Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering.
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.
Hols
P
P
Microbial Ingredients Section, NIZO Food Research, Ede, The Netherlands. hols@gene.ucl.ac.be
Kleerebezem
M
M
Schanck
A N
AN
Ferain
T
T
Hugenholtz
J
J
Delcour
J
J
de Vos
W M
WM
D000409:Alanine / Q000378:metabolism*; D050788:Alanine Dehydrogenase; D000411:Alanine Racemase / Q000235:genetics; D000594:Amino Acid Oxidoreductases / Q000235:genetics; D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D005285:Fermentation; D007536:Isomerism; D007773:Lactates / Q000378:metabolism*; D013294:Lactococcus lactis / Q000378:metabolism*; D008969:Molecular Sequence Data
D017931:DNA Primers; D007773:Lactates; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D000411:Alanine Racemase; D000409:Alanine