Rewiring Lactococcus lactis for ethanol production

Abstract

Lactic acid bacteria (LAB) are known for their high tolerance toward organic acids and alcohols (R. S. Gold, M. M. Meagher, R. Hutkins, and T. Conway, J. Ind. Microbiol. 10:45-54, 1992) and could potentially serve as platform organisms for production of these compounds. In this study, we attempted to redirect the metabolism of LAB model organism Lactococcus lactis toward ethanol production. Codon-optimized Zymomonas mobilis pyruvate decarboxylase (PDC) was introduced and expressed from synthetic promoters in different strain backgrounds. In the wild-type L. lactis strain MG1363 growing on glucose, only small amounts of ethanol were obtained after introducing PDC, probably due to a low native alcohol dehydrogenase activity. When the same strains were grown on maltose, ethanol was the major product and lesser amounts of lactate, formate, and acetate were formed. Inactivating the lactate dehydrogenase genes ldhX, ldhB, and ldh and introducing codon-optimized Z. mobilis alcohol dehydrogenase (ADHB) in addition to PDC resulted in high-yield ethanol formation when strains were grown on glucose, with only minor amounts of by-products formed. Finally, a strain with ethanol as the sole observed fermentation product was obtained by further inactivating the phosphotransacetylase (PTA) and the native alcohol dehydrogenase (ADHE).

Fig 1

An overview of sugar metabolism in L. lactis. The steps involved in maltose and glucose metabolism are indicated. Maltose is taken up by an ABC transport system, whereas glucose is transported mainly by a phosphotransferase system. Maltose_out and Glucose_out indicate extracellular sugars, whereas Maltose and Glucose indicate internalized sugars. Enzymes are indicated by uppercase italicized letters: MAPA, maltose phosphorylase; GK, glucokinase; β-PGM, β-phosphoglucomutase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PDHC, pyruvate dehydrogenase complex; PFL, pyruvate formate lyase; ADHE, native alcohol dehydrogenase. PDC (pyruvate decarboxylase) and ADHB (alcohol dehydrogenase) are both encoded by codon-optimized synthetic genes based on the Zymomonas mobilis sequences. Crosses indicate knockout of enzyme functions in the strains constructed in this work.

Fig 2

Fluxes and specific growth rate as a function of pyruvate decarboxylase expression. Strains containing a transcriptional fusion of pdc and gusA were generated as described in Materials and Methods. The x axis carries Miller units for quantification of GusA activity. The left y axis corresponds to the fluxes, whereas the right y axis corresponds to the specific growth rate. (A) Growth on glucose; (B) growth on maltose. The curves are included as a visual aid and have no physiological relevance. The data shown are averages of three or more experiments, and standard deviations are indicated as error bars (GusA error bars shown for glucose/maltose data only).

Fig 3

Fluxes and specific growth rate as a function of pyruvate decarboxylase and alcohol dehydrogenase expression in the Δ³ldh strain. Strains containing a transcriptional fusion of pdc-adhB and gusA were generated as described in Materials and Methods. The x axis carries Miller units for quantification of GusA activity. The left y axis corresponds to the fluxes, whereas the right y axis corresponds to the specific growth rate. The curves are included as a visual aid and have no physiological relevance. The data shown are averages of three or more experiments, and standard deviations are indicated as error bars. The GusA activities shown are the same as the ones in Fig. 2A.