ARS | Publication request: CONSTRUCTION OF AN OPERON FOR PRODUCTION OF ETHANOL IN LACTOCOCCUS LACTIS

Submitted to: Society of Industrial Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: August 2, 2001
Publication Date: N/A

Technical Abstract: Lactic acid bacteria are candidates for engineered production of ethanol from biomass because they are GRAS organisms that grow at low pH and are tolerant of ethanol and inhibitors. A plasmid was constructed to express the Zymomonas mobilis pdc and adhB genes in Lactococcus lactis. The Z. mobilis pet operon was translationally fused to a synthetic Lactococcal consensus promoter and ribosome binding site. Expression of the pdc gene in L. lactis was confirmed by northern and SDS page analysis. Pyruvate decarboxylase activity was detected in extracts from transformed L. lactis and Escherichia coli cells, using two different assays that are free from interference by lactate dehydrogenase. The rate of conversion of pyruvate to acetaldehyde was 0.13 and 1.2 mmol/min/mg protein for L. lactis and E. coli, respectively, measured in a colorimetric assay. Similar activities were observed in a fluoride release assay, in which fluoropyruvate was the substrate. The transformed L. lactis strain produced low levels of ethanol from glucose. The plasmid complemented the mutations in a nonfermentative E. coli strain, indicating that both pdc and adhB were expressed in E. coli.