatant cultures and cell lysate cultures for all strains were incubated with low-molecular weight PLA beads at 37 °C and 0.5 mL from supernatant culture and cell lysate each were taken after 24 hours for 5 days. Day 5 culture samples for supernatants and cell lysates of S. oneidensis were analyzed with HPLC for acetate and lactate concentrations. Lactate and acetic acid concentrations in Day 5 sample for the supernatant and cell lysate cultures of S. oneidensis containing the four parts and empty vector are shown in Figure 2 and Figure 3, respectively.

In Figure 2, the supernatants' HPLC result does not show any lactate content for all four parts. As the part was expected to cleave PLA to produce the monomer lactic acid, the result shown here indicate that the part was likely non-functional in the extracellular space. Abundance of acetate in all supernatant samples were likely a result of S. oneidensis metabolism in LB broth cultures, indicating that cells were growing and thus all the cells in the respective samples should have been able to express the parts. On the other hand, in Figure 3, the cell lysates' HPLC result for RPA indicated that the sample have the most lactate out of all samples in the treatment group. However, the amount of lactate produced in the negative control pRL814 empty vector was higher than that of ABO, which should have not been the case as the cell lysate negative control had no way of generating lactate in the LB media. Due to this result, a solid conclusion for the activity of the part BBa_K5325002 in the cell lysate samples cannot be made.

References

1. Kawai, F., Nakadai, K., et al. (2011). Different enantioselectivity of two types of poly(lactic acid) depolymerases toward poly(l-lactic acid) and poly(d-lactic acid). Polym. Degrad. Stab. 96(7):1342-1348.
2. Hajighasemi, M., Nocek, B.P., et al. (2016). Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases. Biomacromolecules. 17(6):2027-2039