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Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

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Title: Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics
Authors: Kelwick, RJR
Ricci, L
Chee, SM
Bell, D
Webb, A
Freemont, P
Item Type: Journal Article
Abstract: The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable. Cell-free metabolic engineering strategies have already been used to optimise several biosynthetic pathways and we envisioned that cell-free strategies could be used for optimising PHAs biosynthetic pathways. To this end, we developed several Escherichia coli cell-free systems for in vitro prototyping PHAs biosynthetic operons, and also for screening relevant metabolite recycling enzymes. Furthermore, we customised our cell-free reactions through the addition of whey permeate, an industrial waste that has been previously used to optimise in vivo PHAs production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ∼50%. In cell-free transcription-translation prototyping reactions, GC-MS quantification of cell-free 3-hydroxybutyrate (3HB) production revealed differences between the activities of the Native ΔPhaC_C319A (1.18 ±0.39 µM), C104 ΔPhaC_C319A (4.62 ±1.31 µM) and C101 ΔPhaC_C319A (2.65 ±1.27 µM) phaCAB operons that were tested. Interestingly, the most active operon, C104 produced higher levels of PHAs (or PHAs monomers) than the Native phaCAB operon in both in vitro and in vivo assays. Coupled cell-free biotransformation/transcription-translation reactions produced greater yields of 3HB (32.87 ±6.58 µM) and these reactions were also used to characterise a Clostridium propionicum Acetyl-CoA recycling enzyme. Together, these data demonstrate that cell-free approaches complement in vivo workflows for identifying additional strategies for optimising PHAs production.
Issue Date: 4-Sep-2019
Date of Acceptance: 21-Aug-2018
URI: http://hdl.handle.net/10044/1/63789
DOI: https://dx.doi.org/10.1093/synbio/ysy016
ISSN: 2397-7000
Publisher: Oxford University Press
Journal / Book Title: Synthetic Biology
Volume: 3
Issue: 1
Copyright Statement: © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Sponsor/Funder: Engineering & Physical Science Research Council (E
Engineering & Physical Science Research Council (EPSRC)
Biotechnology and Biological Sciences Research Council (BBSRC)
Engineering & Physical Science Research Council (EPSRC)
Funder's Grant Number: EP/J02175X/1
EP/L011573/1
BB/L027852/1
EP/N023854/1
Publication Status: Published online
Online Publication Date: 2018-09-04
Appears in Collections:Department of Medicine
Faculty of Medicine



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