2012 Annual Report 1a.Objectives (from AD-416): Protect the cellulosic ethanolic fermentations from lactobacilli contaminants that reduce the yield and efficiency of biofuel ethanol production. 1b.Approach (from AD-416): Identify, subclone, and constitutively express lactobacillus peptidoglycan hydrolase antimicrobials on the surface of fermentative yeast. 3.Progress Report: This work supports the National Program 101 mission statement in the area of developing information, tools, and technologies that can be used to improve animal production systems. Significant progress was made on Component 1: Understanding, improving, and effectively using animal genetic and genomic resources. Progress on this project focuses on Problem 1A, the need for developing genome-enabling tools and reagents for livestock (pig and cattle). These tools will not only be useful for traditional animal production research applications (reproduction, growth and development, nutrient intake and utilization, product quality), but will also be used to decrease the environmental footprint of animal production, improve animal health, well-being and resistance to disease, and enhance food safety. The goal of this work is to identify novel enzyme antimicrobials that will kill bacterial contaminants (Lactobacillus species), the major contaminating bacteria that causes huge losses in fermentations that produce ethanol. These enzymes will initially be used to protect ethanol production in fermentations by controlling Lactobacillus contamination, but will eventually be tested for their ability to contribute activities useful for curing infections in dairy cattle mammary glands (mastitis). Specifically we plan to express antimicrobials on the surface of fermentative yeast with the purpose to kill Lactobacillus that contaminate the fermentations and reduce the ethanol produced. There has been significant progress in this project. We have synthesized eight candidate cell wall degrading enzymes and are evaluating their ability to kill the strain Lactobacillus fermentum. L. fermentum is the primary fermentation contaminating strain. These proteins have been expressed and purified using a common bacterial system. At this time, three proteins have been demonstrated to kill the target bacterium, Lactobacillus fermentum and are being tested in three unique expression scenarios designed to produce and target localization of the protein to the surface of the yeast. This agreement will initially help improve productivity of ethanol producing fermentations, and increase biofuels (ethanol) production, but eventually will also help to cure mastitis.