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Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth

L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD(+)-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can cataboliz...

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Autores principales: Niu, Dandan, Tian, Kangming, Prior, Bernard A, Wang, Min, Wang, Zhengxiang, Lu, Fuping, Singh, Suren
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075936/
https://www.ncbi.nlm.nih.gov/pubmed/24884499
http://dx.doi.org/10.1186/1475-2859-13-78
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author Niu, Dandan
Tian, Kangming
Prior, Bernard A
Wang, Min
Wang, Zhengxiang
Lu, Fuping
Singh, Suren
author_facet Niu, Dandan
Tian, Kangming
Prior, Bernard A
Wang, Min
Wang, Zhengxiang
Lu, Fuping
Singh, Suren
author_sort Niu, Dandan
collection PubMed
description L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD(+)-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the E. coli strain to produce L-lactate, three L-lactate dehydrogenase genes from different bacteria were cloned and expressed. The L-lactate producing strains, 090B1 (B0013-070, ΔldhA::diflldD::P(ldh)-ldh(Lca)), 090B2 (B0013-070, ΔldhA::diflldD::P(ldh)-ldh(Strb)) and 090B3 (B0013-070, ΔldhA::diflldD::P(ldh)-ldh(Bcoa)) were developed from a previously developed D-lactate over-producing strain, E. coli strain B0013-070 (ack-ptappspflBdldpoxBadhEfrdA) by: (1) deleting ldhA to block D-lactate formation, (2) deleting lldD to block the conversion of L-lactate to pyruvate, and (3) expressing an L-lactate dehydrogenase (L-LDH) to convert pyruvate to L-lactate under the control of the ldhA promoter. Fermentation tests were carried out in a shaking flask and in a 25-l bioreactor. Strains 090B1, 090B2 or 090B3 were shown to metabolize glucose to L-lactate instead of D-lactate. However, L-lactate yield and cell growth rates were significantly different among the metabolically engineered strains which can be attributed to a variation between temperature optimum for cell growth and temperature optimum for enzymatic activity of individual L-LDH. In a temperature-shifting fermentation process (cells grown at 37°C and L-lactate formed at 42°C), E. coli 090B3 was able to produce 142.2 g/l of L-lactate with no more than 1.2 g/l of by-products (mainly acetate, pyruvate and succinate) accumulated. In conclusion, the production of lactate by E. coli is limited by the competition relationship between cell growth and lactate synthesis. Enzymatic properties, especially the thermodynamics of an L-LDH can be effectively used as a factor to regulate a metabolic pathway and its metabolic flux for efficient L-lactate production. HIGHLIGHTS: The enzymatic thermodynamics was used as a tool for metabolic regulation. ► minimizing the activity of L-lactate dehydrogenase in growth phase improved biomass accumulation. ► maximizing the activity of L-lactate dehydrogenase improved lactate productivity in production phase.
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spelling pubmed-40759362014-07-01 Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth Niu, Dandan Tian, Kangming Prior, Bernard A Wang, Min Wang, Zhengxiang Lu, Fuping Singh, Suren Microb Cell Fact Research L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD(+)-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the E. coli strain to produce L-lactate, three L-lactate dehydrogenase genes from different bacteria were cloned and expressed. The L-lactate producing strains, 090B1 (B0013-070, ΔldhA::diflldD::P(ldh)-ldh(Lca)), 090B2 (B0013-070, ΔldhA::diflldD::P(ldh)-ldh(Strb)) and 090B3 (B0013-070, ΔldhA::diflldD::P(ldh)-ldh(Bcoa)) were developed from a previously developed D-lactate over-producing strain, E. coli strain B0013-070 (ack-ptappspflBdldpoxBadhEfrdA) by: (1) deleting ldhA to block D-lactate formation, (2) deleting lldD to block the conversion of L-lactate to pyruvate, and (3) expressing an L-lactate dehydrogenase (L-LDH) to convert pyruvate to L-lactate under the control of the ldhA promoter. Fermentation tests were carried out in a shaking flask and in a 25-l bioreactor. Strains 090B1, 090B2 or 090B3 were shown to metabolize glucose to L-lactate instead of D-lactate. However, L-lactate yield and cell growth rates were significantly different among the metabolically engineered strains which can be attributed to a variation between temperature optimum for cell growth and temperature optimum for enzymatic activity of individual L-LDH. In a temperature-shifting fermentation process (cells grown at 37°C and L-lactate formed at 42°C), E. coli 090B3 was able to produce 142.2 g/l of L-lactate with no more than 1.2 g/l of by-products (mainly acetate, pyruvate and succinate) accumulated. In conclusion, the production of lactate by E. coli is limited by the competition relationship between cell growth and lactate synthesis. Enzymatic properties, especially the thermodynamics of an L-LDH can be effectively used as a factor to regulate a metabolic pathway and its metabolic flux for efficient L-lactate production. HIGHLIGHTS: The enzymatic thermodynamics was used as a tool for metabolic regulation. ► minimizing the activity of L-lactate dehydrogenase in growth phase improved biomass accumulation. ► maximizing the activity of L-lactate dehydrogenase improved lactate productivity in production phase. BioMed Central 2014-05-29 /pmc/articles/PMC4075936/ /pubmed/24884499 http://dx.doi.org/10.1186/1475-2859-13-78 Text en Copyright © 2014 Niu 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
Niu, Dandan
Tian, Kangming
Prior, Bernard A
Wang, Min
Wang, Zhengxiang
Lu, Fuping
Singh, Suren
Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth
title Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth
title_full Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth
title_fullStr Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth
title_full_unstemmed Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth
title_short Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth
title_sort highly efficient l-lactate production using engineered escherichia coli with dissimilar temperature optima for l-lactate formation and cell growth
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075936/
https://www.ncbi.nlm.nih.gov/pubmed/24884499
http://dx.doi.org/10.1186/1475-2859-13-78
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