Polycyclic aromatic hydrocarbons (PAHs) are a class of hazardous organic contaminants that are widely distributed in the environment. PAHs that undergo long-distance migration and have strong biological toxicity are a great threat to the health of ecosystems [1]. Microbial degradation of PAHs usually targets specific substrates or specific environments [2, 3]. As a result, some PAHS-degrading microorganisms can only play a role in specific types of PAHs. However, once the characteristics of pollutants such as PAHs types and concentrations change, the normal role of microbial flora in the remediation system cannot be guaranteed. So far, many degradation experiments of microorganisms isolated and screened from contaminated areas have focused on studying the degradation of PAHs by single or mixed bacteria in the laboratory [4]. However, when applied to the natural environment, it is often found that the degradation effect of PAHs is limited. This is because these isolated bacteria could not fully adapt to the natural environment, including nutrient status, temperature, pH, type and bioavailability of PAHs, as well as competition and synergism between bacteria, thus affecting the degradation efficiency. This is one of the reasons why the current microbial remediation technology is rarely used in large-scale PAHs contaminated environment. We realized the heterologous expression of degradation enzymes in Escherichia coli through synthetic biological methods, aiming to eliminate this gap, strengthen the adaptability of microorganisms to the environment, and achieve the broad spectrum degradation 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] X. Ma, X. Li, J. Liu, Y. Cheng, J. Zou, F. Zhai, Z. Sun, L. Han, Soil microbial community succession and interactions during combined plant/white-rot fungus remediation of polycyclic aromatic hydrocarbons, The Science of the total environment, 752 (2021) 142224.

[3] K. Dhar, S.R. Subashchandrabose, K. Venkateswarlu, K. Krishnan, M. Megharaj, Anaerobic Microbial Degradation of Polycyclic Aromatic Hydrocarbons: A Comprehensive Review, Reviews of environmental contamination and toxicology, 251 (2020) 25-108.

[4] C. Teerapatsakul, C. Pothiratana, L. Chitradon, S. Thachepan, Biodegradation of polycyclic aromatic hydrocarbons by a thermotolerant white rot fungus Trametes polyzona RYNF13, The Journal of general and applied microbiology, 62 (2017) 303-312.

The ligninolytic enzymes are one of the most-efficient oxidative systems found in nature, playing a pivotal role during wood decay and coal formation [1]. The major groups of ligninolytic enzymes include laccases, lignin peroxidases, manganese peroxidases, and versatile peroxidases [2]. Laccases are multi-copper-containing proteins that catalyze the oxidation of phenolic substrates with concomitant reduction of molecular oxygen to water. Laccases can be divided into three kinds: rhus laccase, bacterial laccase and fungal laccase. At present, bacterial laccase and fungal laccase are more widely used. Studies have found that bacterial laccase are more advantages than fungal laccase, such as copper independence, non-glycosylation, and good thermal stability [3]. Bacterial laccase CueO production in PAHs-degrading bacteria can increase benzo[a]pyrene mineralization [4]. One study found that laccase CotA from Bacillus subtilis exhibit a higher laccase-specific activity than laccase CueO from Escherichia coli, indicating that CotA is a better candidate for the remediation of PAHs than CueO [3]. This is why we chose laccase cotA from Bacillus subtilis as a member of the ligninolytic enzymes system in our project.

Lignin peroxidases have the unique ability to catalyze oxidative cleavage of C–C bonds and ether (C–O–C) bonds in non-phenolic aromatic substrates of high redox potential. Lignin peroxidases present significant potential for application in various industrial sectors, such as second-generation biofuels, cosmetics, food, bio-pulping and biobleaching. However, they are unstable at high temperatures, deactivated by solvents, susceptible to inactivation by hydrogen peroxide and challenging to produce in ample quantities [5]. Several expression systems have been investigated to produce Lignin peroxidases, with fungal hosts that have shown the most promise to date. In our project, Lignin peroxidase LipH8 from Phanerochaete chrysosporium was overexpressed in E. coli for the remediation of PAHs.

LipH8 from Phanerochaete chrysosporium was synthesized after gene and cloned into pET28a(+) plasmid. 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 pET28a(+) vectors carry an N-terminal His•Tag®/thrombin/T7•Tag® configuration plus an C-terminal His•Tag sequence. As the very first section of recombinant protein expression, the whole process of sequence synthesis is of great importance. Gene optimization takes advantage of the degeneracy of the genetic code. Because of degeneracy, one protein can be encoded by many alternative nucleic acid sequences. 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 [6, 7].

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

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

[3] 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.

[4] J. Zeng, X. Lin, J. Zhang, X. Li, M.H. Wong, Oxidation of polycyclic aromatic hydrocarbons by the bacterial laccase CueO from E. coli, Appl Microbiol Biotechnol, 89 (2011) 1841-1849.

[5] 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.

[6] E. Angov, P.M. Legler, R.M. Mease, Adjustment of codon usage frequencies by codon harmonization improves protein expression and folding, Methods in molecular biology, 705 (2011) 1-13.

[7] Y. Xu, K. Liu, Y. Han, Y. Xing, Y. Zhang, Q. Yang, M. Zhou, Codon usage bias regulates gene expression and protein conformation in yeast expression system P. pastoris, Microbial cell factories, 20 (2021) 91.

Although we have no intention of releasing the bacteria into the natural environment this year, the engineered bacteria will eventually move out of the laboratory and into the community, so biosafety is of paramount importance. We chose RelE toxin from the E.coli RelBE toxin-antitoxin system and added an arabinose promoter. The expression of relE was activated by the artificial addition of arabinose, which mediates mRNA cleavage through its association with the ribosome, so as to reduce the population density of E. coli [1].

The suicidal gene relE was cloned into the pBAD43 plasmid which bears the pSC101 replicon to avoid plasmid incompatibility during the subcultures (pET-28a carries the pBR322 replicon) and allowed the expression of RenL protein induced by L-arabinose through a pBAD promoter.

Figure 1. SDS-PAGE analysis of pBAD43-relE mutant strain. SDS-PAGE was used to analyze the expression of RelE. Recombinant vectors pBAD43-relE transformed into BL21 (DE3) competent cells and induced by 10 mg/ml arabinose (Arab) in MSM medium for 30 h at 27 ℃. 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 pBAD43-relE which was induced. Lane 3-4, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pBAD43-relE which was induced. S: Supernant; P: Pellet; Arab: arabinose.

The SDS-PAGE showed that RelE was not successfully expressed. We will adjust the temperature and inducer concentration in the following experiment. If the attempt fails, the New plasmid will be selected as the expression vector. In addition, the molecular weight of the target protein is 11 kD, and SDS-PAGE can't indicate the failure of expression strictly speaking, so changing the concentration of SDS-PAGE is undoubtedly the first choice.

[1] J.M. Hurley, J.W. Cruz, M. Ouyang, N.A. Woychik, Bacterial Toxin RelE Mediates Frequent Codon-independent mRNA Cleavage from the 5 ' End of Coding Regions in Vivo, Journal of Biological Chemistry, 286 (2011) 14770-14778.

Phenanthrene has a molecular formula of C14H10 and a molecular weight of about 178.23. It is insoluble in water, slightly soluble in ethanol, soluble in ethyl ether, hexane, dichloromethane and other organic solvents, with strong stability and hydrophobicity [1]. Phenanthrene is a compound with a benzene ring as its basic structure. Due to its strong stability, it is difficult to migrate independently in the natural environment, and it will cause harm to human life and health through the enrichment of the food chain:

(1) Phenanthrene can be widely found in farmland soil, rivers and lakes, as well as the atmosphere. So, even if the flow of phenanthrene into the natural environment is controlled, the residual phenanthrene in the environment will still pose a long-term threat. Phenanthrene can destroy biological cells and thus is difficult for microorganisms to use in nature [2].

(2) Phenanthrene, as a model compound of PAHs, has the potential toxicity of carcinogenesis, teratogenesis, and mutagenesis [3], and this toxicity is manifested in PAHs as the more rings, the stronger the toxicity [4]. Phenanthrene is mostly found in coal tar, asphalt, and petroleum, and long-term exposure to phenanthrene will increase the risk of cancer [5].

(3) Phenanthelline in nature can enter the food chain through the diet and absorption of animals and plants, and accumulate and enrich step by step in the food chain, damaging human health and even causing genetic defects through biological amplification [6]. Phenanthrene can also exist in the adipose tissue of mammals, which may damage the immune system of animals, produce allergic reactions and further develop into cancer [7].

(4) Phenanthrene can enter the human body and destroy the human immune system by damaging T and B lymphocytes of the human immune system [8]. Detmar et al. [9,10] have shown that pregnant women living in the presence of phenylene for a long time will damage the fetal immune system and also increase the probability of fetal respiratory diseases.

Due to its wide distribution in the environment, phenanthrene poses a serious threat to the natural environment, animals, plants, and human health. Due to its relatively simple structure and easy entry into cells, phenanthrene has also become a model compound for the current study of PAHs [11].

[1] S.M. Chan, T. Luan, M.H. Wong, N.F. Tam, Removal and biodegradation of polycyclic aromatic hydrocarbons by Selenastrum capricornutum, Environ Toxicol Chem, 25 (2006) 1772-1779.

[2] K.C, Jones. Contaminant trends in soils and crops,Environmental Pollution, 69(1991)311-325.

[3] S. Kuppusamy, P. Thavamani, K. Venkateswarlu, Y.B. Lee, R. Naidu, M. Megharaj, Remediation approaches for polycyclic aromatic hydrocarbons (PAHs) contaminated soils: Technological constraints, emerging trends and future directions, Chemosphere, 168 (2017) 944-968.

[4] C.D. Simpson, A.A. Mosi, W.R. Cullen, K.J. Reimer, Composition and distribution of polycyclic aromatic hydrocarbon contamination in surficial marine sediments from Kitimat Harbor, Canada, Sci Total Environ, 181 (1996) 265-278.

[5] K.B. Okona-Mensah, J. Battershill, A. Boobis, R. Fielder, An approach to investigating the importance of high potency polycyclic aromatic hydrocarbons (PAHs) in the induction of lung cancer by air pollution, Food Chem Toxicol, 43 (2005) 1103-1116.

[6] jiboye O O, Yakubu A F, Adams T E, A Review of Polycyclic Aromatic Hydrocarbons and Heavy Metal Contamination of Fish from Fish Farms, Journal of Applied Sciences and Environmental Management, 15(2011)235-238.

[7] A. Chauhan, Fazlurrahman, J.G. Oakeshott, R.K. Jain, Bacterial metabolism of polycyclic aromatic hydrocarbons: strategies for bioremediation, Indian J Microbiol, 48 (2008) 95-113.

[8] S.W. Burchiel, M.I. Luster, Signaling by environmental polycyclic aromatic hydrocarbons in human lymphocytes, Clin Immunol, 98 (2001) 2-10.

[9] A. Rundle, L. Hoepner, A. Hassoun, S. Oberfield, G. Freyer, D. Holmes, M. Reyes, J. Quinn, D. Camann, F. Perera, R. Whyatt, Association of childhood obesity with maternal exposure to ambient air polycyclic aromatic hydrocarbons during pregnancy, Am J Epidemiol, 175 (2012) 1163-1172.

[10] A. Mojiri, J.L. Zhou, A. Ohashi, N. Ozaki, T. Kindaichi, Comprehensive review of polycyclic aromatic hydrocarbons in water sources, their effects and treatments, Sci Total Environ, 696 (2019) 133971.

[11] J. Li, X. Shang, Z. Zhao, R.L. Tanguay, Q. Dong, C. Huang, Polycyclic aromatic hydrocarbons in water, sediment, soil, and plants of the Aojiang River waterway in Wenzhou, China, J Hazard Mater, 173 (2010) 75-81.