Team:BUCT-China/Description
Plastics are the most commonly used materials in many civil and industrial applications. Accordingly, the huge accumulation of plastic waste has been causing serious environmental problems, among which polythene (PE) alone contributes about 64% and is considered as most problematic. Biodegradation has been accepted as the best strategy to cope with this problem.
Abstract
Plastic wraps our food and houses our technology. It is a remarkable substance that has multiple advantages for many applications. But it also brought huge disasters to our ecological environment. By 2025, 250 million tons of plastic waste will be released into the oceans. Over time, that plastic material does not biodegrade, but breaks down into tiny particles known as microplastics, which may be eaten by marine animals and so enter the food chain. Our team is committed to using synthetic biology methods to reuse the plastic waste in the ocean. Last year, we observed a strain Microbulbifer hydrolyticus, which can degrade PE and PS. This year, based on last year's study, we made some improvements and conducted further exploration work. Our project is divided into two parts. The first part is to degrade PE into alkanes. We construct an artificial metabolic pathway and use surface display to express Laccase on the surface of spores, making the reaction more efficient and intuitive. The second part is to use those metabolites to synthesize a new material PHFA (poly hydroxyl fatty acid). Based on genome sequencing and synthetic biology, we constructed another engineered bacteria to utilize plastics to synthetic value-added products such as the new material PHFA. In a word, our research can not only solve the pollution of polyethylene, but also provide a low-cost raw material for the transverse brace of PHFA, making it possible to turn waste into treasure.In our assumption, PHFA will be a new material with good properties, and we will further study it in the future.
Inspiration
The world has witnessed a great growth in the productivity of plastic. From 98.92 million tons in 1990 to 300 million tons in 2019, the productivity almost tripled over 30 years. Plastics are important and convenient chemical material, which used in almost every aspect of our life. The corrosion resistance and durability, of the material because of chemical structure allows it to be used for a long time and not be degraded in 100 years. There are about 8 billion of waste plastic in the environment, and the increasing rate grows from year to year. One of the biggest problem of plastic pollution is that it can be sheared and smashed into tiny pieces called microplastic by wind erosion and water erosion. The report of microplastic pollution can be read from the news every day, which gave us the idea of finding a way to biodegrade microplastic using bacteria. Plastic can be classified into different groups based on their chemical structure and composition. Some of them were found to be biodegrade by various of bacteria, others were not, such as PE. We aim on finding the way to biodegrade PE and biosynthesis it into some environmentally-friendly material.
What is PE
The full name of PE is polyethylene, which is a typical thermoplastic resin prepared by polymerization of ethylene. It is widely used in preservative film, vest type plastic bag, plastic food bag, bottle, pail and other common product in daily life.
What is the advantage and disadvantage of PE
Its fine properties such as low density, great corrosion resistance, good water resistance，non- toxic and odourless make it an great versatile chemical material，which leads to the result that its production rate is much faster than its natural degradation rate.
In fact，its natural degradation rate is slow because its excellent chemical stability, almost unable to dissolve in any solution in normal temperature.
What will waste PE do to the nature
The waste plastic will not be degraded for a long period. Some of them will cover the ground and water, be misidentified as food by animals and do harm to the environment. Others will be deformed into small pieces. If the diameter is less than 1mm, the piece will be called microplastic, which goes into the ocean and is easily ingested by marine creature, finally poses threads to human health through the food chain enrichment.
What is PHFA
The full name of PHFA is poly hydroxyl fatty acid, this is a new material, because its structure is very similar to PHA, we think it may has similar characteristics with PHA and it's also environmentally friendly. At the same time, our material PHFA has longer carbon chain, we speculate that it may be better than PHA in some mechanical properties.
What is the old way to deal with waste plastic
The old ways to deal with plastic can be mostly described as burning and burying. Some of the waste plastic can be burying into the ground far away from city. Others are burning in some certain areas.
Why can’t we use the old way to deal with waste plastic
The burning method will generate harmful, even poisonous gas, such as phenanthrene and pyrene. If the burying method is applied, the waste plastic will blocking the absorption of water and nutrients by plants. In a word, neither of the above method can be a environment-friendly way to deal with waste plastic.
What is our method to deal with PE
In our research, we used synthetic biology method to transform PE into environmental friendly PHA，decompose PE into monomers in Bacillus subtilis, and construct a new cell synthesis pathway in E.coli to synthesize monomers into a new material PHFA.
(1)Pan, L. J. G. . (2011). Decolorization of indigo carmine by laccase displayed on bacillus subtilis spores. Enzyme and Microbial Technology.
(2)Brühlmann, Fredi, Fourage, L. , Ullmann, C. , Haefliger, O. P. , Jeckelmann, N. , & Dubois, Cédric, et al. (2014). Engineering cytochrome p450 bm3 of bacillus megaterium for terminal oxidation of palmitic acid. Journal of Biotechnology, 184, 17-26.
(3)Nuland, Y. M. V. , Vogel, F. A. D. , Eggink, G. , & Weusthuis, R. A. . (2017). Expansion of the ω-oxidation system AlkBGTL of pseudomonas putida GPo1 with AlkJ and AlkH results in exclusive mono-esterified dicarboxylic acid production in E.coli. Microbial Biotechnology.
(4)Murshidul, A. M. , Hyunwoo, J. , Prabhu, N. S. , Taeowan, C. , & Hyungdon, Y. . (2017). Biosynthesis of the Nylon 12 Monomer, ω‐Aminododecanoic Acid with Novel CYP153A, AlkJ, and ω‐TA Enzymes. Biotechnology Journal, 13(4), 1700121.
(5)Junyu, Ziheng, Cui, Kaili, Nie, & Hao. (2019). A quantum mechanism study of the c-c bond cleavage to predict the bio-catalytic polyethylene degradation. Frontiers in microbiology.
(6)Z. L. , R. W. , M. G. , Y. R. , B. Y. , & K. N. , et al. (2020). Biodegradation of low-density polyethylene by microbulbifer hydrolyticus ire-31. Journal of Environmental Management, 263.
(7)Kirmair, L. , & Skerra, A. . (2014). Biochemical analysis of recombinant alkj from pseudomonas putida reveals a membrane-associated, flavin adenine dinucleotide-dependent dehydrogenase suitable for the biosynthetic production of aliphatic aldehydes. Applied & Environmental Microbiology, 80(8), 2468-77.
(8)Beilen, J. B. V. , Eggink, G. , Enequist, H. , Bos, R. , & Witholt, B. . (2010). Dna sequence determination and functional characterization of the oct-plasmid-encoded alkjkl genes of pseudomonas oleovorans. Molecular Microbiology, 6(21), 3121-3136.
(9)Bertram JH, Mulliner KM, Shi K, Plunkett MH, Nixon P, & Serratore NA., et al. (2017). Five fatty aldehyde dehydrogenase enzymes from marinobacter and acinetobacter spp. and structural insights into the aldehyde binding pocket. Applied & Environmental Microbiology,83(12).
(10)Jia, H. , Lee, F. S. , & Farinas, E. T. . (2014). Bacillus subtilis spore display of laccase for evolution under extreme conditions of high concentrations of organic solvent. Acs Combinatorial ence, 16(12), 665-9.
(11)Silu, Sheng, Han, Jia, Sidney, Topiol ., et al. (2017). Engineering cota laccase for acidic ph stability using bacillus subtilis spore display. Journal of microbiology and biotechnology.
(12)Gonzalez, J. M. , Mayer, F. , Moran, M. A. , Hodson, R. E. , & Whitman, W. B. . (1997). Microbulbifer hydrolyticus gen. nov. sp. nov. and marinobacterium georgiense gen. nov. sp. nov. two marine bacteria from a lignin-rich pulp mill waste enrichment community. Int J Syst Bacteriol, 47(2), 369-376.
(13)Li, Fei-Long, Shi, Ying, Zhang, Jiu-Xun., et al. (2018). Cloning, expression, characterization and homology modeling of a novel water-forming nadh oxidase from streptococcus mutans atcc 25175. International Journal of Biological Macromolecules Structure Function & Interactions.
(14)Li, Zhongyu & Wei, Ren & Gao, Meixi & Ren, Yanru & Yu, Bo & Nie, Kaili & Xu, Haijun & Liu, Luo. (2020). Biodegradation of low-density polyethylene by Microbulbifer hydrolyticus IRE-31. Journal of Environmental Management. 263. 110402. 10.1016/j.jenvman.2020.110402.
(15)Andrews, D. , Mattatall, N. R. , Arnold, D. , & Hill, B. C. . (2005). Expression, purification, and characterization of the cua-cytochrome c domain from subunit ii of the bacillus subtilis cytochrome caa3 complex in escherichia coli. Protn Expression & Purification, 42(2), 227-235.
(16)Sirima, M. , & Watanalai, P. . (2018). Display of escherichia coli phytase on the surface of bacillus subtilis spore using cotg as an anchor protein. Applied Biochemistry and Biotechnology, 187, 838-855.
(17)Zhang, G. , An, Y. , Zabed, H. M. , Guo, Q. , & Qi, X. . (2019). Bacillus subtilis spore surface display technology: a review of its development and applications. Journal of Microbiology and Biotechnology.
(18)Kim, J. , & Schumann, W. . (2009). Display of proteins on bacillus subtilis endospores. Cellular & Molecular Life ences Cmls, 66(19), 3127-36.
(19)Lin, P. , Yuan, H. , Du, J. , Liu, K. , & Wang, T. . (2020). Progress in research and application development of surface display technology using bacillus subtilis spores. Applied Microbiology and Biotechnology, 104(6), 2319-2331.
(20)Isticato, R. , & Ricca, E. . (2016). Spore Surface Display. The Bacterial Spore.
(21)Wang, H. , Wang, Y. , & Yang, R. . (2017). Recent progress in bacillus subtilis spore-surface display: concept, progress, and future. Applied Microbiology and Biotechnology, 101(3), 933-949.
(22)Maier, T. , F?Rster, H. H. , Asperger, O. , & Hahn, U. . (2001). Molecular characterization of the 56-kda cyp153 from acinetobacter sp. eb104. Biochemical & Biophysical Research Communications,286(3), 652-658.
(23)Funhoff, E. G. , Bauer, U. , Garcia-Rubio, I. , Witholt, B. , & Van Beilen, J. B. . (2006). Cyp153a6, a soluble p450 oxygenase catalyzing terminal-alkane hydroxylation. Journal of Bacteriology,188(14), 5220-5227.
(24)Van Beilen J B , Funhoff E G , Van Loon A , et al. Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases.[J]. Applied & Environmental Microbiology, 2006, 72(1):59-65.
(25)Malca, S. H. , Scheps, D. , Kuehnel, L. , Venegas-Venegas, E. , Seifert, A. , & Nestl, B. M. , et al. (2012). Bacterial cyp153a monooxygenases for the synthesis of omega-hydroxylated fatty acids. Chemical Communications, 48(42), p.5115-5117.
(26)Process limitations of a whole-cell p450 catalyzed reaction using a cyp153a-cpr fusion construct expressed in escherichia coli. Applied Microbiology & Biotechnology, 100(3), 1197-1208.
(27)Smits, T. H. M. , Seeger, M. A. , Witholt, B. , & Beilen, J. B. V. . (2001). New alkane-responsive expression vectors for escherichia coli and pseudomonas. Plasmid, 46(1), 16-24.
(28)Funhoff, E. G. , Salzmann, J. , Bauer, U. , Witholt, B. , & Beilen, J. B. V. . (2007). Hydroxylation and epoxidation reactions catalyzed by cyp153 enzymes. Enzyme & Microbial Technology, 40(4), 806-812.
(2)Brühlmann, Fredi, Fourage, L. , Ullmann, C. , Haefliger, O. P. , Jeckelmann, N. , & Dubois, Cédric, et al. (2014). Engineering cytochrome p450 bm3 of bacillus megaterium for terminal oxidation of palmitic acid. Journal of Biotechnology, 184, 17-26.
(3)Nuland, Y. M. V. , Vogel, F. A. D. , Eggink, G. , & Weusthuis, R. A. . (2017). Expansion of the ω-oxidation system AlkBGTL of pseudomonas putida GPo1 with AlkJ and AlkH results in exclusive mono-esterified dicarboxylic acid production in E.coli. Microbial Biotechnology.
(4)Murshidul, A. M. , Hyunwoo, J. , Prabhu, N. S. , Taeowan, C. , & Hyungdon, Y. . (2017). Biosynthesis of the Nylon 12 Monomer, ω‐Aminododecanoic Acid with Novel CYP153A, AlkJ, and ω‐TA Enzymes. Biotechnology Journal, 13(4), 1700121.
(5)Junyu, Ziheng, Cui, Kaili, Nie, & Hao. (2019). A quantum mechanism study of the c-c bond cleavage to predict the bio-catalytic polyethylene degradation. Frontiers in microbiology.
(6)Z. L. , R. W. , M. G. , Y. R. , B. Y. , & K. N. , et al. (2020). Biodegradation of low-density polyethylene by microbulbifer hydrolyticus ire-31. Journal of Environmental Management, 263.
(7)Kirmair, L. , & Skerra, A. . (2014). Biochemical analysis of recombinant alkj from pseudomonas putida reveals a membrane-associated, flavin adenine dinucleotide-dependent dehydrogenase suitable for the biosynthetic production of aliphatic aldehydes. Applied & Environmental Microbiology, 80(8), 2468-77.
(8)Beilen, J. B. V. , Eggink, G. , Enequist, H. , Bos, R. , & Witholt, B. . (2010). Dna sequence determination and functional characterization of the oct-plasmid-encoded alkjkl genes of pseudomonas oleovorans. Molecular Microbiology, 6(21), 3121-3136.
(9)Bertram JH, Mulliner KM, Shi K, Plunkett MH, Nixon P, & Serratore NA., et al. (2017). Five fatty aldehyde dehydrogenase enzymes from marinobacter and acinetobacter spp. and structural insights into the aldehyde binding pocket. Applied & Environmental Microbiology,83(12).
(10)Jia, H. , Lee, F. S. , & Farinas, E. T. . (2014). Bacillus subtilis spore display of laccase for evolution under extreme conditions of high concentrations of organic solvent. Acs Combinatorial ence, 16(12), 665-9.
(11)Silu, Sheng, Han, Jia, Sidney, Topiol ., et al. (2017). Engineering cota laccase for acidic ph stability using bacillus subtilis spore display. Journal of microbiology and biotechnology.
(12)Gonzalez, J. M. , Mayer, F. , Moran, M. A. , Hodson, R. E. , & Whitman, W. B. . (1997). Microbulbifer hydrolyticus gen. nov. sp. nov. and marinobacterium georgiense gen. nov. sp. nov. two marine bacteria from a lignin-rich pulp mill waste enrichment community. Int J Syst Bacteriol, 47(2), 369-376.
(13)Li, Fei-Long, Shi, Ying, Zhang, Jiu-Xun., et al. (2018). Cloning, expression, characterization and homology modeling of a novel water-forming nadh oxidase from streptococcus mutans atcc 25175. International Journal of Biological Macromolecules Structure Function & Interactions.
(14)Li, Zhongyu & Wei, Ren & Gao, Meixi & Ren, Yanru & Yu, Bo & Nie, Kaili & Xu, Haijun & Liu, Luo. (2020). Biodegradation of low-density polyethylene by Microbulbifer hydrolyticus IRE-31. Journal of Environmental Management. 263. 110402. 10.1016/j.jenvman.2020.110402.
(15)Andrews, D. , Mattatall, N. R. , Arnold, D. , & Hill, B. C. . (2005). Expression, purification, and characterization of the cua-cytochrome c domain from subunit ii of the bacillus subtilis cytochrome caa3 complex in escherichia coli. Protn Expression & Purification, 42(2), 227-235.
(16)Sirima, M. , & Watanalai, P. . (2018). Display of escherichia coli phytase on the surface of bacillus subtilis spore using cotg as an anchor protein. Applied Biochemistry and Biotechnology, 187, 838-855.
(17)Zhang, G. , An, Y. , Zabed, H. M. , Guo, Q. , & Qi, X. . (2019). Bacillus subtilis spore surface display technology: a review of its development and applications. Journal of Microbiology and Biotechnology.
(18)Kim, J. , & Schumann, W. . (2009). Display of proteins on bacillus subtilis endospores. Cellular & Molecular Life ences Cmls, 66(19), 3127-36.
(19)Lin, P. , Yuan, H. , Du, J. , Liu, K. , & Wang, T. . (2020). Progress in research and application development of surface display technology using bacillus subtilis spores. Applied Microbiology and Biotechnology, 104(6), 2319-2331.
(20)Isticato, R. , & Ricca, E. . (2016). Spore Surface Display. The Bacterial Spore.
(21)Wang, H. , Wang, Y. , & Yang, R. . (2017). Recent progress in bacillus subtilis spore-surface display: concept, progress, and future. Applied Microbiology and Biotechnology, 101(3), 933-949.
(22)Maier, T. , F?Rster, H. H. , Asperger, O. , & Hahn, U. . (2001). Molecular characterization of the 56-kda cyp153 from acinetobacter sp. eb104. Biochemical & Biophysical Research Communications,286(3), 652-658.
(23)Funhoff, E. G. , Bauer, U. , Garcia-Rubio, I. , Witholt, B. , & Van Beilen, J. B. . (2006). Cyp153a6, a soluble p450 oxygenase catalyzing terminal-alkane hydroxylation. Journal of Bacteriology,188(14), 5220-5227.
(24)Van Beilen J B , Funhoff E G , Van Loon A , et al. Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases.[J]. Applied & Environmental Microbiology, 2006, 72(1):59-65.
(25)Malca, S. H. , Scheps, D. , Kuehnel, L. , Venegas-Venegas, E. , Seifert, A. , & Nestl, B. M. , et al. (2012). Bacterial cyp153a monooxygenases for the synthesis of omega-hydroxylated fatty acids. Chemical Communications, 48(42), p.5115-5117.
(26)Process limitations of a whole-cell p450 catalyzed reaction using a cyp153a-cpr fusion construct expressed in escherichia coli. Applied Microbiology & Biotechnology, 100(3), 1197-1208.
(27)Smits, T. H. M. , Seeger, M. A. , Witholt, B. , & Beilen, J. B. V. . (2001). New alkane-responsive expression vectors for escherichia coli and pseudomonas. Plasmid, 46(1), 16-24.
(28)Funhoff, E. G. , Salzmann, J. , Bauer, U. , Witholt, B. , & Beilen, J. B. V. . (2007). Hydroxylation and epoxidation reactions catalyzed by cyp153 enzymes. Enzyme & Microbial Technology, 40(4), 806-812.