Peptidoglycan hydrolase antimicrobials for eradicating lactobacilli that contaminate and reduce ethanol yields in biofuel fermentation
Inventors
Donovan, David M. • Roach, Dwayne R. • Khatibi, Piyum A. • Bischoff, Kenneth M. • Hughes, Stephen R.
Assignees
US Department of Agriculture USDA
Publication Number
US-9068204-B2
Publication Date
2015-06-30
Expiration Date
2033-03-15
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Abstract
Ethanol losses due to bacterial contamination in fermentation cultures weakens the economics of biofuel production. Lactobacillus species are the predominant contaminant. Bacteriophage lytic enzymes are peptidoglycan hydrolases which degrade Gram positive cell walls when exposed externally and are a novel source of antimicrobials. The streptococcal phage λSA2 endolysin construct demonstrated strong lytic activity towards 17 of 22 strains of lactobacilli, staphylococci or streptococci maintaining optimal specific activity under fermentation conditions toward L. fermentum substrates. Lactobacillus bacteriophage endolysin constructs LysA, LysA2 and LysgaY showed exolytic activity towards ˜60% of the lactobacilli tested including four L. fermentum isolates from fuel ethanol fermentations. Presence of ethanol (≦5%) did not affect lytic activity. Lysins were able to reduce both L. fermentum and L. reuteri contaminants in mock fermentations of corn fiber hydrolysates. Recombinant LysA and λSa2 expressed in the yeast Saccharomyces cerevisiae are functional; LysA was shown to reduce lactobacilli in experimentally infected fermentations.
Core Innovation
Ethanol losses caused by bacterial contamination in fermentation cultures detrimentally impact the economics of biofuel production. Lactobacillus species constitute the predominant contaminants, and controlling these contaminants is challenging due to the non-aseptic nature of industrial fermentations. Current methods rely heavily on prophylactic antibiotic treatments, which face issues including antibiotic resistance and potential residue concerns.
Bacteriophage lytic enzymes, specifically endolysins, are peptidoglycan hydrolases capable of degrading Gram positive bacterial cell walls externally. The invention identifies that four endolysin constructs, namely LysA, LysA2, LysgaY, and λSa2, demonstrate significant exolytic activity against various lactobacilli strains under fermentation conditions, including the major contaminant L. fermentum. These enzymes maintain optimal activity in the presence of ethanol (up to 5%) and appropriate pH levels, and when applied, effectively reduce bacterial contamination in mock ethanol fermentations.
Furthermore, LysA and λSa2 endolysin constructs can be functionally expressed in recombinant Saccharomyces cerevisiae, the yeast used in ethanol production, allowing in situ production of antimicrobial agents during fermentation. This genetically engineered yeast expression provides an effective and specific method to control unwanted lactobacilli contamination without inhibiting yeast growth, potentially improving ethanol yields in biofuel fermentation processes.
Claims Coverage
The patent includes three independent claims outlining methods to control bacterial contamination in ethanol fermentation using recombinant bacteriophage lytic endolysins or genetically modified yeast.
Use of recombinant bacteriophage lytic endolysin as antimicrobial in fermentation
A method of controlling bacterial contamination in ethanol fermentation involving introducing yeast S. cerevisiae and a recombinant bacteriophage lytic endolysin into a fermentation vessel. The endolysin has exolytic activity against targeted Gram positive lactic acid bacteria, reducing their growth and colonization during ethanol production without inhibiting yeast growth, thereby increasing ethanol production.
Use of recombinant yeast host cells encoding bacteriophage lytic endolysins in fermentation
A method of controlling lactic acid bacterial contamination in ethanol fermentation by introducing yeast S. cerevisiae and recombinant yeast host cells comprising genes encoding bacteriophage lytic endolysins into a fermentation vessel. The recombinant endolysin reduces targeted Gram positive lactic acid bacterial species growth and colonization without inhibiting fermentative yeast growth, enhancing ethanol yield.
Use of recombinant yeast extracts containing bacteriophage lytic endolysins in fermentation
A method of controlling lactic acid bacterial contamination in ethanol fermentation by introducing yeast S. cerevisiae and recombinant yeast extracts containing bacteriophage lytic endolysins into a fermentation vessel. The recombinant endolysin reduces targeted Gram positive lactic acid bacterial species growth and colonization without affecting yeast growth, resulting in increased ethanol production.
The independent claims focus on employing recombinant bacteriophage lytic endolysins, either directly, via genetically modified yeast cells expressing these enzymes, or via yeast extracts containing them, to specifically reduce Gram positive lactic acid bacterial contaminants during ethanol fermentation processes. These methods aim to control contamination effectively without inhibiting yeast growth, thereby enhancing ethanol production efficiency.
Stated Advantages
Provides effective and specific antimicrobial treatment targeting lactobacilli contaminations in fermentation systems.
Maintains lytic activity under fermentation conditions, including presence of ethanol and relevant pH ranges.
Allows functional expression of endolysins in fermentative yeast, enabling continuous antimicrobial action.
Reduces the need for traditional antibiotics, potentially lowering antibiotic resistance and residue concerns.
Improves ethanol yields by reducing bacterial contamination that competes with yeast or produces inhibitory compounds.
Documented Applications
Control of bacterial contamination, specifically lactobacilli such as L. fermentum, in biofuel ethanol fermentation processes.
Use in ethanol production facilities employing yeast Saccharomyces cerevisiae, including corn fiber hydrolysate fermentations.
Expression of bacteriophage lytic endolysins in recombinant yeast host cells for in situ antimicrobial activity during ethanol fermentation.
Application in fermentation systems derived from various feedstocks including corn, wheat, triticale, barley, cassava, rye, sugar cane, sugar beet, molasses, rice straw, potato waste, wood waste, switch grass, pine, municipal waste, food waste, and beverage industry waste.
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