Abstract
We report the engineering of Lactococcus lactis to produce the amino acid l-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH→l-lactate + NAD+) is performed by l-lactate dehydrogenase (l-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (l-AlaDH; pyruvate + NADH + NH4+→l-alanine + NAD+ + H2O). Expression of l-AlaDH in an l-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of l-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.
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Acknowledgements
We thank G.A. Sprenger for providing plasmid pBM20alaD; P. Renault for sequence information on the alr gene from L. lactis IL1403 preceding publication; and R. Holleman, H. Kosters, and M. Starrenburg for their technical help in HPLC analyses and fermentation. This research was carried out in the framework of the Community Research Programme BIOTECH (contract no. BIO4-CT96-0498). P.H. holds a fellowship of the EC BIOTECH programme (contract no. BIO4-CT96-5093).
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Hols, P., Kleerebezem, M., Schanck, A. et al. Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering. Nat Biotechnol 17, 588–592 (1999). https://doi.org/10.1038/9902
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DOI: https://doi.org/10.1038/9902
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