High-level acetaldehyde production in Lactococcus lactis by metabolic engineering

Abstract

Efficient conversion of glucose to acetaldehyde is achieved by nisin-controlled overexpression of Zymomonas mobilis pyruvate decarboxylase (pdc) and Lactococcus lactis NADH oxidase (nox) in L. lactis. In resting cells, almost 50% of the glucose consumed could be redirected towards acetaldehyde by combined overexpression of pdc and nox under anaerobic conditions.

FIG. 1.

Schematic representation of plasmid constructions for nisin-controlled overexpression of Pdc (pNZ7300), Nox (pNZ7301), and Pdc and Nox (pNZ7302). The nisA promoter is indicated by a grey box, and genes are indicated by arrows. Ligation sites of two compatible cohesive ends or blunt-ended DNA fragments that cannot be digested by either of the two original enzymes used are indicated by the enzyme names in brackets.

FIG. 2.

Nisin-controlled overexpression of pdc and nox in L. lactis NZ9000. Shown are the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude cell extracts of L. lactis NZ9000 harboring plasmids pNZ7300, pNZ7301, and pNZ7302 that were not induced (lanes 1, 6, and 11) and induced with 0.1, 0.2, 0.5, and 1.0 ng of nisin per ml (lanes 2 to 5, 7 to 10, and 12 to 15, respectively). Pdc- and Nox-representing protein bands are indicated by filled and open arrowheads, respectively. Specific activities (SA) of Pdc (filled bars) and Nox (open bars) are shown.