United States of America, As Represented by the Secretary of Agriculture

Lifesensors, Inc.

Stephen R Hughes

Patrick F Dowd

David M Donovan

William R Gibbons

Tauseef R Butt

Kenneth M Bischoff

Piyum A Khatibi

Dwayne R Roach

Ethanol losses due to bacterial contamination in fermentation cultures weakens the economics of biofuel production.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 &#x3bb;SA2 endolysin construct demonstrated strong lytic activity towards 17 of 22 strains of lactobacilli, staphylococci or streptococci maintaining optimal specific activity under fermentation conditions towardsubstrates.bacteriophage endolysin constructs LysA, LysA2 and LysgaY showed exolytic activity towards &#x2dc;60% of the lactobacilli tested including fourisolates from fuel ethanol fermentations. Presence of ethanol (&#x2266;5%) did not affect lytic activity. Lysins were able to reduce bothandcontaminants in mock fermentations of corn fiber hydrolysates. Recombinant LysA and &#x3bb;Sa2 expressed in the yeastare functional; LysA was shown to reduce lactobacilli in experimentally infected fermentations.

Peptidoglycan hydrolase antimicrobials for eradicating lactobacilli that contaminate and reduce ethanol yields in biofuel fermentation

Described are novelstrains NRRL Y-50798 and Y-50799, that were obtained by UV-C irradiation of wild-typeNRRL Y-1109 cultures. The UV-C-mutagenized strains were incubated under anaerobic conditions on xylose or glucose medium for a period of 5 months at 46&#xb0; C. before being selected. These mutagenized strains have potential application in large-scale industrial conversion of lignocellulosic sugars to fuel ethanol given their ability to ferment at temperatures at 46&#xb0; C. and above.

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Described are novelstrains that were obtained by UV-C irradiation of wild-typeNRRL Y-7124 cultures, followed by 5-month anaerobic growth on xylose at 28&#xb0; C. The UV-C-mutagenized strains were able to grow anaerobically on xylose or glucose medium with higher ethanol production than ayeast strain under comparable fermentation conditions. The mutagenized strains were identified by DNA fingerprinting to be unique strains closely related to wild-type. These mutagenized strains have potential application in large-scale industrial conversion of lignocellulosic sugars to fuel ethanol.

Recombinantproduced by transformation with heterologous polynucleotide sequences coding for xylulokinase (XKS) fromand xylose isomerase (XI) are capable of xylose utilization. The transformants express these heterologous polynucleotides at a sufficient functional level to grow aerobically on xylose as the sole carbon source. Further transformation of the recombinant yeasts to overexpress one or more of thegenes PIP2, IMG2, MAK5, VPS9, COX10, ALE1, CDC7, and MMS4, permits the yeast to grow anaerobically on xylose as the sole carbon source. When grown under anaerobic conditions on a culture medium comprising both glucose and xylose, the transformed yeast exhibit increased ethanol productivity, with the yeast growing on the xylose to increase their biomass and fermenting the glucose to ethanol.

Lycotoxin-1 peptide mutant peptides which exhibit increased insecticidal activity are produced by substitution of both a proline for the lysine at amino acid position 24 and a tryptophan for the leucine at amino acid position 25 of the wild-type amino acid sequence for lycotoxin-1. Further substitution of amino acids 8, 9 or 10 of the lycotoxin-1 wild-type amino acid sequence, specifically substituting a histidine for the phenylalanine at amino acid position 8, a glutamine for the glycine at amino acid position 10, or a serine for the leucine at amino acid position 9, provides an even greater increase in insecticidal activity. In addition to changes in the lycotoxin-1 amino acid sequence, the addition of an enterokinase K recognition site, DDDK, to the N-terminus of the lycotoxin-1 peptide increases insecticidal activity further still. Isolated nucleic acid sequences encoding the mutant lycotoxin-1 peptides, expression vectors containing these sequences, microorganisms or other host cells transformed with these vectors, and recombinant methods for producing the peptides, are also disclosed.

Growing community of inventors

Peoria, IL, United States of America

Stephen R Hughes