The Use Of Polyhydroxyalkanoate Synthase (Phacbp-M-Cpf4) And Newly Identified Enoyl-Coa Hydratase (Phajss) For The Production Of Poly[(R)-3-Hydroxybutyrate-Co-(R)-3-Hydroxyhexanoate]

Most industrial plastics are produced using non-renewable resources such as petroleum. Hence, polymer production processes based on renewable resources must be developed to reduce the carbon footprints left by human activities. Polyhydroxyalkanoates (PHAs) are intracellular biopolyesters synthesized...

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Main Author:	Tan, Hua Tiang
Format:	Thesis
Language:	English
Published:	2020
Subjects:	QH1 Natural history (General - Including nature conservation geographical distribution)
Online Access:	http://eprints.usm.my/55070/1/TAN%20HUA%20TIANG.pdf
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id	my-usm-ep.55070
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spelling	my-usm-ep.550702022-10-03T03:09:19Z The Use Of Polyhydroxyalkanoate Synthase (Phacbp-M-Cpf4) And Newly Identified Enoyl-Coa Hydratase (Phajss) For The Production Of Poly[(R)-3-Hydroxybutyrate-Co-(R)-3-Hydroxyhexanoate] 2020-12 Tan, Hua Tiang QH1 Natural history (General - Including nature conservation, geographical distribution) Most industrial plastics are produced using non-renewable resources such as petroleum. Hence, polymer production processes based on renewable resources must be developed to reduce the carbon footprints left by human activities. Polyhydroxyalkanoates (PHAs) are intracellular biopolyesters synthesized by numerous microorganisms as carbon storage under culture conditions of limiting essential nutrients but with excess carbon source. Besides, PHA is biodegradable. Among the various types of PHA, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] has a high potential to serve as a commercial bioplastic due to it having the most identical properties to petroleum-based plastics. PHA synthase of Chromobacterium sp. USM2 (PhaCCs) and PHA synthase isolated from mangrove metagenome (PhaCBP-M-CPF4) have been reported to be able to produce P(3HB-co-3HHx). In this study, PhaCCs and PhaCBP-M-CPF4 as well as enoyl-CoA hydratase of Streptomyces sp. strain CFMR 7 (PhaJSs) were characterized. Thus, five different genotypes of Cupriavidus necator transformants harboring phaCBP-M-CPF4 gene and three different genotypes of C. necator transformants harboring phaCCs gene were developed to evaluate the incorporation efficiency of 3-hydroxyhexanoate (3HHx) monomers. The amount of 3HHx monomer incorporated in the PHA synthesized by these C. necator transformants were examined using palm oil as the sole carbon source. PhaCBP-M-CPF4 enabled the incorporation of higher 3HHx monomer than PhaCCs (up to 18 mol% 3HHx). Besides, the molecular weight (Mw) of P(3HB-co-3HHx) produced by transformants harboring phaCBP-M-CPF4 could reach up to 1.8 × 106 Da, which was six times higher than the P(3HB-co-3HHx) produced by transformants harboring phaCCs. Enoyl-CoA hydratase is crucial for 3HHx accumulation during the production of P(3HB-co-3HHx). This enzyme channels the pathway for supplying (R)-3-hydroxyacyl-CoA monomer units, especially (R)-3-hydroxyhexanoyl-CoA from fatty acid β-oxidation. In this study, phaJSs was identified from the rubber degrading Gram-positive non-PHA producing bacterium, Streptomyces sp. strain CFMR 7. Co-expression of this enoyl-CoA hydratase gene with the chosen PhaC above, phaCBP-M-CPF4, in C. necator PHB¯4, significantly increased 3HHx composition without decreasing the PHA content. This transformant could produce P(3HB-co-3HHx) with 18 mol% of 3HHx and has a Mw of nearly one million Da revealing that both PhaCBP-M-CPF4 and PhaJSs could potentially be used for industrial applications. 2020-12 Thesis http://eprints.usm.my/55070/ http://eprints.usm.my/55070/1/TAN%20HUA%20TIANG.pdf application/pdf en public phd doctoral Perpustakaan Hamzah Sendut Pusat Pengajian Sains Kajihayat
institution	Universiti Sains Malaysia
collection	USM Institutional Repository
language	English
topic	QH1 Natural history (General - Including nature conservation geographical distribution)
spellingShingle	QH1 Natural history (General - Including nature conservation geographical distribution) Tan, Hua Tiang The Use Of Polyhydroxyalkanoate Synthase (Phacbp-M-Cpf4) And Newly Identified Enoyl-Coa Hydratase (Phajss) For The Production Of Poly[(R)-3-Hydroxybutyrate-Co-(R)-3-Hydroxyhexanoate]
description	Most industrial plastics are produced using non-renewable resources such as petroleum. Hence, polymer production processes based on renewable resources must be developed to reduce the carbon footprints left by human activities. Polyhydroxyalkanoates (PHAs) are intracellular biopolyesters synthesized by numerous microorganisms as carbon storage under culture conditions of limiting essential nutrients but with excess carbon source. Besides, PHA is biodegradable. Among the various types of PHA, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] has a high potential to serve as a commercial bioplastic due to it having the most identical properties to petroleum-based plastics. PHA synthase of Chromobacterium sp. USM2 (PhaCCs) and PHA synthase isolated from mangrove metagenome (PhaCBP-M-CPF4) have been reported to be able to produce P(3HB-co-3HHx). In this study, PhaCCs and PhaCBP-M-CPF4 as well as enoyl-CoA hydratase of Streptomyces sp. strain CFMR 7 (PhaJSs) were characterized. Thus, five different genotypes of Cupriavidus necator transformants harboring phaCBP-M-CPF4 gene and three different genotypes of C. necator transformants harboring phaCCs gene were developed to evaluate the incorporation efficiency of 3-hydroxyhexanoate (3HHx) monomers. The amount of 3HHx monomer incorporated in the PHA synthesized by these C. necator transformants were examined using palm oil as the sole carbon source. PhaCBP-M-CPF4 enabled the incorporation of higher 3HHx monomer than PhaCCs (up to 18 mol% 3HHx). Besides, the molecular weight (Mw) of P(3HB-co-3HHx) produced by transformants harboring phaCBP-M-CPF4 could reach up to 1.8 × 106 Da, which was six times higher than the P(3HB-co-3HHx) produced by transformants harboring phaCCs. Enoyl-CoA hydratase is crucial for 3HHx accumulation during the production of P(3HB-co-3HHx). This enzyme channels the pathway for supplying (R)-3-hydroxyacyl-CoA monomer units, especially (R)-3-hydroxyhexanoyl-CoA from fatty acid β-oxidation. In this study, phaJSs was identified from the rubber degrading Gram-positive non-PHA producing bacterium, Streptomyces sp. strain CFMR 7. Co-expression of this enoyl-CoA hydratase gene with the chosen PhaC above, phaCBP-M-CPF4, in C. necator PHB¯4, significantly increased 3HHx composition without decreasing the PHA content. This transformant could produce P(3HB-co-3HHx) with 18 mol% of 3HHx and has a Mw of nearly one million Da revealing that both PhaCBP-M-CPF4 and PhaJSs could potentially be used for industrial applications.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Tan, Hua Tiang
author_facet	Tan, Hua Tiang
author_sort	Tan, Hua Tiang
title	The Use Of Polyhydroxyalkanoate Synthase (Phacbp-M-Cpf4) And Newly Identified Enoyl-Coa Hydratase (Phajss) For The Production Of Poly[(R)-3-Hydroxybutyrate-Co-(R)-3-Hydroxyhexanoate]
title_short	The Use Of Polyhydroxyalkanoate Synthase (Phacbp-M-Cpf4) And Newly Identified Enoyl-Coa Hydratase (Phajss) For The Production Of Poly[(R)-3-Hydroxybutyrate-Co-(R)-3-Hydroxyhexanoate]
title_full	The Use Of Polyhydroxyalkanoate Synthase (Phacbp-M-Cpf4) And Newly Identified Enoyl-Coa Hydratase (Phajss) For The Production Of Poly[(R)-3-Hydroxybutyrate-Co-(R)-3-Hydroxyhexanoate]
title_fullStr	The Use Of Polyhydroxyalkanoate Synthase (Phacbp-M-Cpf4) And Newly Identified Enoyl-Coa Hydratase (Phajss) For The Production Of Poly[(R)-3-Hydroxybutyrate-Co-(R)-3-Hydroxyhexanoate]
title_full_unstemmed	The Use Of Polyhydroxyalkanoate Synthase (Phacbp-M-Cpf4) And Newly Identified Enoyl-Coa Hydratase (Phajss) For The Production Of Poly[(R)-3-Hydroxybutyrate-Co-(R)-3-Hydroxyhexanoate]
title_sort	use of polyhydroxyalkanoate synthase (phacbp-m-cpf4) and newly identified enoyl-coa hydratase (phajss) for the production of poly[(r)-3-hydroxybutyrate-co-(r)-3-hydroxyhexanoate]
granting_institution	Perpustakaan Hamzah Sendut
granting_department	Pusat Pengajian Sains Kajihayat
publishDate	2020
url	http://eprints.usm.my/55070/1/TAN%20HUA%20TIANG.pdf
_version_	1747822309834489856

The Use Of Polyhydroxyalkanoate Synthase (Phacbp-M-Cpf4) And Newly Identified Enoyl-Coa Hydratase (Phajss) For The Production Of Poly[(R)-3-Hydroxybutyrate-Co-(R)-3-Hydroxyhexanoate]

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