Xenobiotics and Microbial and Biotechnological approaches

Xenobiotics- Microbial Biotechnological approach .

1. UNIVERSITY OF AGRICULTURAL SCIENCES, BANGLORE Topic: xenobiotics and biotechnological approaches Department of biotechnology Team : Kings boys

2. Xenobiotics definition A xenobiotic is a chemical substance found within an organism that is not naturally produced. Actual meaning xenobiotic are the compound that are foreign to an organism for its actual nature. Environmental xenobiotic Environmental xenobiotics are xenobiotic compounds with a biological activity that are found as pollutants in the natural environment.

3. INTRODUCTION Environmental xenobiotics is an exciting new topic for the end of the 20th century. We are now all dependent on synthetic substances in agriculture,pharmaceuticals , petrochemicals, colorants, adhesives, preservatives, etc. Xenobiotics such as halogenated solvents undergo photochemical reactions which have been shown to cause depletion of the ozone layer Pesticides, when used for both agricultural and non-agricultural purposes, are transformed to persistent metabolites by soil and water microorganisms.

4. Recalcitrant Xenobiotic The compounds that resist biodegradation and persist in the environment for long period of time are called Recalcitrant Xenobiotic .  Reasons for recalcitrant Xenobiotic They are not recognized as substrate by degradative microorganisms.  Highly stable in nature.  Insoluble in water.  They are highly toxic or release toxic products due to microbial activity.  They have large molecular weight which prevents entry to microbial cells.  

5. TYPES of Recalcitrant Xenobiotic Halocarbons Poly chlorinated biphenyls(PCBs) Synthetic Polymers Alkylhenzyl sulphonates Oil mixtures

6. Sources of xenobiotic compounds Petrochemical industry : oil/gas industry - refineries and the production of basic chemicals e.g. vinyl chloride and benzenes. Plastic industry: - closely related to the petrochemical industry uses a number of complex organic compounds such as anti-oxidants, plasticizers, Pesticide industry: most commonly found central structures are benzene and benzene derivatives, often chlorinated and often heterocyclic. Paint industry: major ingredient are solvents, xylene, toluene, methyl ethyl ketone, methyl isobutyl ketone and preservatives. Others: Electronic industry, Textile industry, Pulp and Paper industry, Cosmetics and Pharmaceutical industry, Wood preservation

7. 1) Pharmaceuticals Pharmaceutically active compounds ( PhACs ) can enter the environment by one route or another as the parent compound or as pharmacologically active metabolites. PhACs can be entered into the environment in two main ways; direct and indirect. PhAC's can be discharged directly by manufacturers of the pharmaceuticals or effluents from hospitals. Indirect source of PhACs into the environment is the passing of antibiotics, anesthetics and Growth promoting hormones by domesticated animals in urine and manure.

8. Fate in environment Once PhACs are entered into the environment they suffer one of three fates: Biodegradation into carbon dioxide and water. Undergo some form of degradation and form metabolites. Persist in the environment unmodified.

9. 2) Petrochemicals Petrochemicals (also known as petroleum distillates) are chemical products derived from petroleum. A hydrocarbon is an organic compound consisting entirely of hydrogen and carbon. The majority of hydrocarbons found on earth naturally occur in crude oil. Aromatic hydrocarbons ( arenes ), alkanes, alkenes, cycloalkanes and alkyne-based compounds are different types of hydrocarbons.

10. Biodegradation of Petroleum compounds Petroleum compounds are categorized into 2 groups Aliphatic hydrocarbon e.g. alkane, alcohol, aldehyde Aromatic hydrocarbon e.g. benzene, phenol, toluene, catechol H.C. (substrate) + O2 H.C.-OH + H2O H.C. (substrate) + O2 H.C. 29 O -H (O H) monooxygenase dioxygenase

11. 3) BIODEGRADATION OF PESTICIDES Pesticides are substances meant for destroying or mitigating any pest. They are a class of biocide . The most common use of pesticides is as plant protection products (also known as crop protection products). It includes: herbicide, insecticide, nematicide, termiticide, molluscicide, piscicide, avicide, rodenticide, insect repellent, animal repellent, antimicrobial, fungicide, disinfectant, and sanitizer.

12. DIFFERENT METHODS Detoxification : Conversion of the pesticide molecule to a non-toxic compound. A single moiety in the side chain of a complex molecule is disturbed(removed), rendering the chemical non-toxic. Degradation : Breakdown or transformation of a complex substrate into simpler products leading to mineralization. E.g. Thirum (fungicide) is degraded by a strain of Pseudomonas and the degradation products are dimethylamine, proteins, sulpholipids , etc

13. c) Conjugation (complex formation or addition reaction): An organism makes the substrate more complex or combines the pesticide with cell metabolites. Conjugation or the formation of addition product is accomplished by those organisms catalyzing the reaction of addition of an amino acid, organic acid or methyl crown to the substrate thereby inactivating the pesticides d) Changing the spectrum of toxicity : Some pesticides are designed to control one particular group of pests, but are metabolized to yield products inhibitory to entirely dissimilar groups of organisms, for e.g. the fungicide PCNB is converted in soil to chlorinated benzoic acids that kill plants.

14. 4) BIODEGRADTION OF PLASTICS Plastic is a broad name given to different polymers with high molecular weight, which can be degraded by various processes. The biodegradation of plastics by microorganisms and enzymes seems to be the most effective process. It consist of two steps- fragmentation and mineralization . But at the core, reaction occurring at molecular level are oxidation and hydrolysis .

15. other HAZARDS OF XENOBIOTICS Highly toxic in nature. Causing skin problems and various types of cancers. They are easily not degraded so they remain in environment for many years. Hazards of xenobiotics in environment Xenobiotics pose a serious issue in sewage treatment plants Some xenobiotics are resistant to degradation for example synthetic organochlorides , PAH , crude oil and coal . Many xenobiotics produce variety of biological effects such as carcinogenic , toxic to humans , ecotoxicity and persistence in environment.

16. Biotechnological approaches in xenobiotics The limitations of conventional remediation technologies include poor environmental compatibility , high cost of implementation and poor public acceptability . Efficiency and performance of bio and phytoremediation approaches can be enhanced by genetically modified microbes and plants. Phytoremediation can also be stimulated by suitable plant-microbe partnerships, i.e. plant-endophytic or plant- rhizospheric associations. Synergistic interactions between recombinant bacteria and genetically modified plants can further enhance the restoration of environments impacted by organic pollutants.

17. Genetic engineering Genetic engineered new strains of microbes that have the unique characteristics compare to the wild type and broad spectrum of catabolic potential for bioremediation of xenobiotics . GEMs have been developed by the identification and manipulation of certain genetic sequences. GEMs exhibit enhanced degradability for a wide range of xenobiotics and have potential for bioremediation from various environmental sources. In 1970 , the first GEMs called “ superbug ” was constructed to degrade oil by the transfer of plasmids which could utilize a number of toxic organic chemicals like octane, hexane, xylene, toluene, camphor and naphthalene .

18. Approaches for the construction of GEM’s The identification of organisms suitable for modification with the relevant genes Microorganisms are well adapted to survive in soil environment may not be able to survive in aquatic environment and hence cannot be used successfully. Therefore, aquatic microbes can be used to develop GEMs for bioremediation of aquatic sources. Scientists have developed Anabaena sp . and Nostoc sp .by the insertion of linA (from P. paucimobilus ) and fcbABC (from Arthrobacter globiformis ) respectively. The gene linA controls the biodegradation of lindane, and fcbABC confers the ability to biodegrade halobenzoates and can be used to remediate these pollutants from water sources.

19. 2. The pathway construction, extension and regulation . GEMs have developed by improving existing catabolic pathways to degrade some more compounds which are not possible to degrade by using wild strain. The complete catabolic pathway may be encoded by a single microorganism, or by a consortium of microorganisms, each performing one or more of the stages of bioremediation of xenobiotics .

20. 3. Modification of enzyme specificity and affinity . GEMs have been developed by hybrid gene clusters which alter their enzymatic activity . These gene clusters encode the enzyme possessing improved transforming capability. E. coli strain is genetically modified to express a hybrid gene cluster for the degradation of trichloroethylene (TCE).

21. 4. bioprocess development, monitoring, control, and bioaffinity , bioreporter, sensor applications for chemical sensing, toxicity reduction, and end point analysis. The use of lux gene-based system has been developed that offers several advantages for monitoring bioremediation processes. Bioluminescence can be easily detected and do not require expensive devices, exogenous addition of chemicals or co-factors. Bioluminescence producing GEMs also help us to understand the spread of microbes in the polluted area and end point of the bioremediation. Further, GEMs possess chemical sensors that allow the monitoring of contaminant bioavailability .

22. Advantages Microbes are confined to aerobic catabolic and co-metabolic pathways and therefore cannot be applied to anaerobic environment. GEMs are developed by inserting genes for oxygenases make it possible to use them in anaerobic environmental conditions. Microbes or GEMs which use xenobiotics as a source of carbon, energy or nitrogen, can get nutrients and grow.

23. Cont ,, Scientists have been developing a novel strategy to construct “Suicidal Genetically Engineered Microorganisms (SGEMs)" by exploring the antisense RNA-regulated plasmid addiction. To design a novel S-GEM is based on the knowledge of killer-anti-killer genes that makes the microbes susceptible to programmed cell death after degradation of xenobiotics. This technology helps in the removal of microbes after bioremediation by their autolysis and therefore, reducing the risks to the human beings and environment.

24. Limitations of GEM’s The information on the genes is very limited which limits the development of GEMs. Second main obstacle in the application of GEM is the regulatory affairs and hazards associated with them.

25. WHITE-ROT FUNGI AND THEIR ENZYMES AS A BIOTECHNOLOGICAL TOOL FOR XENOBIOTIC BIOREMEDIATION MARIEM ELLOUZE AND SAMI SAYADI HTTP://DX.DOI.ORG/10.5772/64145

26. Biological methods, being eco-friendly and cost cheap techniques, were proposed for xenobiotic degradation purposes in order to overcome problems of xenobiotics. Compared to bacteria, most of the fungi are robust organisms and generally more tolerant to high concentrations of pollutants. White-rot fungi (WRF) constitute an eco-physiological group comprising mostly of basi‐diomycetes and litter-decomposing fungi. Recently, there has been a great interest in white-rot fungi and their ligninolytic enzymes, including laccase , manganese peroxidase ( MnP ) and lignin peroxidase ( LiP ), for the degradation of a wide range of xenobiotics .

27. The expression of these enzymes depends on the strain itself: some white-rot fungi produce LiP and MnP , but not laccase , while others produce MnP and laccase , but not LiP , acting simultaneously or separately on xenobiotics released from the environment. The potential of white-rot fungi can be harnessed thanks to emerging knowledge of the physiology and the morphology of these organisms.

28. The importance of high extracellular levels of these enzymes to enable the efficient degradation of recalcitrant compounds under in vivo conditions relates to the sorption and complexation of enzymes in soil and the probable loss of their activity once externalized. Many studies reported the effective degradation of pesticides by fungal strains, including P. chrysosporium and T. versicolor , and involving two different enzyme systems: laccase and peroxidases

29. It is known that white-rot fungi can degrade lignin in the way that the mycelia of the organisms penetrate the cell cavity and release ligninolytic enzymes to decompose materials to a white sponge-like mass. Enzymatic treatment, involving mainly peroxidases and/or laccases , is currently considered as an alternative method for the removal of toxic xenobiotics from the environment.

30. Peroxidase system The lignin degradation system consists on peroxidases, H2O2 -producing enzymes, veratryl alcohol, oxalate, and manganese. All of these enzymes are glycosylated heme proteins that couple the reduction of hydrogen peroxide to water with the oxidation of a variety of sub‐ strates . Lignin peroxidases ( LiPs ) belong to the family of oxidoreductases and were firstly described in the basidiomycete P. chrysosporium in 1983. This enzyme has been recorded for several species of white-rot basidiomycetes .

31. LiP is dependent of H2O2 , with an unusually high redox potential and low optimum pH. This enzyme is able to oxidize a variety of substrates including polymeric ones and has consequently a great potential for application in various industrial treatment processes. MnP catalyzes the oxidation of phenolic structures to phenoxyl radicals. The product Mn3+, being highly reactive, complex with chelating organic acids, such as oxalate, lactate, or malonate . On the other hand, it was reported that MnP may oxidize Mn (II) without H2O2 and with decomposi ‐ tion of acids, and concomitant production of peroxyl radicals.

32. LACCASE SYSTEM Laccases which are blue multicopper oxidases, catalyze the monoelectronic oxidation of a large spectrum of substrates, for example, ortho - and para-diphenols , polyphenols, aminophenols , and aromatic or aliphatic amines, coupled with a full, four electron reduction of O2 to H2O. Laccases act on both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and they can be effectively used in paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation and can act as biosensors.

33. The importance of high extracellular levels of these enzymes to enable the efficient degradation of xenobiotic compounds under in vivo conditions relates to the sorption and complexation of enzymes in soil and the probable loss of much of their activity once externalized. This group may be a useful and a powerful tool for bioremediation purposes thanks to fungal capacities to degrade many xenobiotic substances.

34. RECENT STUDY OF XENOBIOTIC IN FUNGI

35. Biodegradation of Toxic Organic Compounds in Environmental Soil Endophytic Bacteria and Phytoremediation Reported cases of successful bioremediation using endophytic bacteria

36. Rhizospheric Bacteria and Phytoremediation ( Rhizoremediation ) bacteria

37. INTRODUCTION Remediation processes based on plants and on plant–microbe interactions have been proposed to clean up sites contaminated with xenobiotics including polychlorinated biphenyls (PCBs). The extensive root system of plants allows them to pump large amounts of chemicals from soil. In addition, plant roots exude many chemicals that promote rhizobacterial growth and metabolis . 

38. Engineering of the biphenyl catabolic enzymes Bacterial biphenyl catabolic pathway

39. Transgenic plant expressing biphenyl-degrading enzymes In recent years, few investigations have addressed the feasibility of constructing transgenic plants producing active PCB-degrading enzymes. As indicated above, BPDO is a three-component enzyme requiring the participation of BphAE , BphF , and BphG . Analyses of tobacco plants transiently expressing  B. xenovorans  LB400 genes encoding the BPDO components or transformed with them have shown that each of the components can be produced individually as active protein in plants. 

40. Deciphering the molecular mechanisms involved in plant–microbe interactions The choice of plants is likely to impact on the success of the rhizoremediation technology. Some plants such as  Cucurbita pepo  (zucchini) accumulate high level of hydrophobic chemicals, others, such as alfalfa possess extensive root systems that exhibit high affinity toward hydrophobic chemicals a critical criterion is the ability of plants to support the metabolism and survival of the PCB-degrading rhizobacteria  A clear example was provided by Narasimhan  et al . who showed that PCB removal by  Pseudomonas putida  PML2 which is a phenylpropanoid-utilizing and PCB-degrading rhizobacteria was significantly lower in rhizosphere of an  Arabidopsis  mutant exuding less flavonoids than in the rhizosphere of the wild-type strain

41. CONCLUSION PCB-rhizoremediation process is a promising approach to restore contaminated sites. However, challenging issues remain to be overcome. Efforts are required to understand how plants will respond to the presence of high levels of PCB metabolites. New approaches should be conceived that will allow co-ordinate expression of several heterologous genes together in same plant cells and same compartments. Finally we need to have better insight into the mechanisms by which rhizosphere bacteria perceive and modify plant small molecules such as flavonoids that to some extent mimic some of the PCB structural features and how these interactions impact on the catabolic pathways involved in PCB degradation.

42. HIGHLIGHTS Nine genes from different microorganisms were synthesized and modified. Phenol was degraded completely and imported into the tricarboxylic acid cycle. All genes were regulated by monocistronic transcriptional pattern. The engineered E. coli could effectively degrade phenol in coking wastewater

43. INTRODUCTION Phenol is one of the most frequently pollutants found in industrial effluents, landfill runoff waters and rivers and its concentration can reach 10 g/L in some wastewaters. Phenol is potentially carcinogenic to humans, and deaths in adults have resulted after ingestion of 1-32g of phenol. Phenols are categorized as priority hazardous substances due to their proven toxic, mutagenic, carcinogenic, and teratogenic effects

44. MO’S REPORTEDLY UTILISED FOR PHENOL BIODEGRADATION Pseudomonas putida, Rhodococcus erythropolis , Bacillius sp., Alcaligenes faecalis, Ralstonia taiwanensis , Nocardia hydrocarbonoxydans and Candida tropicalis

45. METHOD In this study, two metabolic modules were introduced into Escherichia coli , to elucidate the metabolic capacity of E. coli for phenol degradation. The first module catalyzed the conversion of phenol to catechol. The second module cleaved catechol into the three carboxylic acid circulating intermediates by the ortho- cleavage pathway.

46. PHENOL DEGRADING PATHWAY Phenol is first catalysed to catechol by phenol hydroxylase, which attaches a hydroxyl group to the ortho -position of the aromatic ring under aerobic conditions, to facilitate degradation. Then, catechol is cleaved by dioxygenases, either between the hydroxyl groups or adjacent to one of the hydroxyl groups through ortho - or meta -cleavage. The final products of catechol degradation, namely, acetyl-CoA and succinyl-CoA, were assumed to be imported into the TCA cycle of bacteria.

47. CONSTRUCTION OF PHENOL DEGRADATION STRAIN BL- phe /cat All nine genes used in this study are from different microorganisms, as follows: pheA1 and pheA2 from Rhodococcus erythropolis ; catA , catB and catC from Rhodococcus species AN-22; catD from Rhodococcus jostii ; and pcaI , pcaJ and pcaF from Pseudomonas putida.

48. Scheme of gene clusters in different transformants (BL- phe , BL -cat and BL- phe /cat ). pheA1 , pheA2 (large and small subunits of phenol hydroxylase); catA (catechol 1,2-dioxygenase); catB ( cis,cis -muconate lactonizing enzyme); catC (muconolactone isomerase); catD ( β- ketoadipate enol-lactone hydrolase); pcaI , pcaJ ( β- ketoadipate succinyl-CoA transferase); pcaF ( β- ketoadipyl-CoA thiolase )

49. All synthesized genes were seamlessly connected with T7 promoter and terminator to construct gene expression cassette via the modified overlap-extension PCR method. The PCR products were purified and cloned into pGEM -T easy vector, and sequenced prior to cloning in the expression vector All gene expression cassettes were re-amplified with primer containing different restriction enzyme cutting sites. The PCR fragment was then excised using restriction endonucleases and inserted into pCAMBIA1301 vector in proper order. All the cassettes were transformed to the host E. coli strain BL221-AI after their assembly in the expression vector. The transformant was named BL- phe /cat . The strains BL- phe and BL- cat respectively containing the modules for the conversion of phenol to catechol and catechol degradation.

50. RESULT The key intermediate metabolites and products in the process of phenol degradation can be detected in the chassis cell by stable carbon isotopic tracer(13C6-catechol)). Proteomic analyses showed that low phenol concentrations can activate downstream signalling pathways and these results indicated that the engineered E.coli could completely and quickly degrade phenol. This analysis showed that all genes in the phenol degradation pathway were over-expressed and affected cell division and energy metabolism of the host cells. The engineered E. coli could effectively be used in the remediation of phenol-polluted wastewater.

51. Phenol was completely degraded and imported into the tricarboxylic acid cycle by the engineered bacteria. Phenol in coking wastewater was degraded powerfully by BL- phe /cat . The engineered E. coli can improve the removal rate and shorten the processing time for phenol removal and has considerable potential in the treatment of toxic and harmful pollutants.

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