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United States Department of Agriculture

Agricultural Research Service

Title: FUNCTIONAL EXPRESSION OF BACTERIAL ZYMOBACTER PALMAE PYRUVATE DECARBOXYLASE GENE IN LACTIC ACID BACTERIA

Authors
item Liu, Siqing
item Dien, Bruce
item Cotta, Michael

Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 1, 2005
Publication Date: April 1, 2005
Citation: Liu, S., Dien, B.S., Cotta, M.A. 2005. Functional expression of bacterial Zymobacter palmae pyruvate decarboxylase gene in lactic acid bacteria. Current Microbiology. 50:1-6.

Interpretive Summary: Fuel ethanol used in U.S. gasoline formulations is produced primarily from corn starch. Expanding the use of ethanol beyond that of a fuel oxygenate will require the conversion of materials in addition to corn, particularly renewable agricultural byproducts and residues (biomass). Commercial ethanol fermentation biotechnology is currently based on a relatively small number of microorganisms that are limited in their ability to ferment a variety of sugars. Economic conversion of biomass to ethanol will require the development of new, industrially robust strains that can ferment mixtures of sugars derived from biomass. In this research, we report on our efforts to modify bacterial strains that intrinsically ferment pentose sugars: five carbon sugars present in biomass that are poorly fermented by existing commercial strains. A gene (pdc from the bacterium Zymobacter palmae) that redirects metabolism toward the production of ethanol was introduced into the lactic acid bacterium, Lactococcus lactis. The engineered organism expressed this gene and altered its metabolism toward ethanol production, but did not produce increased levels of alcohol. Additional genes appear to be needed to complete the conversion process. The research also involved the development of a new way to measure the activity of the key enzyme involved in this process (PDC). This assay will be useful in future attempts to engineer additional ethanol producing strains. The results of this research offer valuable insight to us and other researchers to direct future research efforts on the genetic manipulation of organisms to improve ethanol production efficiency.

Technical Abstract: In this study, a pyruvate decarboxylase (PDC) gene from bacterial Zymobacter palmae (Zymopdc) was cloned, characterized, and introduced into lactic acid bacteria (LAB) via a shuttle vector pAK80 as part of a research strategy to develop an efficient ethanol producing LAB. Six constructs containing the Zymopdc gene driven by different Gram-positive promoters were made to express the Zymopdc in Lactococcus lactis subsp LM0230 strain. The Zymopdc gene expression in the host was measured by a novel colorimetric assay based on PDC catalyzed formation of (R)-phenylacetylcarbinol. The transgenes expressed various levels of Zymopdc, attributable to differing strength of the promoter within each specific construct. A constitutive, highly expressed promoter construct (p769Zymopdc) conferred the greatest PDC activity, a moderately active promoter (p772Zymopdc), relatively lower PDC activity, and an acid inducible promoter (p692Zymopdc) demonstrated acid inducible expression. The acid inducible promoter was still active when a 78 bp fragment from 5'side upstream of the ATG of Zymopdc had been included between the promoter and Zymopdc coding sequences (p692EZymopdc), while constructs with this 78 bp sequence in p769EZymopdc and p772EZymopdc displayed lower PDC activities when compared to the corresponding ones without the fragment. The metabolic production of ethanol and other products were examined in flask fermentations. More than eight fold increases in acetaldehyde concentrations were detected in 769Zymopdc and 692Zymopdc recombinant strains. However, the observed ethanol fermentation in these engineered strains showed no detectable difference when compared with that of the recombinant strain carrying lacZ reporter. Further genetic manipulations for ethanol production are discussed.

Last Modified: 9/1/2014
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