Engineering Success

Cycle One

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Design: Finding PLA-degradable enzymes and testing their activities

The mechanism of PLA-degrading enzymes involves the hydrolysis of ester bonds in PLA by enzymes such as proteases, lipases, cutinases, and esterases. This process generates soluble products (lactic acid monomers and oligomers) and partially degraded, insoluble PLA residues, which are analyzed by different biochemical and physical methods (Shalem A, Yehezkeli O, Fishman A. Enzymatic degradation of polylactic acid (PLA). Appl Microbiol Biotechnol. 2024 Jul 10;108(1):413. doi: 10.1007/s00253-024-13212-4. PMID: 38985324; PMCID: PMC11236915.)

To investigate the workflow and degradation efficiency of available enzymes, we first considered the source of three commercially available proteases and lipases, whose enzymatic activities have been well characterized. In addition, we found an enzyme that has higher specificity to PLA. We regarded these four genes as the wild type, and we conducted assays on them to collect data on their efficiency of enzymatic PLA degradation.

Build: gene cloning

The genes of Tritirachium album Proteinase K (PK), Bacillus licheniformis Alcalase (AC), Candida antarctica Lipase B (CA), and Amycolatopsis sp. PLA Depolymerase (DP) were synthesized by Integrated DNA Technologies, Inc., with fragment sizes of 1107 bp, 884 bp, 951 bp, and 671 bp, respectively. First, the synthesized DNA fragments were used as templates for PCR to amplify the target genes, as shown in Figure 2 below.

The gene fragments were cloned into pET-28a for high-level protein expression under IPTG control, driven by the T7 promoter. Following ligation and transformation into E. coli DH5α, the plasmids were extracted and verified by PCR using T7 promoter (T7P) and terminator (T7T) primers, as well as by restriction digestion with EcoRI and PstI, as shown in Figure 3.

Test: pNPB and Lactate production with PLA degradation assay

The pET-28a vectors carrying AC, CA, DP or PK were re-transformed into E. coli BL21 for protein induction along with the empty vector as a negative control for assays and GFP/pET-28a as a positive control for gene expression. First, we picked a single colony for each kind of vector in the 6 ml culture of LB broth supplemented with 50 μg/ml of kanamycin. The overnight cultures were 100x diluted to 300 ml of fresh medium. When the OD600 value reached 0.6-0.8, 0.3 mM of IPTG was added for protein induction at 20°C, 180 rpm for 36 hr. The bacterial cultures were washed and centrifuged, then subjected to sonication using Qsonica Q125 Sonicator (Amplitude 40%, Time 15 min, Pulse 15 sec on and 30 sec off). The lysates were collected and filtered through 0.45 μm PES membrane as crude enzyme extracts for functional assays.

pNPB is a fast and reliable measurement for ester bond hydrolysis, which is used to evaluate esterase or lipase activity. For quick understanding of our enzyme activities, the enzymes were subjected to pNPB assay with 1 mM pNPB in 100μl of 20 mM Tris buffer at pH 8 for 10 min at 37°C. The color change to yellow means the hydrolysis of the ester bond of pNPB, which can be measured at OD420 nm, indicating the release of p-nitrophenol and thus enzyme hydrolytic activity.

To examine the activity of PLA degradation, the enzyme lysates (46 μl) were incubated with PLA powder (0.01 g) at 50°C for 30 min. After centrifugation, the lactate in the supernatants was analyzed by the L-Lactate Assay Kit (Sigma-Aldrich). Lactate production was measured at OD587. The results are shown in Figure 4-7.

As shown in the figures, PLA Depolymerase and Proteinase K exhibited higher enzyme activity in pNPB assay, resulting in greater lactate production from PLA degradation. However, lactate production still faces challenges for practical applications. In contrast, in pNPB assay, Alcalase and Candida antarctica Lipase B exhibited lower enzymatic activity and the reduced PLA degradation efficiency compared to the control groups, including the GFP control and vector-only control.

Learn

We found that the currently available enzymes for PLA degradation in the market are not optimized for PLA. Even the specialized Poly(L-Lactic Acid) Depolymerase from the Amycolatopsis or Paenibacillus strain, which has higher specificity to PLA, still does not optimally degrade certain domains within the PLA structure under specific conditions. Therefore, this project aimed to optimize these enzymes by mutation.

Cycle Two

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Design

This study aimed to generate a large library of mutant enzyme-expressing variants and employ the method of auto-induction to screen potential variants. The gene cloning strategy described above was used to express the mutant enzymes. Following this protocol, we then assessed whether mutagenesis improved the efficiency of enzymatic degradation.

Build

We have found eight approaches to random mutagenesis in the paper of Bitesize Bio.

Throughout the study, we carried out GeneMorph II EZClone Domain Mutagenesis Kit for producing the targeted gene’s mutant variants, so we can delve more into the versatility of a protein. First, we selected the targeted genes as the model, and we adopted the Mutazyme II DNA polymerase to run the error-prone PCR. The part of mutant megaprimer synthesis that amplifies the gene fragment and purifies the targeted fragment with mutations.

This is introduced in the paper as a technique for driving amino acid substitutions in proteins, domains, or promoters by mutation introduced into PCR. (GeneMorph II EZClone Domain Mutagenesis Kit Instruction Manual Catalog #200552 Revision E.0)

Soon, the mutated PCR product would serve as the megaprimer and be annealed to the original donor plasmid to run the EzClone reaction. With the specialized enzyme mix is a 2x formation alongside its high-fidelity DNA polymerase to extend the whole plasmid backbone and insert the mutant into the targeted sites, the completeness of other plasmid sites can be maintained. This can prevent the unwanted secondary mutations to occur in the cloning process. To eliminate the parental DNA template, we added the Dpn I restriction enzyme, specific for methylated and hemimethylated DNA, into the reaction to digest the parental DNA template. That means the final product remains only the new plasmid of the random mutagenesis.

As it is carried out, we would transform the mutant plasmid to the competent cell E.coli DH5α to culture. Then into another competent cell, E.coli BL21. Each colony on the agar plate represents an independent mutant variant. All colonies gather and become a complete mutant library as we proceed with the colony pickup.

However, in cycle 1-3(test) , we found that the LB+IPTG system could not meet the needs of high-throughput measurement. Therefore, we later followed our developed measurement system(For details, please see Measurement) and used the ZYM-5052 auto-induction medium to express the protein. The essence of auto-induction medium is the adoption of the mixture of the carbon substrates. (Mehrnoosh Fathi-Roudsari et al.) It initially began with the glucose to provide fasten growth, lactose for inducing the expression, and glycerol for providing the sustained energy source. This design of the gene expression stage can automatically accomplish the induction, and there is no need to add in the IPTG inducer, which would result differently in the variants.

The lab workflow started by letting the E.coli strain with plasmid inoculated into the LB broth supplemented with the kanamycin antibiotic. The protocol then proceeds into culturing the cell by setting the temperature 37 degrees celsius and spinning in the incubator with 200 rpm for hours. As the cell approached the log phase, they were subcultured at a 1:100 ratio into the ZYM-5052 medium, and we proceeded culturing them. In the beginning, glucose served as their primary source to support fast proliferation, and lactose as an inducer has limited functions because of the absence of cAMP/CAP. When the glucose is fully depleted, lactose will start entering the cell and transfer into allolactose to forbid the Lacl from inhibiting. This contributes to the expression of the T7 RNA polymerase, thereby initiating the transcription and synthesis of the target protein. Likewise, the glycerol continues to provide a sustained energy source, so the induction remains progressive. After 16 to 20 hours, proteins accumulate extensively in the host cells. The culture can only be centrifuged at a low temperature in order to harvest the cells and proceed to the purification.

By following this flow, the culture growth, induction, and expression can all run smoothly. Compared to the induction by IPTG, this approach enables us to achieve a high-level production of proteins at high cell densities. It also increases the stability and efficiency of the lab experiment. For more details, please refer to the measurement webpage.

Test

We followed the same approach mentioned above to run the pNPB assay and the lactate production with the PLA degradation assay. The results are in Figure 8-11.

The results of the pNPB assay showed that after comparing the mutant variants to the GFP and the wild type; we selected a few mutant variants with greater enzymatic activity. These include A6, A13, A14, C5, C6, C11, D3, D13, D16, P2, P7, P15 to test on the lactate production for the PLA degradation assay, and the vector only and wild type served as the control groups in this assay.

Learn

We found that the Proteinase K’s mutant variant (P2 group) has the best degradation efficiency via the data collected by screening. It produced 15 times more lactate than the vector-only control group. This explains that the lactate we’ve tested was produced from PLA, not the metabolism of the host strain. Moreover, the amount of lactate produced is at least 1.6 times higher than the PK wild-type control group. This further justifies that random mutagenesis enhances the ability of the genes to degrade PLA. Although the PLA degrading enzymes in the lab exhibited significant activities on PLA degradation, the condition still needs to be optimized before implementing the application on real-life target fields such as the organic farms. In the future, we hope a work may be carried out to present another way of degrading PLA degradation in real life. The details are presented in the future-work section.