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Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering

Nature Biotechnology volume 17, pages 588–592 (1999)Cite this article

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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Figure 1: Rerouting carbon flux toward l-alanine by overproduction of alanine dehydrogenase (l-AlaDH) in an l-lactate dehydrogenase (l-LDH) and alanine racemase (ALR)-deficient strain.
Figure 2: Nisin-dependent overproduction of alanine dehydrogenase.
Figure 3: Alanine production as a function of nisin concentration and ammonium supply.
Figure 4: End products formation by cell suspensions of the wild-type NZ3900 strain (black bars) and the l-LDH–deficient NZ3950 strain (hatched bars), each harboring pNZ2650.
Figure 5: Time course of glucose consumption and alanine and lactate production by (A) the wild-type NZ3900 strain and (B) the l-LDH–deficient NZ3950 strain, each containing pNZ2650.
Figure 6

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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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Authors and Affiliations

  1. Microbial Ingredients Section, NIZO Food Research, P.O. Box 20, 6710 BA, Ede, The Netherlands

    Pascal Hols, Michiel Kleerebezem, Jeroen Hugenholtz & Willem M. de Vos

  2. Laboratoire de Génétique Moléculaire, Place Croix du Sud 5, Université catholique de Louvain, Louvain-La-Neuve, 1348, Belgium

    Pascal Hols, Thierry Ferain & Jean Delcour

  3. Laboratoire de Chimie Physique et de Cristallographie, Place Louis Pasteur 1, Université catholique de Louvain, Louvain-La-Neuve, 1348, Belgium

    André N. Schanck

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Correspondence to Pascal Hols.

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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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