GENETIC ENGINEERING OF LACTIC ACID BACTERIA TOWARD FERMENTATIVE PRODUCTION OF ETHANOL

Authors: Liu, Siqing; Cotta, Michael

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 5/3/2004
Publication Date: 5/3/2004
Citation: Liu, S., Cotta, M.A. 2004. Genetic engineering of lactic acid bacteria toward fermentative production of ethanol [abstract]. American Society for Microbiology. p. 29.

Interpretive Summary:

Technical Abstract: One of the major tasks of converting renewable biomass to ethanol and other value added products is to create new microbial strains that are capable of fermenting biomass derived sugars (mixture of hexoses and pentoses). Naturally ethanol fermenting Saccharomyces cerevisiae and Zymomonas mobilis only use hexose, not pentose sugars. Several Lactic acid bacteria (LAB) contain genes that enable them to ferment the mixed sugars contained in renewable biomass, and the potential development of these as ethanologens via genetic engineering was explored. In most LAB, pyruvate is converted into lactate via lactate dehydrogenase (encoded by ldh). The Lactobacillus plantarum TF103 strain lacking functional ldh genes produces ethanol, acetoin, 2,3-butanediol, and mannitol. Another ldh mutant of Lactococcus lactis (Neves, Ramos et al. 2002) was also reported to produce ethanol. We aim to engineer LAB by introducing efficient ethanol pathways to convert the lactic acid fermentation capacities into that of ethanol production. In the ethanol producing yeast S. cerevisiae and the bacterium Z. mobilis, the pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) genes function together to convert pyruvate via acetaldehyde to ethanol. The pdc and adh genes from Z. mobilis (assembled as PET operon) enable E.coli to produce ethanol with high yield and rate (Ingram, Conway et al. 1987). Efforts have been made to introduce the PET operon in the more robust Gram-positive Bacillus (Ingram, Barbosa-Alleyne et al. 1999), Lactobacillus casei (Gold, Meagher et al. 1996), and several other LAB (Nichols, Dien et al. 2003). Low efficiency of ethanol fermentation from these studies suggested that the pdc gene of Gram-negative origin did not function in Gram-positive bacteria. To avoid the problems encountered by earlier researchers, we have introduced the Gram-positive Sarcina ventriculi pdc gene into LAB, particularly in a host strain L. plantarum TF103 with both L-ldh and D-ldh inactivated. Western blot analysis, using antiserum against an oligo-peptide for S. ventriculi PDC, confirmed the presence of PDC in transformed LAB. Results from flask fermentation analysis for ethanol production will be presented. Genome shuffling, using the S. vertriculi pdc transformed TF103 strain, will be proposed for advanced strain improvement.