Part:BBa_K4652010

T7-RBS-SpyTag-PCLase1-SpyCatcher-Tr

Polycaprolactone (PCL) is a biodegradable plastic material that has received FDA approval. It's extensively used in various applications such as tissue engineering, drug delivery, food packaging, and agricultural coverings¹. Typically, products made from PCL take 2-3 years to decompose. As a result, there's a growing interest in identifying novel enzymes that can degrade PCL or in optimizing those that are currently available commercially². However, most of them can’t tolerate at extremely high temperatures, including the boiling temperatures encountered during the thermoforming process of shaping PCL plastic.

In Prof. Fan Li's laboratory, novel PCL-degrading enzymes, PCLase I and PCLase II, were identified and purified from Pseudomonas hydrolytica³. This discovery was made possible by cultivating the bacteria in a PCL-emulsified medium. The team conducted an in-depth study of the PCLase enzymes, examining aspects such as enzyme activity, the influence of pH and temperature, substrate specificity, degradation products, as well as the associated gene sequences and protein structures. Learned with this comprehensive data and the enzymes' impressive PCL-degrading efficiency, our aim is to enhance their thermostability.

SpyRing cyclization technique to enhance enzyme thermal resilience was clarified by Dr. Mark Howarth’s team⁴. SpyRing harbors genetically modified SpyTag (13 amino acids) on the N-terminus and SpyCatcher (12kDa) on the C-terminus on the protein of interest. This context spontaneously reacts together through an irreversible isopeptide bond. SpyRing cyclization was demonstrated successfully to increase stress resilience of β-lactamase and some industrially important enzymes. With this synthetic biology tool, we plan to employ SpyRing cyclization techniques and expect to make PCLase thermal resistent, as demonstrated in our work with the SpyTag-GFP-SpyCatcher construct (Part:BBa_K4652002).

PLASMID CONSTRUCTION

PCLase I and PCLase II gene sequences with N-terminal SpyTag and C-terminal SpyCatcher were synthesized by Integrated DNA Technologies, Inc. (IDT) and then cloned into pSB1C3, respectively (SpyTag-PCLase1-SpyCatcher, Part:BBa_K4652008; SpyTag-PCLase2-SpyCatcher, Part:BBa_K4652012). Then, the parts were connected with a T7 promoter (Part:BBa_K1833999), a strong RBS (Part:BBa_B0030), and a double terminator (Part:BBa_B0015). The final construct was verified using colony PCR (Figure 1) and further validated through DNA sequencing. These resultant constructs were designated as T7-SpyTag-PCLase1-SpyCatcher (Part:BBa_K4652010) and T7-SpyTag-PCLase2-SpyCatcher (Part:BBa_K4652013), respectively.





Figure 1. Verification of T7-SpyTag-PCLase1-SpyCatcher (Part:BBa_K4652010) and T7-SpyTag-PCLase2-SpyCatcher (Part:BBa_K4652013) using colony PCR. PCR was performed using a CmR-specific forward primer from the vector and a PCLase-specific reverse primer from the gene. The expected size of the amplified DNA fragments is 2395 bp for T7-SpyTag-PCLase1-SpyCatcher and 2561 bp for T7-SpyTag-PCLase2-SpyCatcher, respectively. The rightmost lane displays a 1 kb DNA ladder. The numbers correspond to selected colonies, with one control (lane 7) derived from a mock pick from a clear zone on the plate.

PCL-DEGRADING LIPASE COMPARISON

To compare the lipase activities of PCLase I, PCLase II, and other commercially available PCR-degrading enzymes such as BCLA10 and CALB11, we conducted a pNPB assay. In this assay, a potential lipase breaks down the ester bond of p-nitrophenylbutyrate (pNPB), producing p-Nitrophenol. The concentration of p-Nitrophenol can be measured at 405nm, and these measurements are corresponding to the lipase activity.

Lysates from E. coli BL21, which carried the T7 promoter-driven expression plasmid with the indicated genes in the same context, were collected after being induced with 0.3 mM IPTG at 25°C for 20 hours. The lipase activities within these lysates were assessed using the pNPB assay10. As shown in Figure 2, PCLase I exhibited the significantly highest readings at 405 nm. This suggests that under our experimental conditions, PCLase I is the most effective lipase, demonstrating potential activity in decomposing PCL through the hydrolysis of the ester bonds between polymers. Consequently, we chose to investigate the characteristics of PCLase I (hereafter referred to as PCLase for short) in terms of its thermostability, protein structure, PCL degradation capability, and its potential use in real-world products.

Figure 2. Comparison between lipase activities of BCLA, CALB, PCLase I, and PCLase II using pNPB assay. E. coli BL21 was transformed using the indicated T7 promoter-driven gene expression plasmid. The bacteria were induced by 0.3 mM of IPTG at 25°C for 20 hours. Subsequently, the lysates were harvested using 0.1 mm Disruptor Beads (Scientific Industries, Inc). A 20 µL aliquot of these lysates was combined with 175 µL of Tris-HCl buffer (20mM, pH=8) and 5 µL of pNPB (40 mM dissolved in 2-methyl-2-butanol). Lipase activity was read at 405 nm based on p-Nitrophenol production. The obtained readings were normalized with the OD600 values at the time of bacterial lysate collection.

THERMOSTABILITY

To assess the thermostability of PCLase, the bacterial lysates, prepared as described earlier, were exposed to a 100°C treatment followed by a pNPB assay to measure lipase activity. PCLase activity decreased to 20% in 5 min but remained stable for up to 30 minutes (Figure 3). The phenomena were consistent with the observations made in lysates containing cyclized GFP (Part:BBa_K4652002)5, but, notably, PCLase exhibited much more prolonged heat resistance at the boiling temperature (i.e., cyclized GFP is tolerate for 5 min, while cyclized PLCase is for 30 min).

Figure 3. Thermostability of PCLase lipase activity. E. coli BL21 was transformed with T7-SpyTag-PCLase1-SpyCatcher (Part:BBa_K4652010). The bacteria were induced by 0.3 mM of IPTG at 25°C for 20 hours. Subsequently, the lysates were harvested using 0.1 mm Disruptor Beads (Scientific Industries, Inc). After treated at 100°C for the indicated time, a 20 µL aliquot of these lysates was combined with 175 µL of Tris-HCl buffer (20mM, pH=8) and 5 µL of pNPB (40 mM dissolved in 2-methyl-2-butanol). Lipase activity was read at 405 nm based on p-Nitrophenol production. All values were divided by the average of the untreated control, with the resulting ratio representing the lipase activity fold change.

PROTEIN STRUCTURE & ACTIVITY

The mechanism in SpyRing for the spontaneous cyclization reaction involves Asp7 on the SpyTag at the N-terminus forming double hydrogen bonds with Glu77 on the SpyCatcher at the C-terminus. This interaction strengthens the creation of an irreversible isopeptide bond between Asp7 on SpyTag and Lys31 on SpyCatcher. As a result, the protein is seamlessly cyclized, enhancing its resistance to chemical, thermal, or enzymatic degradation12.

To verify the protein structure and its correlation to thermal tolerance, in a similar approach on T7-RBS-SpyTag (D7A)-GFP-SpyCatcher-Tr (Part:BBa_K4652003)13, we engineered a SpyTag mutation of PCLase and designated as T7-RBS-SpyTag (D7A)-PCLase1-SpyCatcher-Tr (Part:BBa_K4652011)14. The lysates, as collected previously, were subject to SDS-PAGE and Coomassie Blue staining. In Figure 4, the result showed a distinct band corresponding to the linear form of SpyTag (D7A)-PCLase1-SpyCatcher, with an expected size of 46.53 kDa. However, the lysates from SpyTag-PCLase1-SpyCatcher displayed somewhat fuzzy bands representing incomplete cyclized or fully cyclized forms. This ambiguity might arise from aggregation or polymerization due to isopeptide bond formation15. It's worth noting that the presence of incompletely cyclized PCLase could partially account for the reduced lipase activity after heating observed in the experiment in Figure 3.

Figure 4. Protein structure analysis of cyclized form and linear form of SpyTag-PCLase1-SpyCatcher. 15 µg of IPTG-induced lysates were run on a 4-12% gradient SDS-PAGE (BIO-RAD) and stained with standard Coomassie Blue. The AceColor Prestained Protein Marker (10 - 180 kDa) was used for size reference. Lane 1 refers to the linear proteins derived from lysates of SpyTag (D7A)-PCLase1-SpyCatcher (BBa_K4652011). Lane 2 refers to the cyclized proteins derived from lysates of SpyTag-PCLase1-SpyCatcher (BBa_K4652010). The predicted molecular weight for the SpyTag-PCLase1-SpyCatcher protein is 46.53 kDa.

In addition, we compared the thermostabilities between lipase activities in lysates with linear PCLase (SpyTag (D7A)-PCLase1-SpyCatcher) and cyclized PCLase (SpyTag-PCLase1-SpyCatcher) using the pNPB assay. After being treated at temperatures ranging from 70°C to 100°C for 10 min, the cyclized lipase retained higher activity compared to the linear form of PCLase (Figure 5). It maintained over 50% activity up to 90°C and retained up to 20% at 100°C. It's worth exploring whether purifying and concentrating the fully cyclized PCLase could enhance the lipase activity in the lysates for thermal testing. Taken together, the experimental data demonstrated successfully making a thermostable PCLase by cyclization through SpyRing technique.

Figure 5. Thermostability of PCLase lipase activities between cyclized PCLase (SpyTag-PCLase1-SpyCatcher, BBa_K4652010) and linear PCLase (SpyTag (D7A)-PCLase1-SpyCatcher, BBa_K4652011). E. coli BL21 was transformed with the indicated plasmid and then was induced by 0.3 mM of IPTG at 25°C for 20 hours. Subsequently, the lysates were harvested using 0.1 mm Disruptor Beads (Scientific Industries, Inc). After treated at 100°C for the indicated time, a 20 µL aliquot of these lysates was combined with 175 µL of Tris-HCl buffer (20mM, pH=8) and 5 µL of pNPB (40 mM dissolved in 2-methyl-2-butanol). Lipase activity was read at 405 nm based on p-Nitrophenol production. All values were divided by the average of the untreated control, with the resulting ratio representing the lipase activity fold change.

REFERENCE

1. Ilyas RA, Zuhri MYM, Norrrahim MNF, Misenan MSM, Jenol MA, Samsudin SA, Nurazzi NM, Asyraf MRM, Supian ABM, Bangar SP, Nadlene R, Sharma S, Omran AAB. Natural Fiber-Reinforced Polycaprolactone Green and Hybrid Biocomposites for Various Advanced Applications. Polymers (Basel). 2022 Jan 3;14(1):182. doi: 10.3390/polym14010182. PMID: 35012203; PMCID: PMC8747341.
2. Urbanek AK, Mirończuk AM, García-Martín A, Saborido A, de la Mata I, Arroyo M. Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics. Biochim Biophys Acta Proteins Proteom. 2020 Feb;1868(2):140315. doi: 10.1016/j.bbapap.2019.140315. Epub 2019 Nov 16. PMID: 31740410.
3. Li L, Lin X, Bao J, Xia H, Li F. Two Extracellular Poly(ε-caprolactone)-Degrading Enzymes From Pseudomonas hydrolytica sp. DSWY01T: Purification, Characterization, and Gene Analysis. Front Bioeng Biotechnol. 2022 Mar 18;10:835847. doi: 10.3389/fbioe.2022.835847. PMID: 35372294; PMCID: PMC8971842.
4. Schoene C, Bennett SP, Howarth M. SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Resilience. Methods Enzymol. 2016;580:149-67. doi: 10.1016/bs.mie.2016.05.004. Epub 2016 Jun 16. PMID: 27586332.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]