Research

Polycyclic aromatic hydrocarbons (PAHs) are a large group of chemicals. They represent an important concern due to their widespread distribution in the environment, their resistance to biodegradation, their potential to bioaccumulate and their harmful effects [1, 2]. As a promising option, fungal enzymes are regarded as a powerful choice for degradation of PAHs [3]. Phanerochaete chrysosporium, Pleurotus ostreatus and Bjerkandera adusta are most commonly used for the degradation of such compounds due to their production of ligninolytic enzymes. The ligninolytic enzymes are one of the most-efficient oxidative systems found in nature, playing a pivotal role during wood decay and coal formation [4]. The major groups of ligninolytic enzymes include laccases, lignin peroxidases, manganese peroxidases, and versatile peroxidases [5].

At present, bacterial laccase and fungal laccase are more widely used. One study found that laccase CotA from Bacillus subtilis exhibit a higher laccase-specific activity than laccase CueO from E. coli, indicating that CotA is a better candidate for the remediation of PAHs than CueO [6]. Several expression systems have been investigated to produce lignin peroxidases, with fungal hosts that have shown the most promise to date [7]. In our project, laccase CotA from Bacillus subtilis and lignin peroxidase LipH8 from Phanerochaete chrysosporium were overexpressed in E. coli for the remediation of PAHs.

[1] O.O. Alegbeleye, B.O. Opeolu, V.A. Jackson, Polycyclic Aromatic Hydrocarbons: A critical review of environmental occurrence and bioremediation, Environmental management, 60 (2017) 758-783.

[2] I.A. Titaley, S.L.M. Simonich, M. Larsson, Recent advances in the study of the remediation of polycyclic aromatic compound (PAC)-contaminated soils: transformation products, toxicity, and bioavailability analyses, Environmental science & technology letters, 7 (2020) 873-882.

[3] T. Kadri, T. Rouissi, S. Kaur Brar, M. Cledon, S. Sarma, M. Verma, Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review, Journal of Environmental Sciences, 51 (2017) 52-74.

[4] M. Alcalde, Engineering the ligninolytic enzyme consortium, Trends in biotechnology, 33 (2015) 155-162.

[5] D.W. Wong, Structure and action mechanism of ligninolytic enzymes, Applied biochemistry and biotechnology, 157 (2009) 174-209.

[6] J. Zeng, Q. Zhu, Y. Wu, X. Lin, Oxidation of polycyclic aromatic hydrocarbons using Bacillus subtilis CotA with high laccase activity and copper independence, Chemosphere, 148 (2016) 1-7.

[7] O.D.V. Biko, M. Viljoen-Bloom, W.H. van Zyl, Microbial lignin peroxidases: Applications, production challenges and future perspectives, Enzyme and microbial technology, 141 (2020) 109669.

Build

The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and translation signals. T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell's resources are converted to target gene expression. The cotA and lipH8 sequences were cloned into pET28a (+) expression vector, resulting in vectors pHJ5 and pHJ6, respectively. Then the recombinant plasmids were transformed into BL21 (DE3) competent cells, resulting in mutant strains AE5 and AE6, respectively (Fig. 1). The cotA gene was cloned from the genome of Bacillus subtilis, and the lipH8 gene was synthesized by the Sangon Biotech (Shanghai, China). Codon preference or codon usage bias differs in each organism, and it can create challenges for expressing recombinant proteins in heterologous expression systems, resulting in low and unreliable expression. Therefore, we performed gene optimization before synthesizing the lipH8 gene.

Figure 1. Photographs of mutant strains AE5 (a) and AE6 (b) grown on Luria-Bertani (LB) agar plates with kanamycin-50 ug/ml.

Test

The genetic engineering bacteria was constructed and induced by IPTG. In LB medium with adequate nutrition, we conducted induction expression of CotA and LipH8 proteins at different temperatures and times to observe the distribution of proteins. When the OD600 is between 0.6-0.8, it is time to induce the bacteria to produce CotA (Fig. 2) and LipH8 (Fig. 3) with 0.1 mM IPTG.

Figure 2. SDS-PAGE analysis of AE5 mutant strain CotA. SDS-PAGE was used to analyze the expression of CotA. Recombinant vectors pHJ5 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG for 7 h and 18 h at 16 ℃, 20 ℃, 25 ℃, and 30 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-8, supernatant and pellet fraction of the whole bacterial lysate. S: Supernant; P: Pellet.

Figure 3. SDS-PAGE analysis of AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of LipH8. Recombinant vectors pHJ5 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG for 7 h and 18 h at 20 ℃ and 25 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-8, supernatant and pellet fraction of the whole bacterial lysate. S: Supernant; P: Pellet.

After several rounds of induction and expression, we found that the protein expressed by CotA was mostly distributed in the supernatant at 16 ℃ and 20 ℃, while the protein expressed by LipH8 was well distributed in the supernatant at 20 ℃ in LB medium. We finally chose 20 ℃ as inducing temperature.

Figure 4. SDS-PAGE analysis of AE5 mutant strain CotA and AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of CotA and LipH8. Recombinant vectors pHJ5 and pHJ6 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 20 ℃, respectively. The pellet was then dissolved in MSM medium with 0.1 mM IPTG for 2 d at 20 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli BL21(DE3) contained recombinant pET28a-cotA which was induced. Lane 3-4, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-lipH8 which was induced. Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA and pET28a-lipH8 which were induced. Lane 7-8, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet.

In the degradation experiment of phenanthrene, 10 mg/L phenanthrene was added as the sole organic carbon source in MSM medium. The pellet of the genetic engineering bacteria was dissolved in MSM medium with 0.1 mM IPTG and 10 mg/L phenanthrene for 2 d at 20 ℃ after 20 h induction at 20 ℃ in LB medium with 0.1 mM IPTG. Unfortunately, we observed that most of the protein was present in pellet fraction of the whole bacterial lysate after 2 d of induction in MSM medium (Fig. 4). This gives us some ideas about how to make proteins fold successfully instead of forming inclusion bodies.

Re-design

We carefully combed protocol and found that the proteins expressed by CotA were almost distributed in the supernatant at 16 ℃, so we made a concession and allowed CotA gene to be induced and expressed at 16 ℃. In other experimental groups, the expression was still induced at 20 ℃. We also noted that the high concentration of IPTG was also the main condition for the formation of inclusion bodies, so we did not add IPTG to the MSM medium. In addition, we heated the engineered bacteria prior to the addition of IPTG to produce heat shock proteins to help the target proteins fold successfully.

Re-test

Figure 5. SDS-PAGE analysis of AE5 mutant strain CotA and AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of CotA and LipH8. Recombinant vectors pHJ5 and pHJ6 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 16 ℃ and 20 ℃, respectively. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA which was induced. Lane 3-4, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-lipH8 which was induced. Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet.

We followed the idea of Re-Design and changed the temperature and the concentration of the inducer. The figure above clearly shows that the target protein induced in LB medium is present in the supernatant (Fig. 5). This result was encouraging, and we then completed the induction of expression in MSM medium.

Figure 6. SDS-PAGE analysis of AE5 mutant strain CotA and AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of CotA and LipH8. Recombinant vectors pHJ5 and pHJ6 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 16 ℃ and 20 ℃, respectively. The pellet was then dissolved in MSM medium without IPTG for 2 d at 20 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA which was induced. Lane 3-4, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-lipH8 which was induced. Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA and pET28a-lipH8 which were induced. Lane 7-8, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet.

After 2 d of induction in MSM medium without IPTG, the target proteins CotA and LipH8 can be clearly visualized in supernatant fraction of the whole bacterial lysate, which proves that the engineering iteration is effective (Fig. 6).

Figure 7. Oxidation of phenanthrene with the whole bacteria of CotA and LipH8 at 20 ℃ for 1 d, 3d, and 5d. The experiment was carried out in MSM medium without IPTG, and the oxidation was determined using noncellular components as the control. The differences in the PAH oxidation were determined by comparing the controls based on one-way ANOVA followed by Dunnett’s test (* P < 0.05).

SDS-PAGE results showed that the constructed expression system was successful. In order to verify whether the protein had biological activity, the concentration of phenanthrene was detected by HPLC. Both CotA and LipH8 could degrade phenanthrene, and coexpression of CotA and LipH8 was more effective (Fig. 7).