A novel framework for the cell-free enzymatic production of glucaric acid.

Petroll, K; Care, A; Bergquist, PL; Sunna, A

A novel framework for the cell-free enzymatic production of glucaric acid.

Petroll, K Care, A

Bergquist, PL Sunna, A

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Publisher:: ACADEMIC PRESS INC ELSEVIER SCIENCE
Publication Type:: Journal Article
Citation:: Metabolic engineering, 2020, 57, pp. 162-173
Issue Date:: 2020-01

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Field	Value	Language
dc.contributor.author	Petroll, K
dc.contributor.author	Care, A https://orcid.org/0000-0002-0035-7961
dc.contributor.author	Bergquist, PL
dc.contributor.author	Sunna, A
dc.date.accessioned	2021-01-18T21:55:00Z
dc.date.available	2019-11-08
dc.date.available	2021-01-18T21:55:00Z
dc.date.issued	2020-01
dc.identifier.citation	Metabolic engineering, 2020, 57, pp. 162-173
dc.identifier.issn	1096-7176
dc.identifier.issn	1096-7184
dc.identifier.uri	http://hdl.handle.net/10453/145468
dc.description.abstract	Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ± 0.9 mM (3.0 ± 0.2 g l-1) and a molar yield of 35.2 ± 2.3% using non-immobilised enzymes, and a titre of 8.1 ± 0.2 mM (1.70 ± 0.04 g l-1) and a molar yield of 20.2 ± 0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ± 0.02 g/h/l for free enzymes and 0.170 ± 0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.
dc.format	Print-Electronic
dc.language	eng
dc.publisher	ACADEMIC PRESS INC ELSEVIER SCIENCE
dc.relation.ispartof	Metabolic engineering
dc.relation.isbasedon	10.1016/j.ymben.2019.11.003
dc.rights	info:eu-repo/semantics/closedAccess
dc.subject	1003 Industrial Biotechnology
dc.subject.classification	Biotechnology
dc.title	A novel framework for the cell-free enzymatic production of glucaric acid.
dc.type	Journal Article
utslib.citation.volume	57
utslib.location.activity	Belgium
utslib.for	1003 Industrial Biotechnology
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
pubs.organisational-group	/University of Technology Sydney
utslib.copyright.status	closed_access	*
dc.date.updated	2021-01-18T21:54:35Z
pubs.publication-status	Published
pubs.volume	57

Abstract:

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ± 0.9 mM (3.0 ± 0.2 g l-1) and a molar yield of 35.2 ± 2.3% using non-immobilised enzymes, and a titre of 8.1 ± 0.2 mM (1.70 ± 0.04 g l-1) and a molar yield of 20.2 ± 0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ± 0.02 g/h/l for free enzymes and 0.170 ± 0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.

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