Progress 10/01/08 to 09/30/11
Outputs
OUTPUTS: During the first year (2008-2009), we obtained from our international collaborators in China a bacterial strain that showed an exceptional ability to degrade solid feather or hair into soluble proteins or amino acids. We first determined various conditions for the maximal degradation of feather leaf or mill by the strain in hope for finding the key time-point and enzymatic reaction for the process. Then, we compared the extracellular enzyme protein profiles of the strain between before and after induction. Using the Cornell Proteomics Core Facility, we conducted two-dimensional gel electrophoresis of the culture supernatant and identified more than 70 proteins. Because most of these proteins are unique or novel proteins, we could not identify the protein annotation with the existing database. Thus, we started the full genome sequencing of the strain using the Cornell DNA Sequencing Core Facility. However, the initial attempt of Illumina single-end sequencing rendered only a partial success. Thus, we needed to apply the paired-end sequencing to re-sequence the genome and used the Velvet assembly software to draft the genome to approximately 90% accuracy during 2009-2010. With this new genome assembly, we re-analyzed the proteomic data on the induced secretory proteins collected during the previous year. We also applied the same approach to conduct quantitative 2D-LC MS/MS proteomics of non-secretory proteins and identified several pathways up-regulated by feather fermentation. During 2010-2011, we used the Cornell Bioinformatics Core Facility to sort out key regulatory and functional protein candidates from the up-regulated pathways for feather degradation. This project has been continuously funded by a New York State Center for Advanced Technology grant (Cornell Biotechnology) for three years. Meanwhile, we expressed and characterized 11 new mutants of a thermostable serine protease (TfpA) from Thermomonospora fusa YX that was previously studied in our laboratory. The feather-hydrolytic ability and environmental impacts (pH, temperature, reductant, and protease inhibitors) on their function were investigated. We also collaborated with Dr. Gary Harmon to determine if microbes could be used to process feathers as a high-quality fertilizer and filed a patent application. PARTICIPANTS: Jeremy Weaver, Ph.D. student Yan Liu, visiting Ph.D. student Krystal Lum, Undergraduate researcher (honors thesis) TARGET AUDIENCES: Livestock producers and feed companies in New York State and elsewhere will benefit greatly from an effective keratinase complex that can be used to improve nutritional values of low-quality proteins and reduce nitrogen excretion of animal waste to environment. The development of a new keartinase complex may allow Biotechnology companies in the US to launch a novel and competitive enzyme product. Currently, there is no such enzyme complex product available. We have already received industrial matching funding for the project. We will present our results at Cornell Nutrition Conference and national meetings of American Society of Animal Science, and inform the stakeholders of the progress. We will consult with feed companies about the appropriate sources and levels of low-quality feed proteins such as feather meal for our experimental design. We will develop formal collaborations with feed and biotechnology companies in evaluating the effectiveness of the keratinase complex and in applying the technology to the field. PROJECT MODIFICATIONS: Application of the cutting-edge genome sequencing, proteomics of proteins associated with feather-degradation, and bioinformatics of the metabolic pathways will give us unique opportunities to seek for key feather-degrading proteases. The animal feeding trial needs to be postponed before the desired protease complex is developed.
Impacts
Our long-term goal is to develop effective keratinases (a special type of protease) to enhance nutritional values of low-quality proteins and to reduce their environmental pollution. Feather is one of the most underutilized by-products from poultry processing, representing 5 to 7% of the total weight of mature chickens. Over one million tons of feather waste is produced annually in the US. However, only a small portion of feather is processed into animal feed and the rest is dumped or land filled, causing environmental concerns. Feathers are almost pure keratin containing more than 90% crude protein content. Despite, it is not considered a rich protein source for animal feed because of its insolubility and poor digestibility. Because autoclaving or alkali treatment of feather is energy-consuming and does not render the complete hydrolysis of feather protein, enzymatic hydrolysis becomes the most promising method to improve digestibility of feather meal. Our project has three specific objectives: 1) to express the identified new keratinase enzyme genes in high efficient yeast system and to characterize biochemical properties of the expressed extracellular enzymes; 2) to test keratin-degrading capacity of different combinations of the expressed enzymes; and 3) to determine the efficacy and the environmental benefit of the selected enzyme complexes for animal feeding. Our progress will help us in identifying unique keratinases to achieve these goals. The genomic approach and the well-established protein engineering applied by us will increase our success chance. The main intended beneficiaries are the feed industry and animal producers in New York State and elsewhere in the US. The intended technology will help the feed industry develop novel protease enzyme complex to compete for the world market. This project will integrate at least three major activities associated with the NIFA's five national goals. The results will help an agricultural production system that is highly competitive in the global economy by producing new animal feed supplements and increasing the global competitiveness of the US animal feed and production systems. Making low-quality protein sources as value-added animal feed will improve harmony between agriculture and environment and preserve valuable natural resource of proteins. With new products for the feed industry and efficient production systems for producers, our findings will enhance economic opportunity and quality of life for Americans.
Publications
- Lei, X. G. and J. M. Porres. 2011. Invited chapter: Phytase: an enzyme to improve soybean nutrition. Soybean/Book 5, InTech - Open Access Publisher, http://www.intechweb.org/.
- Huang, J. Q., D. L. Li, H. Zhao, L. H. Sun, X. J. Xia, K. N. Wang, X. G. Luo, and X. G. Lei. 2011. The selenium deficiency disease exudative diathesis in chicks is associated with down-regulation of seven common selenoprotein genes in liver and muscle. J. Nutr. 141:1605-10.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: During the previous period (2008-2009), we had started to sequence the full genome of the feather-degrading bacterial strain obtained from our international collaborators in China in order to decode the secreted proteins induced by feather fermentation. However, the initial attempt of Illumina single-end sequencing rendered only a partial success. Thus, we have applied Illumina paired-end sequencing to re-sequence the genome and used the Velvet assembly software to draft the genome to approximately 90% accuracy. With this new genome assembly, we have re-analyzed the proteomic data on the induced secretory proteins collected during 2008-2009. We have also applied the same approach to conduct quantitative 2D-LC MS/MS proteomics of non-secretory proteins and identified several pathways up-regulated by feather fermentation. At the present time, we are using the Cornell Bioinformatics Core Facility to sort out key regulatory and functional protein candidates from the up-regulated pathways for feather degradation. This project has been continuously funded by a New York State Center for Advanced Technology grant (Cornell Biotechnology). At the same time, we have re-activated expression and characterization of a thermostable serine protease (TfpA) from Thermomonospora fusa YX that was previously studied in our laboratory. A total of 11 new mutants have been prepared. The feather-hydrolytic ability and environmental impacts (pH, temperature, reductant, and protease inhibitors) on their function have been systematically investigated. PARTICIPANTS: Jeremy Weaver, Ph.D. student; Yan Liu, visiting Ph.D. student; Krystal Lum, Undergraduate researcher (honors thesis) TARGET AUDIENCES: Livestock producers and feed companies in New York State and elsewhere will benefit greatly from an effective keratinase complex that can be used to improve nutritional values of low-quality proteins and reduce nitrogen excretion of animal waste to environment. The development of a new keartinase complex may allow Biotechnology companies in the US to launch a novel and competitive enzyme product. Currently, there is no such enzyme complex product available. We have already received industrial matching funding for the project. We will present our results at Cornell Nutrition Conference and national meetings of American Society of Animal Science, and inform the stakeholders of the progress. We will consult with feed companies about the appropriate sources and levels of low-quality feed proteins such as feather meal for our experimental design. We will develop formal collaborations with feed and biotechnology companies in evaluating the effectiveness of the keratinase complex and in applying the technology to the field. PROJECT MODIFICATIONS: Application of the cutting-edge genome sequencing, proteomics of proteins associated with feather-degradation, and bioinformatics of the metabolic pathways will give us unique opportunities to seek for key feather-degrading proteases. The animal feeding trial needs to be postponed still the designed protease complex is developed.
Impacts
Our long-term goal is to develop effective keratinases (a special type of protease) to enhance nutritional values of low-quality proteins and to reduce their environmental pollution. Feather is one of the most underutilized by-products from poultry processing, representing 5 to 7% of the total weight of mature chickens. Over one million tons of feather waste is produced annually in the US. However, only a small portion of feather is processed into animal feed and the rest is dumped or landfilled, causing environmental concerns. Feathers are almost pure keratin containing more than 90% crude protein content. Despite, it is not considered a rich protein source for animal feed because of its insolubility and poor digestibility. Because autoclaving or alkali treatment of feather is energy-consuming and does not render the complete hydrolysis of feather protein, enzymatic hydrolysis becomes the most promising method to improve digestibility of feather meal. In this proposal, we have three specific objectives: 1) to express the identified new keratinase enzyme genes in high efficient yeast system and to characterize biochemical properties of the expressed extracellular enzymes; 2) to test keratin-degrading capacity of different combinations of the expressed enzymes; and 3) to determine the efficacy and the environmental benefit of the selected enzyme complexes for animal feeding. Our progress during 2009-2010 will help us in identifying unique keratinases to achieve these goals. The genomic approach and the well-established protein engineering applied by us will increase our success chance. The main intended beneficiaries are the feed industry and animal producers in New York State and elsewhere in the US. The intended technology will help the feed industry develop novel protease enzyme complex to compete for the world market. This project will integrate at least three major activities associated with the CSREES' five national goals. The results will help an agricultural production system that is highly competitive in the global economy by producing new animal feed supplements and increasing the global competitiveness of the US animal feed and production systems. Making low-quality protein sources as value-added animal feed will improve harmony between agriculture and environment and preserve valuable natural resource of proteins. With new products for the feed industry and efficient production systems for producers, our findings will enhance economic opportunity and quality of life for Americans.
Publications
- X. G. Lei. 2010. Developing biotechnology to convert poultry feathers into high-quality protein. Proceedings of the Cornell Nutrition Conference for Feed Manufacturers, the 72 meeting, October 19-21, 2010, East Syracuse, NY, page 94.
- J. K. Patterson, K. Yasuda, R. M. Welch, D. D. Miller, and X. G. Lei. 2010. Supplemental dietary inulin of variable chain lengths alters intestinal bacterial populations in young pigs. J. Nutr. 140:2158-2161.
- L. Zheng, Z. Q. Cheng, X. Bei, Y. Zheng, X. H. Jiang, R. P. Glahn, R. M. Welch, D. D. Miller, X. G. Lei, and H. X. Shou. 2010. Nicotianamine, a novel promoter of iron absorption from rice grain. PLoS One Apr 16;5(4):e10190.
- E. J. Mullaney, A. H.J. Ullah, K. Sethumadhavan, and X. G. Lei. 2010. Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization. Appl. Microbiol. Biotech. 87(4):1367-72.
- T. Qin, H. Zhao, X. Xia and X. G. Lei. 2010. Cloning of porcine pancreatic α-amylase gene and characterization of the enzyme over-expressed in Pichia pastoris. J. Anim. Sci. 88 (Suppl. 1).
- L. H. Sun, H. Zhao, M.Y. Xie, J. Xing, X. J. Xia and X. G. Lei. 2010. Heterologous expression of a truncated bovine lactoferrin gene in E. coli to produce a novel antimicrobial peptide. J. Anim. Sci. 88 (Suppl. 1).
- F. Wang, H. Zhao, X. J. Xia, and X. G. Lei. 2010. Cloning of a porcine trypsinogen gene and over-production of the protein as a feed additive. J. Anim. Sci. 88 (Suppl. 1).
- M. Y. Xie, L.H. Sun, H. Zhao, X. J. Xia and X. G. Lei. 2010. Developing an efficient E. coli expression system for producing a recombinant antimicrobial peptide plectasin. J. Anim. Sci. 88 (Suppl. 1).
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: From our international collaborators in China, we have obtained a bacterial strain that showed an exceptional ability to degrade solid feather or hair into soluble proteins or amino acids. During the past year, we have first determined various conditions for the maximal degradation of feather leaf or mill by the strain in hope for finding the key time-point and enzymatic reaction for the process. Then, we have compared the extracellular enzyme protein profiles of the strain between before and after induction. Using the Cornell Proteomics Core Facility, we have conducted two-dimensional gel electrophoresis of the culture supernatant and identified more than 70 proteins. Because most of these proteins are unique or novel proteins, we could not find the protein annotation with the existing database. Thus, we have recently completed the full genome sequencing of the strain using the Cornell DNA Sequencing Core Facility. At the present time, we are using the Cornell Bioinformatics Core Facility to assemble the genome data and to identify the extracellular proteins induced by the feather degradation. Another significant progress of the project has been the success to attract an innovation grant, a biotechnology CAT grant, and an industrial sponsor, along with a major collaboration between Cornell University and the Chinese Academy of Agricultural Sciences. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Livestock producers and feed companies in New York State and elsewhere will benefit greatly from an effective keratinase complex that can be used to improve nutritional values of low-quality proteins and reduce nitrogen excretion of animal waste to environment. The development of a new keartinase complex may allow Biotechnology companies in the US to launch a novel and competitive enzyme product. Currently, there is no such enzyme complex product available. We have already received industrial matching funding for the project. We will present our results at Cornell Nutrition Conference and national meetings of American Society of Animal Science, and inform the stakeholders of the progress. We will consult with feed companies about the appropriate sources and levels of low-quality feed proteins such as feather meal for our experimental design. We will develop formal collaborations with feed and biotechnology companies in evaluating the effectiveness of the keratinase complex and in applying the technology to the field. PROJECT MODIFICATIONS: With a powerful bacterial strain to degrade feather from our international collaborators, we have devoted a significant amount of efforts in exploring the mechanism of feather-degradation using the cutting-edge genomics, proteomics, and bioinformatics. The animal feeding trial needs to be postponed still the designed protease complex is developed.
Impacts
Our long-term goal is to develop effective keratinases (a special type of protease) to enhance nutritional values of low-quality proteins and to reduce their environmental pollution. Feather is one of the most underutilized by-products from poultry processing, representing 5 to 7% of the total weight of mature chickens. Over one million tons of feather waste is produced annually in the US. However, only a small portion of feather is processed into animal feed and the rest is dumped or landfilled, causing environmental concerns. Feathers are almost pure keratin containing more than 90% crude protein content. Despite, it is not considered a rich protein source for animal feed because of its insolubility and poor digestibility. Because autoclaving or alkali treatment of feather is energy-consuming and does not render the complete hydrolysis of feather protein, enzymatic hydrolysis becomes the most promising method to improve digestibility of feather meal. In this proposal, we have three specific objectives: 1) to express the identified new keratinase enzyme genes in high efficient yeast system and to characterize biochemical properties of the expressed extracellular enzymes; 2) to test keratin-degrading capacity of different combinations of the six expressed enzymes; and 3) to determine the efficacy and the environmental benefit of the selected enzyme complexes for animal feeding. The main intended beneficiaries are the feed industry and animal producers in New York State and elsewhere in the US. The intended technology will help the feed industry develop novel protease enzyme complex to compete for the world market. This project will integrate at least three major activities associated with the CSREES' five national goals. The results will help an agricultural production system that is highly competitive in the global economy by producing new animal feed supplements and increasing the global competitiveness of the US animal feed and production systems. Making low-quality protein sources as value-added animal feed will improve harmony between agriculture and environment and preserve valuable natural resource of proteins. With new products for the feed industry and efficient production systems for producers, our findings will enhance economic opportunity and quality of life for Americans.
Publications
- K. Yasuda, H. D. Dawson, E. V. Wasmuth, C. A. Roneker, K. M. Kohan, C. Chen, J. F. Urban, R. M. Welch, D. D. Miller, and X. G. Lei. 2009. Supplemental dietary inulin suppresses expression of iron-related inflammation genes in young pigs. J. Nutr. 2009 Sep 23. [Epub ahead of print]
- J. D. Weaver, A. H.J. Ullah, K. Sethumadhavan, E. J. Mullaney, and X. G. Lei. 2009. Impact of assay conditions on activity estimate and kinetics comparison of Aspergillus niger PhyA and Escherichia coli AppA2 phytases. J. Agr. Food Chem. 24:57(12):5315-20.
- J. K. Patterson, M. A. Rutzke, S. L. Fubini, R. P. Glahn, R. M. Welch, X. G. Lei, and D. D. Miller. 2009. Dietary inulin supplementation does not promote colonic iron absorption in a porcine model. J. Agr. Food Chem. 2009 Jun 24;57(12):5250-6.
- E. Tako, R. P. Glahn, j. M. Laparra, R. M. Welch, X. G. Lei, J. D. Kelly, M. A. Rutzke, and D. D. Miller. 2009. Iron and zinc bioavailabilities to pigs from red and white beans (Phaseolus vulgaris L.) are similar. J. Agr. Food Chem. 2009 Apr 22; 57(8):3134-40.
- E Tako. J. M. Laparra, R. P. Glahn, R. M. Welch, X. G. Lei, S. Beebe, and D. D. Miller. 2009. Biofortified black beans in a maize and bean diet provide more bioavailable iron to piglets than standard black beans. J. Nutr. 139(2):305-9. Epub 2008 Dec 23.
- J. D. Weave1, D. R. Ripol2 and X. G. Lei, 2008. Demolition and construction on the road to a better understanding of phytase. Proceedings of the Cornell Nutrition Conference for Feed Manufacturers.
- L. E. Denmark, J. D. Weaver, K. R. Roneker, and X. G. Lei. 2009. Genetic engineering of an Escherichia coli mutant phytase for thermostability does not affect the enzymatic efficacy in a diet for young pigs. J. Anim. Sci. 86 (Suppl. 1).
- C. E. Mills, C. A. Faber, K. R. Roneker, and X. G. Lei. 2009. Comparative effects of Escherichia coli AppA2 and Aspergillus niger PhyA phytases on bone property of weanling pigs fed a high phosphorus diet. J. Anim. Sci. 87 (Suppl. 1).
- Y. J. Zhang, H. Zhao, J. C. Zhou, X. J. Xia, and X. G. Lei. 2009. Heterologous expression of recombinant porcine elastase 2 as a feed enzyme. J. Anim. Sci. 87 (Suppl. 1).
- Y. Zhao, H. Zhao, J. C. Zhou, X. J. Xia, and X. G. Lei. 2009. Cloning and expression of porcine carboxypeptidase A1 for feed application. J. Anim. Sci. 87 (Suppl. 1).
- Y. Liu, H. Zhao, J. C. Zhou, X. J. Xia, and X. G. Lei. 2009. Expression and purification of porcine pancreatic carboxypeptidase B in a yeast system. J. Anim. Sci. 87 (Suppl. 1).
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