Cargando…

Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol

BACKGROUND: Lactobacillus reuteri converts glycerol to 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO) via 3-hydroxypropionaldehyde (3HPA) as an intermediate using enzymes encoded in its propanediol-utilization (pdu) operon. Since 3HP, 1,3PDO and 3HPA are important building blocks for the...

Descripción completa

Detalles Bibliográficos
Autores principales: Dishisha, Tarek, Pereyra, Luciana P, Pyo, Sang-Hyun, Britton, Robert A, Hatti-Kaul, Rajni
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045878/
https://www.ncbi.nlm.nih.gov/pubmed/24886501
http://dx.doi.org/10.1186/1475-2859-13-76
_version_ 1782319400531001344
author Dishisha, Tarek
Pereyra, Luciana P
Pyo, Sang-Hyun
Britton, Robert A
Hatti-Kaul, Rajni
author_facet Dishisha, Tarek
Pereyra, Luciana P
Pyo, Sang-Hyun
Britton, Robert A
Hatti-Kaul, Rajni
author_sort Dishisha, Tarek
collection PubMed
description BACKGROUND: Lactobacillus reuteri converts glycerol to 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO) via 3-hydroxypropionaldehyde (3HPA) as an intermediate using enzymes encoded in its propanediol-utilization (pdu) operon. Since 3HP, 1,3PDO and 3HPA are important building blocks for the bio-based chemical industry, L. reuteri can be an attractive candidate for their production. However, little is known about the kinetics of glycerol utilization in the Pdu pathway in L. reuteri. In this study, the metabolic fluxes through the Pdu pathway were determined as a first step towards optimizing the production of 3HPA, and co-production of 3HP and 1,3PDO from glycerol. Resting cells of wild-type (DSM 20016) and recombinant (RPRB3007, with overexpressed pdu operon) strains were used as biocatalysts. RESULTS: The conversion rate of glycerol to 3HPA by the resting cells of L. reuteri was evaluated by in situ complexation of the aldehyde with carbohydrazide to avoid the aldehyde-mediated inactivation of glycerol dehydratase. Under operational conditions, the specific 3HPA production rate of the RPRB3007 strain was 1.9 times higher than that of the wild-type strain (1718.2 versus 889.0 mg/g(CDW).h, respectively). Flux analysis of glycerol conversion to 1,3PDO and 3HP in the cells using multi-step variable-volume fed-batch operation showed that the maximum specific production rates of 3HP and 1,3PDO were 110.8 and 93.7 mg/g(CDW).h, respectively, for the wild-type strain, and 179.2 and 151.4 mg/g(CDW).h, respectively, for the RPRB3007 strain. The cumulative molar yield of the two compounds was ~1 mol/mol glycerol and their molar ratio was ~1 mol(3HP)/mol(1,3PDO). A balance of redox equivalents between the glycerol oxidative and reductive pathway branches led to equimolar amounts of the two products. CONCLUSIONS: Metabolic flux analysis was a useful approach for finding conditions for maximal conversion of glycerol to 3HPA, 3HP and 1,3PDO. Improved specific production rates were obtained with resting cells of the engineered RPRB3007 strain, highlighting the potential of metabolic engineering to render an industrially sound strain. This is the first report on the production of 3HP and 1,3PDO as sole products using the wild-type or mutant L. reuteri strains, and has laid ground for further work on improving the productivity of the biotransformation process using resting cells.
format Online
Article
Text
id pubmed-4045878
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-40458782014-06-20 Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol Dishisha, Tarek Pereyra, Luciana P Pyo, Sang-Hyun Britton, Robert A Hatti-Kaul, Rajni Microb Cell Fact Research BACKGROUND: Lactobacillus reuteri converts glycerol to 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO) via 3-hydroxypropionaldehyde (3HPA) as an intermediate using enzymes encoded in its propanediol-utilization (pdu) operon. Since 3HP, 1,3PDO and 3HPA are important building blocks for the bio-based chemical industry, L. reuteri can be an attractive candidate for their production. However, little is known about the kinetics of glycerol utilization in the Pdu pathway in L. reuteri. In this study, the metabolic fluxes through the Pdu pathway were determined as a first step towards optimizing the production of 3HPA, and co-production of 3HP and 1,3PDO from glycerol. Resting cells of wild-type (DSM 20016) and recombinant (RPRB3007, with overexpressed pdu operon) strains were used as biocatalysts. RESULTS: The conversion rate of glycerol to 3HPA by the resting cells of L. reuteri was evaluated by in situ complexation of the aldehyde with carbohydrazide to avoid the aldehyde-mediated inactivation of glycerol dehydratase. Under operational conditions, the specific 3HPA production rate of the RPRB3007 strain was 1.9 times higher than that of the wild-type strain (1718.2 versus 889.0 mg/g(CDW).h, respectively). Flux analysis of glycerol conversion to 1,3PDO and 3HP in the cells using multi-step variable-volume fed-batch operation showed that the maximum specific production rates of 3HP and 1,3PDO were 110.8 and 93.7 mg/g(CDW).h, respectively, for the wild-type strain, and 179.2 and 151.4 mg/g(CDW).h, respectively, for the RPRB3007 strain. The cumulative molar yield of the two compounds was ~1 mol/mol glycerol and their molar ratio was ~1 mol(3HP)/mol(1,3PDO). A balance of redox equivalents between the glycerol oxidative and reductive pathway branches led to equimolar amounts of the two products. CONCLUSIONS: Metabolic flux analysis was a useful approach for finding conditions for maximal conversion of glycerol to 3HPA, 3HP and 1,3PDO. Improved specific production rates were obtained with resting cells of the engineered RPRB3007 strain, highlighting the potential of metabolic engineering to render an industrially sound strain. This is the first report on the production of 3HP and 1,3PDO as sole products using the wild-type or mutant L. reuteri strains, and has laid ground for further work on improving the productivity of the biotransformation process using resting cells. BioMed Central 2014-05-27 /pmc/articles/PMC4045878/ /pubmed/24886501 http://dx.doi.org/10.1186/1475-2859-13-76 Text en Copyright © 2014 Dishisha et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Dishisha, Tarek
Pereyra, Luciana P
Pyo, Sang-Hyun
Britton, Robert A
Hatti-Kaul, Rajni
Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol
title Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol
title_full Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol
title_fullStr Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol
title_full_unstemmed Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol
title_short Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol
title_sort flux analysis of the lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045878/
https://www.ncbi.nlm.nih.gov/pubmed/24886501
http://dx.doi.org/10.1186/1475-2859-13-76
work_keys_str_mv AT dishishatarek fluxanalysisofthelactobacillusreuteripropanediolutilizationpathwayforproductionof3hydroxypropionaldehyde3hydroxypropionicacidand13propanediolfromglycerol
AT pereyralucianap fluxanalysisofthelactobacillusreuteripropanediolutilizationpathwayforproductionof3hydroxypropionaldehyde3hydroxypropionicacidand13propanediolfromglycerol
AT pyosanghyun fluxanalysisofthelactobacillusreuteripropanediolutilizationpathwayforproductionof3hydroxypropionaldehyde3hydroxypropionicacidand13propanediolfromglycerol
AT brittonroberta fluxanalysisofthelactobacillusreuteripropanediolutilizationpathwayforproductionof3hydroxypropionaldehyde3hydroxypropionicacidand13propanediolfromglycerol
AT hattikaulrajni fluxanalysisofthelactobacillusreuteripropanediolutilizationpathwayforproductionof3hydroxypropionaldehyde3hydroxypropionicacidand13propanediolfromglycerol