Progress 02/15/09 to 02/14/12
Outputs
Progress Report Objectives (from AD-416): The objective of this Cooperative Research and Development Agreement (CRADA) is to develop and scale-up novel methods for the production of xylitol from agricultural feedstocks, such as corn fiber and wheat straw. Approach (from AD-416): Fermentation variables such as cost (as a function of components and time) , substrate concentration, enzyme expression, media composition, carbon source, temperature, pH, and seed inoculum quality will be optimized using recombinant Escherichia coli strains such as ZUC 140, ZUC170, and ZUC99/pATX210 which were developed under a previous Cooperative Research and Development Agreement (CRADA) between ARS, zuChem, and Biotechnology Research and Development Corporation (BRDC). These strains will be independently improved/optimized and then engineered to combine both the pathways for conversion of xylose to xylitol and arabinose to xylitol, mutagenized to improve flux of the system to produce xylitol, and directed evolution will be performed in order to improve activities of individual enzymes. In general, improvement will be made in substrate specificity, thermal stability, and kinetic performance of the various enzymes, as well as overall xylitol yield from arabinose and xylose. New enzymes relevant to the conversion of xylose and/or arabinose to xylitol with improved activities will be isolated as necessary for the project. In addition, corn fiber and wheat straw feedstocks will be evaluated for xylitol production and compared to other hemicellulose hyrdolysate sources which are available during the project. This will include evaluation of different purification and production methods for making hemicellulose hydrolysates or the feedstocks; mutagenesis of the E. coli strains for adaptation to the feedstocks and inhibitors which may be present in the feedstock; and testing and optimization of the feedstocks (which may have different percentages of arabinose and xylose) for higher yields of pentose sugars. Feedstock pre-treatment will include dilute acid pre-treatments at moderate temperature. Under this agreement, both ARS and zuChem, Incorporated, Chicago, IL, have worked together to develop a commercially feasible bioprocess for making xylitol, a low calorie anticariogenic sweetener. This collaborative effort was established to enhance ongoing ARS research in this area. Microbial production of xylitol using hemicellulosic biomass such as corn fiber and wheat straw is attractive for reducing the manufacturing cost. Research was focused on using molecular biology and pathway engineering to create a recombinant microorganism with the ability to produce xylitol from xylose and arabinose simultaneously because the hemicellulosic hydrolyzates are generally rich in those sugars. During previous cooperative agreement, we constructed a recombinant Escherichia coli strain that can produce xylitol from xylose with very high productivity from a mixture of xylose and arabinose without producing any arabitol from arabinose. We also constructed a recombinant Escherichia coli strain that converted arabinose to xylitol. We now have developed a sequential fermentation process of converting both xylose and arabinose to xylitol using these two recombinant E. coli strains � one for conversion of arabinose to xylitol and the other one for conversion of xylose to xylitol. We have also developed a temperature- inducible expression system for xylose reductase enzyme. We have developed processes for generating xylose from various hemicellulose sources and remediating the fermentation inhibitors. We have optimized the fermentative conversion of xylose to xylitol by an improved recombinant E. coli strain along with a xylitol recovery process. The complete process of making xylitol from hemicellulose was scaled up to 30 liters and commercialization agreements have been negotiated. The project ended on February 14, 2012.
Impacts
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Publications
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) The objective of this Cooperative Research and Development Agreement (CRADA) is to develop and scale-up novel methods for the production of xylitol from agricultural feedstocks, such as corn fiber and wheat straw. Approach (from AD-416) Fermentation variables such as cost (as a function of components and time) , substrate concentration, enzyme expression, media composition, carbon source, temperature, pH, and seed inoculum quality will be optimized using recombinant Escherichia coli strains such as ZUC 140, ZUC170, and ZUC99/pATX210 which were developed under a previous Cooperative Research and Development Agreement (CRADA) between the Agricultural Research Service (ARS), zuChem, and Biotechnology Research and Development Corporation (BRDC). These strains will be independently improved/optimized and then engineered to combine both the pathways for conversion of xylose to xylitol and arabinose to xylitol, mutagenized to improve flux of the system to produce xylitol, and directed evolution will be performed in order to improve activities of individual enzymes. In general, improvement will be made in substrate specificity, thermal stability, and kinetic performance of the various enzymes, as well as overall xylitol yield from arabinose and xylose. New enzymes relevant to the conversion of xylose and/or arabinose to xylitol with improved activities will be isolated as necessary for the project. In addition, corn fiber and wheat straw feedstocks will be evaluated for xylitol production and compared to other hemicellulose hyrdolysate sources which are available during the project. This will include evaluation of different purification and production methods for making hemicellulose hydrolysates or the feedstocks; mutagenesis of the E. coli strains for adaptation to the feedstocks and inhibitors which may be present in the feedstock; and testing and optimization of the feedstocks (which may have different percentages of arabinose and xylose) for higher yields of pentose sugars. Feedstock pre-treatment will include dilute acid pre-treatments at moderate temperature. Under this CRADA, research has been focused on using molecular biology and pathway engineering to create a recombinant microorganism with the ability to produce xylitol from xylose and arabinose simultaneously because the hemicellulosic hydrolyzates are generally rich in these two pentose sugars. First, we have worked on combining the two previously developed pathways to convert xylose to xylitol and arabinose to xylitol. In this combined system, the expression of the xylose reductase that converts xylose to xylitol must be tightly controlled because it will also convert arabinose to arabitol � an undesired co-product. We have worked on engineering a system that put the xylose reductase under the control of a temperature sensitive promoter. This would allow for the initial conversion of all the arabinose to xylitol at low temperature followed by a temperature shift that would induce the xylose reductase enzyme to complete the conversion of xylose to xylitol. A significant amount of time was spent constructing appropriate expression vectors and strains. The wild-type repressor of the bacterial lactose operon (lacI) was deleted from strains, and mutants of the lacI gene were made by site- directed mutagenesis. Multiple temperature-sensitive mutants were made, which were used to construct an expression system in which the enzyme (xylose reductase) expression was controlled by temperature. After troubleshooting the system, we were able to effect a temperature controlled expression of the xylose reductase, although some leaky expression still exists which will ultimately need to be reduced. The second task we worked on during this timeframe was the improvement of the pathway to convert arabinose to xylitol. In this instance, protein expression levels were analyzed of the three genes involved in the conversion process and it was determined that the final step, l-xylulose reductase (lxr), appeared to be rate limiting in the conversion of arabinose to xylitol. When we substituted the lxr gene from a bacterium for the lxr gene from a yeast we had been using, we noticed a significant improvement in overall conversion of arabinose to xylitol (>1.5X improvement). These results are very promising and further improvements are currently the main focus of our efforts since the more rapidly arabinose is converted to xylitol the less problematic conversion of arabinose to arabitol is when combined with a xylose reductase technology. Finally, we have tested and worked on optimization of the xylose to xylitol conversion route on a variety of different hemicellulose hydrolyzate sources. These include sources such as corn fiber, wheat straw, sugar cane bagasse, and sugar beet hemicelluloses. We have achieved very high levels of xylitol production and productivities that exceed 3 g per liter per hour, depending on the hemicellulose source tested. The cooperator meets biweekly in person with the Authorized Departmental Officer's Designated Representative (ADODR).
Impacts
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Publications
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The objective of this Cooperative Research and Development Agreement (CRADA) is to develop and scale-up novel methods for the production of xylitol from agricultural feedstocks, such as corn fiber and wheat straw. Approach (from AD-416) Fermentation variables such as cost (as a function of components and time) , substrate concentration, enzyme expression, media composition, carbon source, temperature, pH, and seed inoculum quality will be optimized using recombinant Escherichia coli strains such as ZUC140, ZUC170, and ZUC99/pATX210 which were developed under a previous Cooperative Research and Development Agreement (CRADA) between the Agricultural Research Service (ARS), zuChem, and Biotechnology Research and Development Corporation (BRDC). These strains will be independently improved/optimized and then engineered to combine both the pathways for conversion of xylose to xylitol and arabinose to xylitol, mutagenized to improve flux of the system to produce xylitol, and directed evolution will be performed in order to improve activities of individual enzymes. In general, improvement will be made in substrate specificity, thermal stability, and kinetic performance of the various enzymes, as well as overall xylitol yield from arabinose and xylose. New enzymes relevant to the conversion of xylose and/or arabinose to xylitol with improved activities will be isolated as necessary for the project. In addition, corn fiber and wheat straw feedstocks will be evaluated for xylitol production and compared to other hemicellulose hyrdolysate sources which are available during the project. This will include evaluation of different purification and production methods for making hemicellulose hydrolysates or the feedstocks; mutagenesis of the E. coli strains for adaptation to the feedstocks and inhibitors which may be present in the feedstock; and testing and optimization of the feedstocks (which may have different percentages of arabinose and xylose) for higher yields of pentose sugars. Feedstock pre-treatment will include dilute acid pre-treatments at moderate temperature. Under this CRADA, research has been focused on using molecular biology and pathway engineering to create a recombinant microorganism with the ability to produce xylitol from xylose and arabinose simultaneously because the hemicellulosic hydrolyzates are generally rich in these sugars. We have worked to optimize a two-stage fermentation process using two recombinant bacterial strains � the first one to convert arabinose to xylitol and the second one to convert xylose to xylitol with a goal of converting both xylose and arabinose (typically present in a dilute acid pretreated lignocellulosic biomass hydrolyzate) to xylitol sequentially. In both cases, glucose was used as co-substrate. Initially, we have observed inferior performance at stage 2 due to inhibition of growth by acetate formed in stage 1. By using glucose-limited fed-batch fermentation, we have reduced the acetate formation in stage 1. We engineered a bacterium for conversion of arabinose to xylitol by a pathway using arabitol as an intermediate. This pathway did not efficiently convert arabinose to xylitol, but it remains a possible conversion route if we can find an enzyme to efficiently convert arabitol to xylulose. The wild-type repressor of the bacterial lactose operon (lacI) was deleted from strains, and mutants of the lacI gene were made by site-directed mutagenesis. Multiple temperature-sensitive mutants were made, which were used to construct an expression system in which the enzyme (xylose reductase) expression was controlled by temperature. A mutant with a less effective lac repressor was used to construct an expression system in which gene expression was controlled by the presence of arabinose in the culture medium. The cooperator meets regularly in person with the Authorized Departmental Officer's Designated Representative.
Impacts
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Publications
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The objective of this Cooperative Research and Development Agreement (CRADA) is to develop and scale-up novel methods for the production of xylitol from agricultural feedstocks, such as corn fiber and wheat straw. Approach (from AD-416) Fermentation variables such as cost (as a function of components and time) , substrate concentration, enzyme expression, media composition, carbon source, temperature, pH, and seed inoculum quality will be optimized using recombinant Escherichia coli strains such as ZUC 140, ZUC170, and ZUC99/pATX210 which were developed under a previous Cooperative Research and Development Agreement (CRADA) between ARS, zuChem, and Biotechnology Research and Development Corporation (BRDC). These strains will be independently improved/optimized and then engineered to combine both the pathways for conversion of xylose to xylitol and arabinose to xylitol, mutagenized to improve flux of the system to produce xylitol, and directed evolution will be performed in order to improve activities of individual enzymes. In general, improvement will be made in substrate specificity, thermal stability, and kinetic performance of the various enzymes, as well as overall xylitol yield from arabinose and xylose. New enzymes relevant to the conversion of xylose and/or arabinose to xylitol with improved activities will be isolated as necessary for the project. In addition, corn fiber and wheat straw feedstocks will be evaluated for xylitol production and compared to other hemicellulose hyrdolysate sources which are available during the project. This will include evaluation of different purification and production methods for making hemicellulose hydrolysates or the feedstocks; mutagenesis of the E. coli strains for adaptation to the feedstocks and inhibitors which may be present in the feedstock; and testing and optimization of the feedstocks (which may have different percentages of arabinose and xylose) for higher yields of pentose sugars. Feedstock pre-treatment will include dilute acid pre-treatments at moderate temperature. Significant Activities that Support Special Target Populations Under this new 2-year CRADA, both ARS and zuChem are working together to develop a commercially feasible bioprocess for making xylitol, a low calorie anticariogenic sweetener since February 2009. This collaborative effort was established to enhance ongoing ARS research in this area. Microbial production of xylitol, using hemicellulosic biomass such as agricultural and forestry residues, is attractive for reducing the manufacturing cost. Under a previous 5-year CRADA that ended in February 2008, research was focused on using molecular biology and pathway engineering to create a recombinant microorganism with the ability to produce xylitol from xylose and arabinose simultaneously because the hemicellulosic hydrolyzates are generally rich in these sugars. We constructed a recombinant bacterium that produces xylitol from xylose with very high productivity from a mixture of xylose and arabinose without producing any arabitol from arabinose. We also engineered a xylose reductase enzyme to have increased specificity towards xylose. Finally, we constructed a recombinant bacterial strain that converts arabinose to xylitol. The new work has been targeted to generate a recombinant bacterium that will convert both xylose and arabinose to xylitol. The cooperator meets regularly in person with the Authorized Departmental Officer's Designated Representative.
Impacts
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