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Development and characterisation of a recombinant Escherichia coli for the production of Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHA) are biodegradable polymers synthesized by a variety of microorganisms as an intracellular carbon and energy storage materials. The nature of Escherichia coli, which is devoid of PHA-degrading enzymes, and the extensive genome studies on this microbe that can be easily ma...

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Main Author: Yee, Lian Ngit
Format: Thesis
Language:English
Published: 2013
Online Access:http://psasir.upm.edu.my/id/eprint/42823/1/FBSB%202013%2011R.pdf
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spelling my.upm.eprints.428232016-06-24T03:34:03Z http://psasir.upm.edu.my/id/eprint/42823/ Development and characterisation of a recombinant Escherichia coli for the production of Polyhydroxyalkanoates Yee, Lian Ngit Polyhydroxyalkanoates (PHA) are biodegradable polymers synthesized by a variety of microorganisms as an intracellular carbon and energy storage materials. The nature of Escherichia coli, which is devoid of PHA-degrading enzymes, and the extensive genome studies on this microbe that can be easily manipulated and improved have led it to be a powerful host for PHA accumulation. In order to overcome the low cell density, instability of PHA production and inability of glucose uptake for wild type, Comamonas sp. EB172, a high acid-tolerant strain isolated from POME sludge in the open digester, recombinant E. coli was constructed. In this study, the unknown PHA biosynthesis genes of Comamonas sp. EB172 were successfully isolated. It was found that the isolated acetyl-CoA acetyltransferase (phaACo), acetoacetyl-CoA reductase (phaBCo) and PHA synthase, class I (phaCCo) genes were clustered together as phaCCo-phaACo-phaBCo. The modified pGEM’ vector with PHA biosynthetic genes express under the control of native promoter from Cupriavidus necator. Recombinant E. coli harbouring isolated PHA biosynthesis genes were then compared with recombinant E. coli harbouring C. necator PHA biosynthesis genes as a control in shake flask fermentation. Approximately 41% of PHA content was detected in recombinant E. coli JM109 harbouring pGEM’-phaCABCo and it was comparable to that recombinant E. coli JM109 containing phbCABRe with 46% PHA content (as a control). The effect of carbon and nitrogen on cell growth and PHA accumulation of recombinant E. coli was evaluated. The use of glucose can increase cell growth and PHA accumulation with nitrogen supplementation. However, mixed organic acids failed to increase cell growth but the PHA accumulated in the cell can be improved by supplying the nitrogen. However, E. coli JM109 transformant harbouring pGEM’-phaCABCo successfully polymerised P(3HB-co-3HV) when fed with mixed organic acids without nitrogen source. Overall,supplementation of nitrogen source in the medium improved the cell dry weight with glucose as carbon source, and increased the 3HV monomer for the polymer produced from mixed organic acids. Fed-batch fermentation not only improved the cell growth using 20 g/L glucose and 1 g/L (NH4)2SO4, but also improved the productivity using mixed organic acids for P(3HB-co-3HV) copolymer production. The productivity with 0.1 g PHA/(L.h) was achieved using 10 g/L mixed organic acids as feeding carbon source. The constant feeding of 10 g/L mixed organic acids triggered 3HV monomer formation started after 16 h and enhanced PHA content to more than 70% and P(3HB-co-3HV) copolymer with about 2 mol% 3HV monomer. The fed-batch cultivation increased the cell growth, PHA accumulation and improved the molecular weight of polymer in the range of 850 - 1490 kDa. The overall results in this study indicated that the isolated PHA biosynthesis genes from wild type bacteria can serve as PHA production system using glucose and mixed fatty acids. This finding can contribute towards the accumulation of PHA using natural or renewable carbon sources. 2013-03 Thesis NonPeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/42823/1/FBSB%202013%2011R.pdf Yee, Lian Ngit (2013) Development and characterisation of a recombinant Escherichia coli for the production of Polyhydroxyalkanoates. PhD thesis, Universiti Putra Malaysia.
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description Polyhydroxyalkanoates (PHA) are biodegradable polymers synthesized by a variety of microorganisms as an intracellular carbon and energy storage materials. The nature of Escherichia coli, which is devoid of PHA-degrading enzymes, and the extensive genome studies on this microbe that can be easily manipulated and improved have led it to be a powerful host for PHA accumulation. In order to overcome the low cell density, instability of PHA production and inability of glucose uptake for wild type, Comamonas sp. EB172, a high acid-tolerant strain isolated from POME sludge in the open digester, recombinant E. coli was constructed. In this study, the unknown PHA biosynthesis genes of Comamonas sp. EB172 were successfully isolated. It was found that the isolated acetyl-CoA acetyltransferase (phaACo), acetoacetyl-CoA reductase (phaBCo) and PHA synthase, class I (phaCCo) genes were clustered together as phaCCo-phaACo-phaBCo. The modified pGEM’ vector with PHA biosynthetic genes express under the control of native promoter from Cupriavidus necator. Recombinant E. coli harbouring isolated PHA biosynthesis genes were then compared with recombinant E. coli harbouring C. necator PHA biosynthesis genes as a control in shake flask fermentation. Approximately 41% of PHA content was detected in recombinant E. coli JM109 harbouring pGEM’-phaCABCo and it was comparable to that recombinant E. coli JM109 containing phbCABRe with 46% PHA content (as a control). The effect of carbon and nitrogen on cell growth and PHA accumulation of recombinant E. coli was evaluated. The use of glucose can increase cell growth and PHA accumulation with nitrogen supplementation. However, mixed organic acids failed to increase cell growth but the PHA accumulated in the cell can be improved by supplying the nitrogen. However, E. coli JM109 transformant harbouring pGEM’-phaCABCo successfully polymerised P(3HB-co-3HV) when fed with mixed organic acids without nitrogen source. Overall,supplementation of nitrogen source in the medium improved the cell dry weight with glucose as carbon source, and increased the 3HV monomer for the polymer produced from mixed organic acids. Fed-batch fermentation not only improved the cell growth using 20 g/L glucose and 1 g/L (NH4)2SO4, but also improved the productivity using mixed organic acids for P(3HB-co-3HV) copolymer production. The productivity with 0.1 g PHA/(L.h) was achieved using 10 g/L mixed organic acids as feeding carbon source. The constant feeding of 10 g/L mixed organic acids triggered 3HV monomer formation started after 16 h and enhanced PHA content to more than 70% and P(3HB-co-3HV) copolymer with about 2 mol% 3HV monomer. The fed-batch cultivation increased the cell growth, PHA accumulation and improved the molecular weight of polymer in the range of 850 - 1490 kDa. The overall results in this study indicated that the isolated PHA biosynthesis genes from wild type bacteria can serve as PHA production system using glucose and mixed fatty acids. This finding can contribute towards the accumulation of PHA using natural or renewable carbon sources.
format Thesis
author Yee, Lian Ngit
spellingShingle Yee, Lian Ngit
Development and characterisation of a recombinant Escherichia coli for the production of Polyhydroxyalkanoates
author_facet Yee, Lian Ngit
author_sort Yee, Lian Ngit
title Development and characterisation of a recombinant Escherichia coli for the production of Polyhydroxyalkanoates
title_short Development and characterisation of a recombinant Escherichia coli for the production of Polyhydroxyalkanoates
title_full Development and characterisation of a recombinant Escherichia coli for the production of Polyhydroxyalkanoates
title_fullStr Development and characterisation of a recombinant Escherichia coli for the production of Polyhydroxyalkanoates
title_full_unstemmed Development and characterisation of a recombinant Escherichia coli for the production of Polyhydroxyalkanoates
title_sort development and characterisation of a recombinant escherichia coli for the production of polyhydroxyalkanoates
publishDate 2013
url http://psasir.upm.edu.my/id/eprint/42823/1/FBSB%202013%2011R.pdf
http://psasir.upm.edu.my/id/eprint/42823/
_version_ 1643833395627687936
score 13.211869

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