Escherichia coli Strains Engineered for Homofermentative Production of D-Lactic Acid from Glycerol

Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the ef...

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Veröffentlicht in:Applied and Environmental Microbiology 2010-07, Vol.76 (13), p.4327-4336
Hauptverfasser: Mazumdar, Suman, Clomburg, James M, Gonzalez, Ramon
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creator Mazumdar, Suman
Clomburg, James M
Gonzalez, Ramon
description Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to D-lactic acid (D-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for D-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of D-lactate from pyruvate (D-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic D-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of D-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of D-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for D-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of D-lactate and is redox balanced, thus representing a viable metabolic pathway.
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On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic D-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of D-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of D-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for D-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. 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On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic D-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of D-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of D-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for D-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. 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source American Society for Microbiology; MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Alma/SFX Local Collection
subjects Alcohol
Anaerobiosis
ATP
Biotechnology
Biotechnology - methods
Chemical synthesis
Culture Media
E coli
Enzymes
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Escherichia coli - growth & development
Escherichia coli - metabolism
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
Fermentation
Genetic Engineering - methods
Glycerol - metabolism
Lactic Acid - biosynthesis
Metabolic Networks and Pathways
Metabolism
Raw materials
title Escherichia coli Strains Engineered for Homofermentative Production of D-Lactic Acid from Glycerol
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