Progress 09/01/09 to 08/31/13
Outputs
Target Audience: The target audience for this research is primarily researchers interested in biomass feedstock modifications for cellulosic processing. Changes/Problems: The initial workplan included a collobaration as part of the processing assessments; however, we were unable to finalize the research agreement with that group and completed the processing internally. We did not use the funds that were budgeted for that contract. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact. Agrivida has produced transgenic sorghum plants that contain enzymes required for converting biomass into biofuels. The enzymes have been modified to be inactive to protect plant growth with the capability to be activated after harvest. Processing experiments have demonstrated production of intermediate sugars using moderate conditions and lower energy and enzyme inputs, an important step in reducing the economic cost of cellulosic ethanol production by 20% below the industry standard. Objective 1. Engineer enzyme switches that have efficient temperature and pH induced splicing activity. This objective was completed in the previous year. Objective 2. Create switch-modified cellulase and xylanase enzymes and demonstrate their inducible activity. This objective was completed in the previous year. Objective 3. Generate sorghum lines that express switch-modified enzymes, select events that accumulate high enzyme levels, and demonstrate enzyme maturation and activity when produced in sorghum. Outputs. The transformation work has been optimized in one sorghum genotype - TX430. We performed over 100 transformation experiments with 15 different constructs, comprising controls and combinations of enzymes. The transformation efficiency remained low, but increased over the previous year with 50 events produced from about 8,000 sorghum immature embryos involved in transformation experiments. These plants were grown to maturity in our greenhouse, and both grain and stover was harvested for analysis. Outcomes / Impacts. We finalized our efforts to optimize sorghum transformation, but it is still very low compared to maize, 1% vs 40%. We were able to produce transgenic plants with our key enzymes discovered in Objectives 1 and 2 in previous years. For XylO43, we were able to utilize the dimerization domain approach to transform the plants, significantly improving the plant phenotype and poor germination characteristics associated with the unmodified enzyme. For CelNteg, we performed transformations with two modified approaches – 1) plant expression with localization that was achieved without intein modification, and 2) using the intein modified XylEU59 as the switching mechanism. Using the latter approach we were able to demonstrate splicing in the plant with both activity testing and western blots. Objective 4. Test the release of fermentable sugars from transgenic sorghum. Outputs. We finished the processing experiments of transgenic biomass from the events generated in Objective 3. Harvested greenhouse maize and sorghum was dried at 40oC and milled using a 1mm screen. These plants were evaluated in a consolidated process with moderate pretreatment and saccharification, using 20 mg of milled biomass. The pretreatment occurred at temperatures from 45oC-95 oC using a modified bisulfite pulping process. The saccharification was performed at 50oC using 0.2 ml AccelleraseTM 1500/g biomass + 0.1 ml AccelleraseTM XY/g biomass as a full enzyme cocktail for 72 hours. Monosaccharide concentrations in the hydrolysates were determined by both YSI and HPLC. Data from the resulting sugar production was used in a technoeconomic model to estimate the cost of producing cellulosic ethanol. Outcomes / Impacts. During the year, the process development group analyzed biomass samples from nearly 250 unique transgenic events. For the top candidate plants expressing two or more enzymes, we were able to achieve glucose and xylose production levels of 85% and 70%, respectively, with an enzyme loading of 20% the industry standard. Combined with the more moderate pretreatment, we estimate that cellulosic ethanol production costs with these feedstocks would be 20% lower than conventional processing.
Publications
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Progress 09/01/11 to 08/31/12
Outputs
Target Audience: The target audience for this report is primarily researchers interested in biomass feedstock modifications for cellulosic processing. Changes/Problems: The primary objectives for the project involve working with sorghum, not switchgrass. This was changed at project inception, but we were unable to change the documentation. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? We will complete the project by finalizing our sorghum transformation optimization and generating a set of transgenic sorghum events which embed our intein-modified enzymes. We will continue to evaluating the release of sugars from the transgenic materials.
Impacts
What was accomplished under these goals?
Impact. Agrivida has produced transgenic plants that contain enzymes required for converting biomass into biofuels. The enzymes have been modified to be inactive to protect plant growth with the capability to be activated after harvest. Processing experiments have demonstrated production of intermediate sugars using moderate conditions and lower energy and enzyme inputs, an important step in reducing the economic cost of cellulosic ethanol production by 15% below the industry standard. Agrivida has also developed transformation techniques for integrating the enzyme technology into forage sorghum. Objective 1. Engineer enzyme switches that have efficient temperature and pH induced splicing activity. This objective was completed in the previous year. Objective 2. Create switch-modified cellulase and xylanase enzymes and demonstrate their inducible activity. Outputs. We completed the intein-modification of the first intein-modified xylanase (XynB-CD) in the previous project year. This year, we continued the development of the second xylanase (XynO43) and one cellulose (CelNteg) pro-enzymes. For these two enzymes, we continued to construct new intein modified enzyme candidates using a panel of different inteins in at least ten different sites of the enzymes. Enzyme activities were determined at low temperatures and after heat treatments. We assessed the pro-enzymes via western blots to test intein splicing functionality. We selected candidates that demonstrated low activity initially at low temperatures but regained increased activity levels after heat treatment. For the top candidates, we created mutagenesis libraries to evolve their ability to regain activity at high temperature. We screened the libraries in either a phage expression system or robotically in a microbial host. Outcomes / Impacts. For XynO43, we discovered a number of candidates which exhibited spontaneous splicing and recovered enzyme activity; however, these candidates did not display pre-splicing control on activity. Alternatively, we discovered candidates where enzyme activity was initially within 3X background activity, but the highest post-slicing activity we could recover was 30% of the native, well below our metric or 50%. After considerable effort, we ended our attempts at intein-modification and attempted a dimerization domain approach. For CelNteg, we discovered a number of permissive intein locations where the intein didn’t mechanistically splice, but enzyme activity was still recovered. In one such location, rather than using a lone intein, we embedded a whole intein-modified enzyme. The resulting characterization experiments demonstrated that we were able to meet the pre- and post-slicing metrics defined above. Objective 3. Generate sorghum lines that express switch-modified enzymes, select events that accumulate high enzyme levels, and demonstrate enzyme maturation and activity when produced in sorghum. Outputs. The transformation work has focused on two sorghum genotypes - B.Wheatland and TX430 using the protocols optimized in the previous year of the project. We performed over 300 transformation experiments with 13 different constructs, comprising controls and combinations of enzymes. The transformation efficiency remained very low with 48 events produced from about 25,500 sorghum immature embryos involved in transformation experiments. Due to the low efficiency, we also used maize as a model system to test the constructs containing the enzymes developed in Objective 2. We generated transgenic plants with both wild type and intein-modified enzymes. These plants were grown to maturity in our greenhouse, and both grain and stover was harvested for analysis. Outcomes / Impacts. We have made considerable efforts to further improve sorghum transformation, and we recently identified a sorghum genotype and specific tissue culture conditions, which have improved our throughput and efficiency using test vectors that contain only the selectable marker. We have also assembled a set of new vectors with different genetic elements that we believe will improve sorghum selection after transformation and thereby improve the production of transgenic sorghum events. These vectors are progressing into the transformation pipeline and will hopefully help to further increase our ability to generate transgenic sorghum events containing our intein-modified enzymes. In maize transformation, we have generated approximately 500 events for preliminary processing analyses. We have confirmed that unmodified xylanase enzymes lead to significant phenotypes in both stover and grain development, necessitating the intein modification step. Objective 4. Test the release of fermentable sugars from transgenic sorghum. Outputs. We began processing experiments of transgenic maize biomass from the events generated in Objective 3. Harvested greenhouse maize was dried at 40oC and milled using a 1mm screen. These plants were evaluated in a consolidated process with moderate pretreatment and saccharification, using 20 mg of milled biomass. The pretreatment occurred at temperatures from 45oC-95 oC using a modified bisulfite pulping process. The saccharification was performed at 50oC using 0.2 ml AccelleraseTM 1500/g biomass + 0.1 ml AccelleraseTM XY/g biomass as a full enzyme cocktail for 72 hours. We also performed experiments using lower enzyme loadings. Monosaccharide concentrations in the hydrolysates were determined by both YSI and HPLC. Data from the resulting sugar production was used in a technoeconomic model to estimate the cost of producing cellulosic ethanol. Transgenic sorghum will be tested in the following year. Outcomes / Impacts. During the year, the process development group analyzed biomass samples from nearly 500 unique transgenic events. For the top candidate plants expressing two or more enzymes, we were able to achieve glucose and xylose production levels of 80% and 60%, respectively, with an enzyme loading of 40% the industry standard. Combined with the more moderate pretreatment, we estimate that cellulosic ethanol production costs with these feedstocks would be 15% lower than conventional processing.
Publications
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Objective I. We completed the intein engineering project and identified several engineered inteins that maintain splicing functionality. We synthesized recombinant intein sequences that contained 4 domains derived from each of 10 distinct inteins. We combined domains in common polypeptide backbones yielding a set of ~10,000 unique recombinant inteins. These were introduced into a tester thermostable cellulase. Libraries of yeast expressing and secreting the intein-modified cellulases were evaluated for protein expression and splicing. Results showed that exchanging elements between highly divergent intein sequences rarely resulted in a functional intein. We constructed recombinant inteins from parent sequences with ~50% amino acid conservation and identified 21 hybrid inteins that were functional. We inferred that certain domains within inteins are more tolerant of substitutions, marking these as more promising targets for subsequent mutagenesis. The hybrid inteins will be used in Objective II. Objective II. We are developing two xylanase and one cellulase pro-enzymes. We constructed libraries of intein-modified enzymes with different inteins in different sites of the enzymes. Enzyme activities were determined at low temperatures and after heat treatments. We assessed the pro-enzymes via western blots to test intein splicing functionality. We selected candidates that demonstrated low activity initially at low temperatures but regained increased activity levels after heat treatment. For top candidates, we created mutagenesis libraries to evolve their ability to regain activity at high temperature. We screened the libraries in either a phage expression system or robotically in a microbial host. For xylanase XynB-CD we successfully evolved an intein-modified xylanase that met our activity metrics and demonstrated splicing on a western blot. The native and intein-modified XynB-CD will be transformed into sorghum in Objective III. We continue to work on xylanase and cellulase candidates. Objective III. Multiple varieties of sorghum were grown in a greenhouse with tight environmental controls. Immature somatic embryos from each genotype were collected, sterilized, plated on embryo induction medium, and incubated to produce somatic embryogenic clusters. The best genotypes were used for transformation experiments to explore optimal conditions for induction, incubation, selection, and regeneration. Agrobacterium tumefaciens transformation vectors were constructed using standard molecular techniques. The plasmids were introduced into Agrobacterium strains and cultures were grown on plates. For plant transformation, the sorghum somatic embryogenic clusters were infected by mixing with bacterial suspension and placed on co-cultivation medium, followed by induction medium and incubation. Resulting somatic ebryogenic clusters were transferred onto selection medium and subsequently on regeneration medium. Variables such as inoculation level, the concentration of the selective agent, and several variations on the regeneration medium were tested to arrive at a system for sorghum transformation. We're currently transforming sorghum with genes of interest. PARTICIPANTS: R. Michael Raab, Jeremy Schley Johnson, Gabor Lazar, Mary Ross, Christine Feulner, Humberto de la Vega, Derek Sturtevant, Phil Lessard, Oleg Bougri, Matthew Parker, Vladimir Samoylov, Katie White, and Kelly Bondanza each contributed more than one person month to the first year of this project. Dr. Raab was project director and provided technical oversight and team management. Dr. Schley Johnson provided project and resource management. Dr. Lazar contributed to the intein engineering project and led the development of one of the proenzyme targets. Ms. Ross provided technical assistance to Dr. Lazar. Dr. de la Vega contributed to the intein project and led the development of one of the proenzyme targets. Mr. Sturtevant provided technical assistance to Dr. de la Vega. Dr. Lessard led the domain swapping project for the intein engineering objective. Ms. Feulner provided technical assistance to Dr. Lessard. Dr. Bougri constructed plant transformation vectors for the project. Dr. Samoylov led optimization and performance of sorghum transformation. Ms. Bondanza and Ms. White provided technical assistance to Dr. Samoylov. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective I. Methods for carrying out in vitro and in vivo recombination among sequences encoding domains from divergent inteins have been developed. From high throughput screens, pro-enzymes that carry recombinant inteins have been identified that are able to recover a portion of the wild type enzyme activity after in vitro intein activation. A comparison of the domain composition of the inteins that have been recovered from both active and inactive recombinant clones has provided insights into the types of interactions within an intein that promote or prevent protein activation in a context-specific manner. Based on this work, we have identified a number of recombinant inteins that maintain functional splicing. Most of these resulted from recombinations between sequences that were more closely related, while recombination of sequences that were more distantly related was considerably less successful. We continue to explore the application of these inteins in Objective II, as we develop new intein-modified pro-enzymes. Objective II. We developed an intein-modified XynB-CD pro-enzyme that had significant activity regulation. Compared to the wt xylanase (XynB), both XynB-CD constructs showed improved expression with high catalytic activity and thermostability in E.coli. A total of 42 intein modified pro-enzymes (iXynb-CD) were constructed cloned and expressed in lambda phage. Screening for heat inducible switching of xylanase activity was performed on xylanase diagnostic plates using a solid substrate, AZCL-xylan. Phagemid rescued candidates were validated for heat inducible enzyme activity in liquid assays. Xylanase activity of the two lead candidates have met our goal for less than 10% of that of the wild type XynB-CD at low temperature, while after a heat induction pretreatment, activity increases over 70% of the wild type. Each iXynB-CD lead shows superior thermostability with no apparent loss in activity. For the other xylanase and cellulase target enzymes, pro-enzyme lead candidates have been developed and further mutagenesis will be performed to reach the metrics. Within this set we have several leads for the O68 xylanase enzyme, which we are further developing. Objective III. We have found specific sorghum varieties which perform best in terms of health, size of seed head, and predictable in terms of time between pollen shed and embryo production. Based on efficiency of somatic embryo induction and proliferation of somatic cultures in general, four genotypes were selected for further transformation studies, which led to the final selection of two genotypes for transformation. Procedures for cultivation, incubation, and induction of embryogenic cultures and transformation of those clusters have been developed. We have also optimized Agrobacterium inoculation levels, concentration levels for our selective agent, selection procedures, and regeneration, and have now demonstrated successful sorghum transformation. We are continuing to optimize our plant expression vectors to improve transformation efficiency, and are beginning to transform enzymes into plants.
Publications
- No publications reported this period
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Objective I: During the first year, we have initiated intein engineering by designing and synthesizing recombinant plasmids encoding elements (domains) derived from 14 distinct inteins, representing varying levels of sequence-relatedness to other inteins in the set. Individual clones were prepared such that domains from distinct inteins were juxtaposed in common polypeptide support structures (backbones) yielding a set of ~125 different pairwise combinations of domains. This set represents the initial diversity of our libraries, from which we can generate further diversity through intein domain shuffling. Coding sequences for a subset of these inteins were introduced into the coding sequences of our tester thermostable cellulase clone. Recombination among the coding sequences for these intein-modified pro-enzyme and expression in microbial hosts has allowed us to create libraries of recombinant inteins with up to 10,000 possible combinations of intein domains. Objective II: We focused on developing pro-enzymes of two xylanases and one cellulase. For each of the enzyme candidates, we constructed libraries of pro-enzymes with different inteins in different sites within the enzymes. Initial screens were performed to evaluate the activity of these enzymes at low temperatures and after heat treatments. We selected candidates that demonstrated low activity initially with increasing activity after the heat treatment. For these candidates, we created mutagenesis libraries to evolve their ability to regain activity at high temperature. For one of the xylanases, we also removed the carbohydrate binding domain and tested the differently truncated versions for catalytic activity and thermostability to select a catalytic domain construct (XynB-CD) for pro-enzyme development. We generated intein modified catalytic domain constructs (iXynB-CD) and screened the iXynB-CD pro-enzymes for heat and/or pH shift inducible activity. We used automation to select and validate leads. Objective III: Multiple varieties of sorghum were grown in a greenhouse with a computerized control system that allows tight control over the indoor conditions despite the season. Immature somatic embryos from each genotype were collected, sterilized, plated on embryo induction medium, and incubated to produce somatic embryogenic clusters. The most promising genotypes were used for further transformation experiments which explored optimal conditions for co-cultivation, induction, and incubation. Agrobacterium tumefaciens transformation vectors were constructed using standard molecular techniques. The plasmids were introduced into Agrobacterium strains, and cultures were grown on plates. For plant transformation, the sorghum somatic embryogenic clusters were infected by mixing the explants with bacterial suspension and placed on co-cultivation medium, followed by induction medium and incubation. Resulting somatic ebryogenic clusters were transferred onto selection medium. PARTICIPANTS: R. Michael Raab, Jeremy Schley Johnson, Gabor Lazar, Mary Ross, Humberto de la Vega, Derek Sturtevant, Phil Lessard, Sarah Dohle, Matthew Parker, Vladimir Samoylov, and Kelly Bondanza each contributed more than one person month to the first year of this project. Dr. Raab was project director and provided technical oversight and team management. Dr. Schley Johnson provided project and resource management. Dr. Lazar contributed to the intein engineering project and led the development of one of the proenzyme targets. Ms. Ross provided technical assistance to Dr. Lazar. Dr. de la Vega contributed to the intein project and led the development of one of the proenzyme targets. Mr. Sturtevant provided technical assistance to Dr. de la Vega. Dr. Lessard led the domain swapping project for the intein engineering objective. Ms. Dohle provided technical assistance to Dr. Lessard. Dr. Somoylov led optimization and performance of sorghum transformation. Ms. Bondanza provided technical assistance to Dr. Samoylov. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective I: Methods for carrying out in vitro and in vivo recombination among sequences encoding domains from divergent inteins have been developed. Thorough testing of these methods has identified conditions that ensure the maximum possible diversity among libraries of recombinant inteins, a factor that is critical to the objective of thoroughly screening possible recombinants for splicing activity. Furthermore, methods were developed and tested for superimposing random mutagenesis upon recombinant inteins with the best activity, enabling further exploration of the structure/activity relationships among intein domains. From high throughput screens, pro-enzymes that carry recombinant inteins have been identified that are able to recover a portion of the wild type enzyme activity after in vitro intein activation. A comparison of the domain composition of the inteins that have been recovered from both active and inactive recombinant clones has provided insights into the types of interactions within an intein that promote or prevent protein activation in a context-specific manner. Objective II: Two versions of the catalytic domain construct were selected for enzyme development. Compared to the wt xylanase (XynB), both XynB-CD constructs showed improved expression with high catalytic activity and thermostability in E.coli. A total of 42 intein modified pro-enzymes (iXynb-CD) were constructed cloned and expressed in lambda phage. Screening for heat inducible switching of xylanase activity was performed on xylanase diagnostic plates using a solid substrate, AZCL-xylan. Phagemid rescued candidates were validated for heat inducible enzyme activity in liquid assays. Several candidates demonstrated heat inducible high activity with thermostability. Xylanase activity of the two lead candidates have met our goal for less than 10% of that of the wild type XynB-CD at low temperature, while after a heat induction pretreatment, activity increases over 60% of the wild type. Each iXynB-CD lead shows superior thermostability with no apparent loss in activity. For the other xylanase and cellulase target enzymes, pro-enzyme lead candidates have been developed and further mutagenesis will be performed to reach the metrics. Objective III: We have found specific sorghum varieties which perform best in terms of health, size of seed head, and predictable in terms of time between pollen shed and embryo production. Based on efficiency of somatic embryo induction and proliferation of somatic cultures in general, four genotypes were selected for further transformation studies. Procedures for cultivation, incubation, and induction of embryogenic cultures and transformation of those clusters have been developed.
Publications
- No publications reported this period
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