Progress 10/01/12 to 09/30/16
Outputs
Target Audience:The scientific community interested in lactic acid bacteria and/or the use of lignocellulosic feed stocks for fermentation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?There has been, at minimum, weekly meeting between the graduate students (Jesse Heidenreich and Kara Hulce) and the Principal Investigator (James Steele). These meetings provided significant opportunities for one-on-one mentoring. Additionally, Ms. Heidenreich wrote and defended her Ph.D. thesis during this year which provided extensive mentoring opportunities. How have the results been disseminated to communities of interest?The results were disseminated to the University of Wisconsin-Madison research committee through Ms. Heidenreich's Ph.D. seminar and the publication of her Ph.D. thesis. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In silico analysis of the Lactobacillus casei 12A genome for cellobiose utilization clusters, defined as the presence of a β-glucosidase and cellobiose related transporter(s), resulted in the identification of six putative cellobiose clusters. RNA was isolated from L. casei 12A grown either with glucose or cellobiose as the carbohydrate sourse. RNA-Seq analysis indicated that 2 of the clusters are up-regulated during growth on cellobiose. To determine which of these clusters was required for cellobiose utilization, gene replacement was utilized to inactivate the β-glucosidase genes in these cultures. One of these, designated Lca12A_0108, resulted in a strain incapable of growth on cellobiose. These results indicated that this cluster is essential for cellobiose utilization by this organism. Additionally, the gene encoding the catabolite-control protein A (ccpA) was inactivated. The results from the characterization of this mutant indicated that Inactivation of ccpA relieved catabolite repression of Lca12A_0108 during growth on a mixture of glucose and cellobiose. These results indicated that only one of the putative cellobiose utilization clusters in L. casei 12A is required for cellobiose utilization and that this cluster is regulated, in part, by catabolite repression. We evaluated an endoglucanase from Clostridium celluloyticum that had been engineered to contain a carbohydrate binding module (CBM), designated CelECBM3a, for the ability to produce growth substrates (i.e. cellobiose) for a L. casei ethanologen. This enzyme exhibited activity on phosphoric acid swollen cellulose (PASC) under conditions that allowed for growth and ethanol production by a L. casei ethanologen; Limited ethanol production was observed, most likely differences in temperature optimums for CelECBM3a and L. casei limited ethanol production. Additionally, we evaluated an endoglucanase, designated CMXCBM5, from a nonculturable organism that has been modified by the addition of a CBM, for compatibility with a L. casei ethanologen. This enzyme had activity on PASC and xylan, however neither yielded a significant quantity of fermentable carbohydrates. This was likely due to the products of CMXCBM5 activity on these substrates being too large for L. casei ethanologens to utilize. The results demonstrate the importance of matching enzymes utilized to deconstruct lignocellulosic biomass with the biocatalyst that will be used to ferment these substrates. These results contribute of the knowledge base required for the production of biofuels from plant biomass. The development of biofuel production methods from plant biomass is necessary if we are going to develop processes for fuel production that do not compete with food production.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2015
Citation:
Heidenreich, J.M. 2015. PhD thesis: Utilization of carbohydrates derived from cellulose and hemicellulose by Lactobacillus casei and a Lb. casei ethanologen. Department of Food Science, University of Wisconsin-Madison.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience is scientific communities interested in lactic acid bacteria and/or the use of lignocellulosic fermentation feed stocks. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?There has been, at minimum, weekly meetings between the graduate students (Jesse Heidenreich and Kara Hulce) and the Principal Investigator (James Steele). These meeting have provided significant opportunities for one-on-one mentoring. Additionally, Ms. Heidenreich wrote and defended her Ph.D. thesis during this year, which provided extensive opportunities for mentoring. How have the results been disseminated to communities of interest?The results were disseminated to the University of Wisconsin-Madison research committee through Ms. Heidenreich's Ph.D. seminar and the publication of her Ph.D. thesis. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete some additional analysis of microbial substrates produced and utilized, as well as the metabolic end products formed. These results are required to publish the cellobiose utilization research and to better understand how the activity of different endoglucanases on cellulose influences microbial growth.
Impacts
What was accomplished under these goals?
We evaluated an endoglucanase from Clostridium celluloyticum that had been engineered to contain a carbohydrate binding module (CBM), designated CelECBM3a, for compatibility with a L. casei ethanologen. This enzyme exhibited activity on phosphoric acid swollen cellulose (PASC) under conditions that allowed for growth and ethanol production by a L. casei ethanologen. Limited ethanol production was observed, most likely differences in temperature optimums for CelECBM3a and L. casei limited ethanol production. Additionally, we evaluated an endoglucanase, designated CMXCBM5, from a nonculturable organism that has been modified by the addition of a CBM, for compatibility with a L. casei ethanologen. This enzyme had activity on PASC and xylan, however, neither yielded a significant quantity of fermentable carbohydrates. This was likely due to the products of CMXCBM5 activity on these substrates being too large for L. casei ethanologens to utilize.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The target audience is the scientific community interested in lactic acid bacteria. This work was presented at the Eleventh Symposium on Lactic Acid Bacteria. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? There has been, at minimum, weekly meeting between the graduate student (Jesse Heidenreich) and the Principal Investigator (James Steele). These meeting have provided significant opportunities for one-on-one mentoring. Additionally, Jesse presented her work at the Eleventh Symposium on Lactic Acid Bacteria in 2014, which provided her with the opportunity to interact with other scientist in the field and attend a number of related scientific presentations. How have the results been disseminated to communities of interest? Our results to date were presented as a poster entitled "Metabolism of β (1,4) glycoside substrates by Lactobacillus casei 12A" at the Eleventh Symposium on Lactic Acid Bacteria in 2014. What do you plan to do during the next reporting period to accomplish the goals? We plan to write a manuscript describing the cellobiose results described above. Additionally, we plan to screen a variety of cellulases for their ability to enhance the growth of Lactobacillus casei 12A on cellulose.
Impacts
What was accomplished under these goals?
The Lactobacillus casei 12A genome for contains six putative cellobiose utilization clusters, defined as the presence of a β-glucosidase and cellobiose related transporter. Two of these have been determined to be up-regulated during growth on cellobiose. The inactivation of one of these, designated Lca12A_0108, resulted in a strain incapable of growth on cellobiose. These results indicated that this cluster is essential for cellobiose utilization by this organism. Additionally, the gene encoding the catabolite-control protein A (ccpA) has been inactivated. Inactivation of ccpA was demonstrated to relieve catabolite repression during growth on a mixture of glucose and cellobiose. Lactobacillus casei has the potential to be the biocatalyst of choice for the production of biofuels from lignocellulosic materials (i.e corn stover or switch grass). However, it lacks enzymes to degrade lignocellulosic materials and uses the sugars produced by these enzymes relatively slowly. This report addresses the later issue, specifically the ability to rapidly utilize the sugars generated from cellulose, glucose and cellobiose.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The scientific community interested in microbiology and the production of biofuels. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? There has been, at minimum, weekly meetings between the graduate student (Jesse Heidenreich) and the Principal Investigator (James Steele). These meetings have provided significant opportunities for one-on-one mentoring. Additionally, Jesse presented her work at the 2013 Annual Meeting of the American Society for Microbiology, which provided her with the opportunity to interact with other scientist in the field and attend a number of related scientific presentations. How have the results been disseminated to communities of interest? Our results to date were presented as a poster entitled “Ethanol production by Lactobacillus casei 12A by utilization of plant biomass” at the 2013 annual meeting of the American Society for Microbiology. What do you plan to do during the next reporting period to accomplish the goals? We plan to inactivate the two a β-glucosidase genes present in the cellobiose clusters that were up-regulated during growth on cellobiose and determine if they are required for growth on cellobiose. Additionally, we plan to express a L. casei codon optimized cellulose gene in 12A.
Impacts
What was accomplished under these goals?
In silico analysis of the Lactobacillus casei 12A genome for cellobiose utilization clusters, defined as the presence of a β-glucosidase and cellobiose related transporter(s), resulted in the identification of six putative cellobiose clusters. RNA has been isolated from L. casei 12A grown either with glucose or cellobiose as the carbohydrate sourse. RNA-Seq analysis has been conducted and the data indicates that 2 of the clusters are up-regulated during growth on cellobiose. Efforts to inactivate these two clusters are underway.
Publications
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Progress 10/01/12 to 12/31/12
Outputs
OUTPUTS: We are only three months into this project and hence do not have results to disseminate. PARTICIPANTS: To date, the research group that has participated in this research includes Jesse Heidenreich and Dr. James Steele. TARGET AUDIENCES: The target audience is researchers working on the conversion of carbohydrates to biofuels and co-products in academic and industrial laboratories. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have developed the necessary methods to pursue objective 1 and will be utilizing these methods to identify the cellobiose clusters that are essential for cellobiose utilization by Lactobacillus casei 12A.
Publications
- No publications reported this period
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