Escherichia coli W was genetically engineered to produce l-alanine as the primary fermentation product from sugars by replacing the native d-lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native d-lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of l-alanine to d-alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l−1 glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g−1 glucose) with a chiral purity greater than 99.5% l-alanine.
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The authors thank Lorraine Yomano and Sean York for their instruction in molecular cloning and fermentation and the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) for sequencing and amino acid analysis. This research was supported by grants from the US Department of Energy (FG02-96ER20222 and FG36-04GO14019) and BioEnergy International, LLC.
Electronic supplementary material
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Metabolic evolution of XZ112 to select XZ113. Strain XZ113 was isolated after 12 serial transfers at 24-h intervals in NBS mineral salts medium containing 50 g l−1 glucose and 1 mM betaine (inoculum of 0.017 mg CDW l−1; pH controlled with 5 N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. A Cell mass (g l−1). B Alanine production (3 days) (DOC 33 kb)
Effect of pH on cell growth and alanine production by strain XZ123 (NBS mineral salts medium containing 80 g l−1 glucose and 1 mM betaine; inoculum of 0.017 mg CDW l−1). Broth pH was automatically controlled by the addition of 5 N ammonium hydroxide. A Cell mass. B Alanine production. Symbols: □, pH 6.5; △, pH 7.0; ▽, pH 7.5; and ◇, pH 8.0 (DOC 32 kb)
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Zhang, X., Jantama, K., Moore, J.C. et al. Production of l-alanine by metabolically engineered Escherichia coli . Appl Microbiol Biotechnol 77, 355–366 (2007). https://doi.org/10.1007/s00253-007-1170-y
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