Sunday, July 26, 2009
P105
L-LACTATE PRODUCTION USING METABOLICALLY ENGINEERED Escherichia coli
Key words: Escherichia coli, L-lactate, lactate dehydrogenase
Lactate is being used to obtain the renewable and biodegradable polymer called PoliLactic Acid (PLA). PLA requires higher proportions of L-lactate to confer adequate physical and biodegradation properties. Currently, lactate is produced mainly by Lactobacillus species requiring complex media. Inexpensive media and simple conditions might be used with E. coli as biocatalyst. Under fermentative conditions, E. coli produces a mixture of compounds including D-lactate. The aim of this work was to engineer the E. coli metabolism to obtain a homolactic strain, with L-lactate as main product. For this purpose, a D-lactate producing strain was engineered using E coli MG1655 as the progenitor strain. Pathways that compete for carbon availability were eliminated and the gene that codes for the L-lactic acid dehydrogenase gene (ldhE) from B. subtilis was used to replace the D-ldhA native gene. Previous works, using multicopy and inducible plasmids, had proven a cell toxicity effect when lactate dehydrogenase genes from Bacillus species are over-expressed in E. coli. The resulting strain (MG1655 DpflB, DadhE, DfrdA, DldhA::ldhE) was evaluated in non-aerated cultures using mineral medium containing 40 g/L of glucose. This strain converts 95% of glucose in optically pure L-lactate in 48 h with only 1.4 g/L of cells with a productivity of 0.8 g/ L.h. Our single copy construction into the E. coli chromosome showed very similar growth rates compared to the wild type strain, demonstrating non toxic effects and reliability for the production of the L-isomer.
Lactate is being used to obtain the renewable and biodegradable polymer called PoliLactic Acid (PLA). PLA requires higher proportions of L-lactate to confer adequate physical and biodegradation properties. Currently, lactate is produced mainly by Lactobacillus species requiring complex media. Inexpensive media and simple conditions might be used with E. coli as biocatalyst. Under fermentative conditions, E. coli produces a mixture of compounds including D-lactate. The aim of this work was to engineer the E. coli metabolism to obtain a homolactic strain, with L-lactate as main product. For this purpose, a D-lactate producing strain was engineered using E coli MG1655 as the progenitor strain. Pathways that compete for carbon availability were eliminated and the gene that codes for the L-lactic acid dehydrogenase gene (ldhE) from B. subtilis was used to replace the D-ldhA native gene. Previous works, using multicopy and inducible plasmids, had proven a cell toxicity effect when lactate dehydrogenase genes from Bacillus species are over-expressed in E. coli. The resulting strain (MG1655 DpflB, DadhE, DfrdA, DldhA::ldhE) was evaluated in non-aerated cultures using mineral medium containing 40 g/L of glucose. This strain converts 95% of glucose in optically pure L-lactate in 48 h with only 1.4 g/L of cells with a productivity of 0.8 g/ L.h. Our single copy construction into the E. coli chromosome showed very similar growth rates compared to the wild type strain, demonstrating non toxic effects and reliability for the production of the L-isomer.
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