Progress 01/15/11 to 01/14/16

Outputs
Target Audience:Target audiences: The target audience in the last year was undergraduate and graduate students at Michigan State University as well as our collaborators at MBI International, a subsidiary of the Michigan State University Foundation. It also included graduate students at the Catholic University of Korea, and a group of Korean microbiologists. Efforts: In 2015, the research activities conducted as part of this project were highlighted in the graduate Integrative Microbial Biology course (MMG 801) as part of a lecture on metabolic flux analysis, in a seminar directed at graduate students of the Catholic University of Korea, and in a seminar offered to Korean microbiologists at the 42nd Annual Meeting & International Symposium of the Korean Society for Microbiology and Biotechnology. Undergraduates in my laboratory were trained in different aspects of the microbiology work associated with this project, in particular with aseptic techniques, cloning, gene knockout technology, gene expression assays, and anaerobic techniques. Both graduate students learned to extract total RNA from bacterial cultures and learned to process and analyze RNA sequencing data. The postdoctoral fellow learned anaerobic techniques, as well as new statistical methods to analyze proteomics and RNA sequencing data. I met several times with the scientists at MBI International to transfer gene knockout technology, results on identification of potential glucose and succinate transporters, and exchange ideas for future work. MBI International and I collaborated on a grant pre-proposal to DOE, which was not allowed to move forward as a full proposal. Changes/Problems:Major problems: We had to make changes in our objectives based on the difficulties encountered with engineering A. succinogenes. These changes are described in the Accomplishment section already. Only a few genetic tools exist for A. succinogenes which have been developed in the PD's laboratory. The genetics of Pasteurellaceae is poorly understood in general. For example, the structure of promoters is not understood. In contrast to the situation with Escherichia coli,it is almost impossible to identify a promoter sequence just by looking at the sequence, and E. coli promoters typically do not function in A. succinogenes, making genetic tools available in E. coli non-functional in A. succinogenes. This lack of knowledge of the genetics of Pasteurellaceae explains the multiple difficulties encountered in the project, the repeated changes in objectives for each of the aims, and the slow progress on individual aims. Unexpected outcomes: We expected the ΔpflB strain to produce more succinate than the wild-type strain in glycerol microaerobic chemostats. We also expected that decreasing the strength of the promoter guiding the expression of the PDO pathway genes would decrease PDO production and increase succinate production. Finally we expected to be able to knock out the acetate production pathway, knowing that succinate production generates ATP in A. succinogenes, contrary to the situation in E. coli. What opportunities for training and professional development has the project provided?Training: The PI trained her graduate students in science practices, including data interpretation and analysis, evidence-based explanation and argumentation, development of scientific questions, experimental design, and hypothesis generation and evaluation. She also trained them in oral and written science communication. The PI and graduate students trained a total of twelve undergraduate students working on the project in many experimental methods used in this research, including microbiology techniques (in particular aseptic and anaerobic techniques),molecular biology (cloning, PCR), enzyme assays, and HPLC. They trained the undergraduate students in maintaining good lab notebooks as well. Most of the training was done one-on-one. The PI, postdoctoral fellow, graduate students trained one undergraduate student to give professional science presentations. The student presented his project in lab meeting, received extensive feedback and presented his work twice more, until his presentation (communication skills and content) was of high quality. Professional development: in year 2 of the project one graduate student attended a two-week long workshop on genome assembly. Since then, the two graduate students have attended day-long workshops on RNA seq analysis and the use of MatLab to benefit their research. They are using these new computational skills to analyze their RNA seq data. 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: This project focused on sustainable bioenergy research, particularly on developing enhanced-value co-products. The Energy Independence and Security Act of 2007 mandates the use of one billion gallons of biodiesel by 2012. The production of this drop-in fuel comes along with the production of 100 million gallons of glycerol, as waste. Our goal was to develop a microbial process that converts glycerol into a high-value product, succinate. Succinate production from glycerol would have several benefits. It would increase the economic sustainability of biodiesel production. It could also replace oil-based maleic anhydride as the feedstock in a ~$15 billion/yr bulk chemical market. Actinobacillus succinogenes is one of the best natural succinate producers, and it grows on glycerol by respiration. The best succinate yields (75% of all the glycerol consumed, or 75% of the maximum theoretically possible) were in cultures grown under 5% oxygen. Because these growth conditions are challenging and expensive to maintain in industrial settings, we have been engineering A. succinogenes to grow on glycerol with no oxygen. We first engineered a strain that could grow without oxygen by forcing it to produce 1,3-propanediol (PDO) as a side-product. PDO is another molecule with many industrial applications of its own. This strain produced PDO efficiently (up to 60% of the glycerol used), but produced little succinate (at most 8%). We are now working on redirecting most of the glycerol used for PDO production toward succinate production. In our studies, we noticed that A. succinogenes does not consume glycerol in the presence of glucose. We are now engineering A. succinogenes to allow it to consume both carbon sources simultaneously. We should be able to balance glucose and glycerol consumption to simultaneously maximize anaerobic growth and succinate production. Because purifying succinate away from other fermentation products adds a major cost to succinate production, we have been working on reducing the number and amounts of byproducts secreted during succinate production. We blocked formate production and have been working toward blocking acetate production as well. To further increase succinate production, we identified the proteins that import sugars into the cell for growth, and the proteins that likely export succinate out of the cell, with the goal of increasing transport in and out of the cell. We have shared our results, methods, and strains with MBI International. This partnership guides the de-risking of succinate production technology to accelerate transfer to market. ACCOMPLISHMENTS IN REPORTING PERIOD Aim 1, characterization of A. succinogenes's growth and succinate production on glycerol. Because A. succinogenes does not grow fermentatively on glycerol, we studied its growth and succinate production during respiratory growth. The succinate yield was highest (75% of maximum theoretical yield) in a microaerobic. To our disappointment, our ΔpflB strain (aim 4) strain that produced 1.7-fold more succinate than the wild-type strain on glucose produced the same succinate yield as the wild-type strain in glycerol microaerobic chemostats. Our multiple attempts to vary oxygen input and chemostat flow rate did not increase succinate production by the ΔpflB strain. Aim 2, developing a strain that grows anaerobically on glycerol. Because the best succinate production took place in microaerobic conditions, and because microaerobic conditions are not ideal for industrial scale up, we narrowed our aim 2 to developing a strain that produces succinate fermentatively. We initially used two approaches. First, we tried to express the Escherichia coli hydrogen-evolving hydrogenase 3 in A. succinogenes, to remove the extra electrons through hydrogen evolution. This strategy was abandoned early, because expressing these genes in A. succinogenes was toxic to the cells. Second, we expressed the Clostridium butyricum 1,3-propanediol (PDO) pathway in A. succinogenes to remove the excess electrons by producing PDO. A strain expressing the PDO genes under a strong promoter on a plasmid produced PDO efficiently (up to 60% of the maximum theoretical yield), but produced little succinate (at most 8% of the maximum theoretical yield). Our attempts to substitute the pflB gene with the PDO pathway genes on the chromosome using a ΔpflB::pdo cassette failed. Placing the PDO genes under the control of promoters showing 4-fold and 10-fold lower activity than the pckA promoter yielded the same high PDO and low succinate production. Work past the grant period will involve testing promoters with 15- to 50-fold lower activity than the pckA promoter (as identified in our RNA sequencing results). In a third approach, we noticed that A. succinogenes does not consume glycerol when glucose is present. We constructed a synthetic operon expressing the glycerol metabolism genes under a constitutive promoter. The goal with this strategy is to develop a strain that grows on a combination of lignocellulosic hydrolysates and glycerol. The wild-type strain expressing the glycerol genes grew on glucose alone, but grew poorly on glucose-glycerol and stopped growing after 2-3 transfers to fresh media. The same synthetic operon expressing a glycerol kinase mutated to remove two types of feedback inhibition did not allow co-utilization of glucose and glycerol either. Construction of a glpR knockout to remove the last possible inhibition by the glycerol repressor is in progress. Aim 2c: I had established a collaboration with Dr. D.H. Park from Seokyeong University, Korea, to accomplish this aim last winter. Dr. Park had previously shown that a microbial fuel cell cathode could serve as electron donor to enhance growth and succinate production by glucose-grown A. succinogenes. But the collaboration fell through. Dr. Park stopped answering my e-mails in the fall, and I haven't heard from him in 6 months. Aim 3, quantification of metabolic fluxes in glycerol-grown A. succinogenes. Aim 3 was designed to help us compare the engineering approaches used in aim 2, and to predict which further modifications were needed to further improve succinate production on glycerol. The preliminary work needed for MFA studies (i.e., establishing the biomass composition of glycerol-grown cultures) was done, but we could not proceed further with this aim because of our lack of success in constructing strains able to grow fermentatively on glycerol. . Aim 4, Optimization of our knockout methodology and engineering of A. succinogenes for increased succinate production from glycerol. Early on, we established a markerless knockout method that allowed us to block formate production (ΔpflB mutant strain). We have since demonstrated that markerless mutations can be directly introduced in A. succinogenes using multiplex transformation. Acetate is the major remaining by-product in the ΔpflB strain. We made numerous attempts to inactivate the acetate pathway, but all approaches failed, even when trying to delete the pta and ackA genes in a strain expressing the two genes on a plasmid. The next strategy was to partially inhibit the expression of pta and ackA with an inhibitory RNA. To test this approach, we synthetized a small inhibitory RNA based on model synthetic E. coli inhibitory RNAs using lacZ as the target gene. No decrease in LacZ activity in the recombinant strain was detected. We have since purified small RNAs from A. succinogenes and are characterizing the structure of these small RNAs. This knowledge will be used past the grant period to synthetize an inhibitory RNA targeting the acetate pathway genes.

Publications

Progress 01/15/14 to 01/14/15

Outputs
Target Audience: Target audiences: At this point of our project, the target audience is undergraduate and graduate students at Michigan State University as well as our collaborators at MBI International, a subsidiary of the Michigan State University Foundation. Efforts: The research activities conducted as part of this project are regularly highlighted in the freshman Frontiers in Biochemistry (BMB 101) course, the senior undergraduate Biochemistry Seminar (BMB 495 course), the graduate Integrative Microbial Biology course (MMG 801), and the graduate Multidisciplinary Bioprocessing Laboratory course (CHE 883). In CHE883, teams of two-three interdisciplinary students work part-time for one semester in the laboratory to accomplish a small project in relationship to this research effort. We have transferred our gene knockout technology to MBI International, which is also interested in constructing mutant derivatives of A. succinogenes. We have an agreement with MBI International. MBI International focuses on developing and commercializing sustainable bio-based technologies, and already has several patents on succinate production by A. succinogenes. We expect that the commercial viability of the strains and processes developed in this project will be demonstrated in larger scale by MBI International, with further technology transfer to commercial partners. Changes/Problems: We have made changes in our objectives based on the difficulties encountered with engineering A. succinogenes. These changes ar described in the Accomplishment section already. Only a few genetic tools exist for A. succinogenes which have been developed in the PD's laboratory. The genetics of Pateurellaceae is poorly understood in general. For example, the structure of promoters is not understood. In constrast to the situation with Escherichia coli,it is almost impossible to identify a promoter sequence just by looking at the sequence, and E. coli promoters typically do not function in Pasteurellaceae species, making genetic tools available in E. coli non-functional in A. succinogenes and other Paterurellaceae. This lack of knowledge of the genetics of Pasteurellaceae explain the multiple difficulties encountered in the project and the repreated changes in objectives for each of the aims. What opportunities for training and professional development has the project provided? Training activities The PD and graduate students Nik McPherson and Rajasi Joshi have trained the three new undergraduate students working on the project in many experimental methods used in this research. They trained the undergraduate students in maintaining good lab notebooks as well. Most of the training was done one-on-one. The PD taught both graduate students (Nik McPherson and Rajasi Joshi) working on the project to work with RNA. Professional development Both graduate students learned to extract total RNA and small RNAs from cultures of A. succinogenes. They both learned to quantify RNAs using RT-qPCR. Nik McPherson also learned to purify elongation factors and ribosomes to perform translation elongation assays. Undergraduate students Vladislav Jdanov, Jack Peleman, and Aaron Walters have learned and now master the most common molecular biology techniques used in the project (genomic and plasmid DNA extractions, restrictions, PCR, cloning, transformations). They have also learned anaerobic techniques, and how record growth curves. Vladislav Jdanov has learned to use the HPLC and he can now do fermentation balances on A. succinogenes cultures. Aaron Walters has learned to perform enzyme assays to measure beta-galactosidase activity. Jack Peleman has learned to purify proteins, to do protein assays, run SDS-PAGE gels, and to do far-western blots. How have the results been disseminated to communities of interest? April 2014: Invited talk on "Redox power at work: microbial engineering and enzyme catalysis" In the Department of Microbiology and Molecular Genetics at Michigan State University. October 2014: Poster: R. Joshi and C. Vieille. "Development of tools and strategies to increase succinate production by Actinobacillus succinogenes on glycerol." USDA 2014 PD workshop for Sustainable Bioenergy: Enhanced-value Co-Product Development; Washington, DC. What do you plan to do during the next reporting period to accomplish the goals? Aim 1, characterization of A. succinogenes's growth and succinate production on glycerol. Completed. Aim 2, developing a strain that grows anaerobically on glycerol. Starting from a strong promoter, we have constructed a mutant promoter library by randomly mutating the -10 region. This library has been cloned in front of the reporter gene, lacZ, and is ready for transformation into a ΔlacZ A. succinogenes strain (already constructed). We plan to screen a few hundred independent transformants for lacZ activity in 96-well plates (already optimized) to rank the different promoters by strength. A selection of promoters of different strengths will be tested in front of the PDO genes to express them at a level optimum for growth and succinate production. Screening of this library will start once we have determined the level of PDO production in a strain expressing the PDO genes under the non-mutagenized promoter. The nature of promoters in Pasteurellaceae species is not well understood. Comparing the strength of hundreds of promoters will help the help the community of scientists working on these bacterial species (animal pathogens in particular) understand what features make a good promoter. We have started constructing a synthetic operon expressing the glycerol metabolism genes (i.e., permease, kinase, and dehydrogenase) under a constitutive promoter. The five genes are cloned in the order the proteins are used in the pathway. At first, this synthetic operon will be expressed from a strong promoter on a plasmid to demonstrate the proof of concept that A. succinogenes can consume glycerol and glucose simultaneously. Based on the fermentation balance of the first test strain, promoters of different strengths will then be tested to balance glycerol and glucose consumption to increase succinate production. The PDO production and glucose-glycerol co-utilization approaches will be tested in the wild-type and the ΔpflB strains. Aim 3, quantification of metabolic fluxes in glycerol-grown A. succinogenes in the strains built in Aim 2. While we have finished the preliminary work for MFA studies, we have not finished Aim 2, developing strains growing anaerobically on glycerol. Because accomplishing Aim 3 would not directly translate into immediate increases in succinate production, we will likely put it on hold until we have better performing strains. Aim 4, construction of an A. succinogenes strain that produces succinate as sole fermentation product from glycerol. We will continue our approach to inhibit the acetate production pathway with a synthetic RNA. To maximize our chances of success, we will base the design on the structure of sequenced A. succinogenes small RNAs. The A. succinogenes small RNAs are ready for sequencing, but we plan to also sequence small RNAs from two other Pasteurellaceae species (Haemophilus influenzae and Actinobacillus pleuropneumoniae) to increase the applicability of this study to the entire Pasteurellaceae family, including animal pathogens. These two species are available in the laboratory from a colleague in the PD's department. We will be able to identify small RNAs common to multiple species, which will make future designs of synthetic inhibitory RNAs that more successful. The RNAseq data will be analyzed using a computational pipeline available from another laboratory on the PD's department. The RNAseq data will be used to design several small RNAs that will be expressed in A. succinogenes from a plasmid. The recombinant strains will be tested for acetate production in glucose- and glycerol-based media.

Impacts
What was accomplished under these goals? IMPACT: This project focuses on sustainable bioenergy research, particularly on developing enhanced-value co-products. The Energy Independence and Security Act of 2007 mandates the use of one billion gallons of biodiesel by 2012. The production of this drop-in fuel comes along with the production of 100 million gallons of glycerol, its direct byproduct. The goal of this project is to develop a microbial process that converts glycerol into a high-value product, succinate. Succinate as a co-product of biodiesel production would have several benefits. It would increase the economic sustainability of biodiesel production. It could also replace petroleum-based maleic anhydride as the feedstock in a ~$15 billion bulk chemical market. Actinobacillus succinogenes is one of the best natural succinate producers, and it grows on glycerol by respiration. In our first aim, we characterized A. succinogenes's growth on glycerol in different conditions. The best succinate yields (75% of the maximum theoretical yield) were in cultures grown microaerobically under 5% oxygen. Because microaerobic cultures are challenging and expensive to maintain in industrial settings, in our second aim, we are engineering A. succinogenes to grow anaerobically on glycerol. In a first approach, we engineered a strain that could grow anaerobically by forcing it to produce 1,3-propanediol (PDO) as a fermentation byproduct. This strain produced PDO efficiently (up to 60% of the maximum theoretical yield), but produced little succinate (at most 8% of the maximum theoretical yield). We are pursuing this strategy by trying to minimize how much glycerol is used for PDO production. In a second approach, we have noticed that A. succinogenes does not use glycerol when glucose is present in the growth medium. We are now engineering A. succinogenes to allow it to consume both carbon sources simultaneously. We should be able to balance glucose and glycerol consumption to simultaneously maximize anaerobic growth and succinate production. We might not pursue our third aim, which would provide information on the way sugars are preferentially consumed in the cell, but which would not translate into immediate increases in succinate production. Instead, we are focusing on our fourth aim of reducing the number and amounts of byproducts secreted during succinate production to increase succinate yields, productivity, and titers on glycerol. We share our results, methods, and strains with MBI International, which has an established relationship with an industry partner. This partnership guides the derisking of succinate production technology to accelerate transfer to market. ACCOMPLISHMENTS IN REPORTING PERIOD Aim 1, characterization of A. succinogenes's growth and succinate production on glycerol. Most of this aim was completed in year 3. One journal article was published on our results. Because the best conditions for succinate production from glycerol were microaerobic, and because microaerobic conditions are not ideal for industrial scale up, we revised our Aim 2 to focus on developing a strain that produces succinate in anaerobic conditions. In year 3 we had determined that a ΔpflB mutant strain that produced 1.7-fold more succinate than the wild-type strain on glucose produced the same succinate yield (78 mmol/mol glycerol) as the wild-type strain in glycerol-grown microaerobic chemostat cultures. In year 4, our attempts to vary oxygen input and medium flow rate to maximize succinate production by the ΔpflB strain did not produce any yield increases. We will go back to studying this mutant strain once we have developed a strain that can grow anaerobically on glycerol in Aim 2. Aim 2, developing a strain that grows anaerobically on glycerol. In years 3 and 4, we tried two separate approaches to develop a strain able to grow anaerobically on glycerol. In one approach, we attempted to express the Escherichia coli hydrogen-evolving hydrogenase 3 in A. succinogenes, to get rid of the extra electrons through hydrogen evolution. This strategy was abandoned early in year 4, because expressing these genes in A. succinogenes was toxic to the cells .The second approach consists of expressing the Clostridium butyricum 1,3-propanediol (PDO) pathway in A. succinogenes to remove the excess electrons by producing PDO. Our proof-of-concept strain expressing the PDO genes under a strong promoter on a plasmid produced 1,3-propanediol efficiently (up to 60% of the maximum theoretical yield), but produced little succinate (at most 8% of the maximum theoretical yield). In year 4, to bring the PDO gene copy number down to one, we built a recombination cassette that substituted the pflB gene with the PDO genes under the same strong promoter. All our attempts to build a new ΔpflB strain by selecting for anaerobic growth on glycerol using this construct failed. We have since placed the PDO genes under a very weak promoter in the plasmid used in the initial construct. The recombinant wild-type strain carrying this plasmid has not been able to grow anaerobically on glycerol, and no PDO is detected in glucose-grown cultures, suggesting that this promoter is too weak. Our next strategy is to build a promoter library to identify promoters of intermediary levels and test them for PDO gene expression. Because of the difficulties met in our other approaches, we are starting a third approach. We have noticed that A. succinogenes does not consume glycerol when glucose is present. We started constructing a synthetic operon expressing the glycerol metabolism genes under a constitutive promoter. Expressing the glycerol utilization genes constitutively should allow A. succinogenes to grow anaerobically on a mixture of glycerol and glucose. Aim 3, quantification of metabolic fluxes in glycerol-grown A. succinogenes in the strains built in Aim 2. Aim 3 was designed to help us compare the engineering approaches used in Aim 2, and to predict which further modifications (knockouts or knock-ins) are needed to further improve succinate production on glycerol. Metabolic flux analysis (MFA) require that we know the exact biomass composition and the dry cell weight in relationship to optical density. Repeat experiments in glycerol-grown cultures indicated that dry cell weight and protein contents were identical in glucose- and glycerol-grown cultures. We are assuming that the rest of the cell composition is also identical in glucose- and glycerol-grown cultures. We will be able to use the same energetic and intermediate metabolite requirement calculations for biomass synthesis in glycerol-grown cultures as we used for glucose-grown cultures. These studies completed the preliminary work needed prior to MFA studies. Aim 4, construction of an A. succinogenes strain that produces succinate as sole fermentation product from glycerol. Acetate is the major by-product in succinate production by A. succinogenes. In year 4, we made numerous attempts to inactivate the acetate pathway, but all our approaches failed. We attempted to knockout the two genes (pta and ackA) separately or as a double deletion. We attempted the deletions in a strain expressing the two genes on a plasmid, in case the two genes were essential. The next approach is to partially inhibit the expression of pta and ackA with an inhibitory RNA. To test this approach with a reporter gene, we constructed a small inhibitory RNA based on model synthetic inhibitory RNAs in Escherichia coli using lacZ as target gene. No decrease in LacZ activity in the recombinant strain could be detected. We since purified small RNAs from A. succinogenes grown in different conditions. We will use RNAseq to identify the structure of small inhibitory RNAs in A. succinogenes, and use this knowledge to synthetize an inhibitory RNA targeting the acetate pathway genes.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Schindler, B.D., R.V. Joshi, and C. Vieille. 2014. Respiratory glycerol metabolism of Actinobacillus succinogenes 130Z for succinate production. J. Ind. Microbiol. Biotechnol. 41:13391352. doi: 10.1007/s10295-014-1480-x.

Progress 01/15/13 to 01/14/14

Outputs
Target Audience: At this point of our project, the target audience is undergraduate and graduate students at Michigan State University plus our collaborators at MBI International, a subsidiary of the Michigan State University Foundation. The research activities conducted as part of this project are regularly highlighted in the freshman Frontiers in Biochemistry (BMB 101) course, the senior undergraduate Biochemistry Seminar (BMB 495 course), and the graduate Integrative Microbial Biology (MMG 801) course. We have started to transfer our gene knockout technology to MBI International, which is also interested in constructing mutant derivatives of A. succinogenes. We have an agreement with MBI International. MBI International focuses on developing and commercializing sustainable bio-based technologies, and already has several patents on succinate production by A. succinogenes. We expect that the commercial viability of the strains and processes developed in this project will be demonstrated in larger scale by MBI International, with further technology transfer to commercial partners. To our knowledge, no one has built a library of artificial promoters for any species of Pasteurellaceae. Pasteurellaceae encompass many animal pathogens. Developing a library of promoters of incrementally increasing strength that have so similarity to Pasteurellaceae DNA could help the community of scientists developing vaccines against these animal pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two graduate students have been working on the project since its inception. They are constantly learning new methods and deepening their understanding of microbial physiology and genetics. They are learning to write scientific manuscripts and to communicate science in general. Five undergraduate students have been trained and have helped in the experimental work in 2013, including a visiting student from Germany and a visiting student from India, and three MSU students. All the undergraduate students learned to work with anaerobic and aseptic methods and they learned multiple common molecular biology methods. Because of her valued contribution to the project, the Indian visiting student is included as a coauthor on our manuscript submitted to the Journal of Industrial Microbiology and Biotechnology. As part of their training, the two graduate students attended the 35th Symposium on Biotechnology for Fuels and Chemicals in April-May 2013 in Portland, OR, where they got the opportunity to network with people in their field and to discuss many related research endeavors. How have the results been disseminated to communities of interest? We transferred our gene deletion technology (i.e., knowledge and genetic constructs) to MBI International, which whom we have a cooperative agreement, and which is also interested in constructing mutant derivatives of A. succinogenes. MBI International focuses on developing and commercializing sustainable bio-based technologies, and already has several patents on succinate production by A. succinogenes. We expect that the commercial viability of the strains and processes developed in this project will be demonstrated in larger scale by MBI International, with further technology transfer to commercial partners. We shared our just-accepted manuscript on our knockout technology with a large commercial company possibly interested in licensing the succinate production process. Our results were written in two papers. One published online on March 7th, 2014 in Applied and Environmental Microbiology, the second under review by the Journal of Industrial Micribology and Biotechnology. What do you plan to do during the next reporting period to accomplish the goals? Aim 1 is completed. Aim 2. Engineer A. succinogenes or the growth conditions for anaerobic growth on glycerol. We initiated the construction of a library of synthetic promoters. The strength of these promoters will be screened using lacZ as the reporter gene in an A. succinogenes strain containing a lacZ deletion (already constructed). Promoters of incrementally increasing strength will be used to express the PDO genes in a DNA recombination cassette that will substitute the pflB gene with the PDO genes in the A. succinogenes chromosome. We expect to find a promoter that will express the PDO pathway genes at a level optimum for growth and succinate production. As many promoters (at least 200) as possible will be ranked in strength and their sequences will be used to identify sequence features associated with promoter strength. Such study has never been accomplished for any Pasteurellaceae species, including pathogens of farm animals. Our results could be used as tools to engineer serovars of these pathogens into live vaccines. Aim 3. Quantify metabolic fluxes in glycerol-grown A. succinogenes. 13C-based metabolic flux analysis (MFA) experiments necessitate that we know the exact composition of the biomass and the dry cell weight in relationship to the optical density of the bacterial culture. In 2014, we will start by completing the preliminary work of establishing the exact composition of the biomass in glycerol-grown A. succinogenesand perform the first 13C-based MFA studies. Aim 4. Optimization of our gene deletion (knockout) methodology and engineering of A. succinogenes for increased succinate production from glycerol. While we have completed our aim of optimizing our gene knockout method for A. succinogenes, certain genes that we planned to delete appear to be essential for growth. To get around this problem, we plan to use synthetic inhibitory RNAs to partially inhibit expression of these genes. The target genes with be the two genes involved in acetate production. We expect the best recombinant strain to have a decreased acetate yield and a correspondingly increased succinate yield to compensate for the loss in ATP synthesis. Part of our approach will be to generate different constructs of incrementally increasing inhibitory capacity to identify what sequence and structural parameters control inhibitory capacity in inhibitory RNAs in A. succinogenes. Almost nothing is known about small inhibitory RNAs in Pasteurellaceae. This study will also help the scientific community that studies Pasteurellaceae species pathogenic to farm animals.

Impacts
What was accomplished under these goals? IMPACT. Actinobacillus succinogenes is a promising bacterial catalyst to develop a succinate production process from glycerol and create a high-value use for crude glycerol, the major byproduct of biodiesel production. A sustainable succinate production process from glycerol could have two major economic impacts: (i) A chemicals and industrial materials market based on petroleum would be replaced with a market based on a renewable resource. Such a change would decrease the US's dependency on foreign oil. (ii) Transforming crude glycerol into a higher value-added product would increase the sustainability of biodiesel production. We have made great strides toward developing a sustainable succinate production process from glycerol by reaching two milestones. First, we have identified bacterial growth conditions (i.e., microaerobic conditions) in which the original A. succinogenes strain produces succinate from pure glycerol with a yield reaching 75% of the maximum theoretical yield. We also demonstrated that succinate yields on crude glycerol obtained from the biodiesel industry are similar to those reached with pure glycerol, indicating that crude glycerol is an excellent feedstock for succinate production. Second, we have established a method to delete genes of A. succinogenes that will allow us to engineer A. succinogenes for even higher succinate yields from glycerol. We transferred our gene deletion technology to MBI International, which whom we have a cooperative agreement, and which is also interested in constructing mutant derivatives of A. succinogenes. MBI International focuses on developing and commercializing sustainable bio-based technologies, and already has several patents on succinate production by A. succinogenes. We expect that the commercial viability of the strains and processes developed in this project will be demonstrated in larger scale by MBI International, with further technology transfer to commercial partners. AIM BY AIM ACCOMPLISHMENTS. Aim 1. Characterize the parameters of A. succinogenes’ growth on glycerol and the metabolic pathways involved. One of the first genes we deleted in A. succinogenes was pflB, responsible for formate production. We characterized the product balance of our pflB deletion mutant in continuous microaerobic cultures and tested different culture parameters to increase succinate production by this mutant strain. We determined that the pflB deletion mutant produced the same succinate yield (78 mmol/mol glycerol) as the wild-type strain in identical culture conditions. To our disappointment, our attempts to modify culture conditions (e.g., flow rate and oxygen concentration) to find conditions that maximize succinate production in the mutant strain have not increased succinate yields so far. One manuscript was submitted for publication on the characterization of A. succinogenes respiratory growth on glycerol. Aim 2. Engineer A. succinogenes or the growth conditions for anaerobic growth on glycerol. Two parallel strategies had been initiated in 2012 to satisfy the redox requirements of A. succinogenes's anaerobic growth on glycerol. (i) Because expressing the PDO pathway genes under control of a strong promoter on a plasmid led to poor growth and mostly PDO production, in 2013 we planned to express the Clostridium butyricum 1,3-propanediol (PDO) pathway genes stably by inserting them in the A. succinogenes chromosome. We initiated the construction of a library of synthetic promoters. The strength of these promoters will be screened using lacZ as the reporter gene in an A. succinogenes strain containing a lacZ deletion (already constructed). Promoters of incrementally increasing strength will be used to express the PDO genes in a DNA recombination cassette that will substitute the pflB gene with the PDO genes in the A. succinogenes chromosome. We expect to find a promoter that will express the PDO pathway genes at a level optimum for growth and succinate production. (ii) The strategy of expressing the Escherichia coli hydrogen-evolving hydrogenase 3 in A. succinogenes was abandoned because expressing theses genes in A. succinogenes was toxic to the cells. Transformation of a plasmid that expresses the Escherichia coli hydrogen-evolving hydrogenase 3 under control of a strong promoter in A. succinogenes yielded cultures that started aggregating early after inoculation and were unable to grow to high densities. To our knowledge, no one has built a library of artificial promoters for any species of Pasteurellaceae. Pasteurellaceae encompass many animal pathogens. Developing a library of promoters of incrementally increasing strength that have no similarity to Pasteurellaceae DNA could help the community of scientists developing vaccines against these animal pathogens. Aim 3. Quantify metabolic fluxes in glycerol-grown A. succinogenes. 13C-based metabolic flux analysis (MFA) experiments necessitate that we know the exact composition of the biomass and the dry cell weight in relationship to the optical density of the bacterial culture. Because our previous MFA studies were on glucose-grown cultures, in 2013 we started determining biomass composition in continuous glycerol-grown cultures. An indication that these measurements are required is that the dry cell weight increased from 0.535 g/unit OD660 in glucose-grown cultures to 0.770 g/unit OD660 in glycerol-grown cultures. Measures of protein and RNA contents are in progress. Aim 4. Optimization of our gene deletion (knockout) methodology and engineering of A. succinogenes for increased succinate production from glycerol. After developing a knockout technology for A. succinogenes, in 2013 we determined how reproducible the knockout method is, what the impact of the deletion cassette size on knockout frequency is, and whether the same method can be used to introduce successive deletions in the same strain. We demonstrated that our knockout method is high reproducible, as long as the DNA flanking the selection marker gene is at least 600-bp long and as long as it contains at least 200 bp of homologous DNA. We also demonstrated that at least two consecutive deletions can be introduced into the same strain using the same method and selection marker (published in Applied and Environmental Microbiology in 2014). But we have still been unable to delete the two genes responsible for acetate production from acetyl-CoA (phosphate acetyltransferase and acetate kinase genes), likely because these two genes are essential for A. succinogenes growth. We now plan to decrease the expression of these genes using synthetic inhibitory RNAs. For many years we had faced the problem that cultures of A. succinogenes could not be numerated on plate, as serial dilutions led to a drastic drop in CFUs dilution as low as 10-3. After testing multiple hypotheses, last year we identified polyvinyl alcohol as a non-toxic additive that we could add to dilution buffers and to agar plates, which allows perfect numerations of serial dilutions. EVENTS. The PD gave invited talks on this project in May 2013 at the 35th Symposium on Biotechnology for Fuels and Chemicals in Portland, OR and in October 2013 at the 25th Anniversary Meeting of the Association for the Advancement of Industrial Crops in Washington, DC. One poster on this project was also presented in May 2013 at the 35th Symposium on Biotechnology for Fuels and Chemicals in Portland, OR.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Joshi, R.V., B.D. Schindler, N. McPherson, K. Tiwari, and C. Vieille. 2014. A markerless gene knockout method for Actinobacillus succinogenes 130Z based on natural transformation. Appl. Environ. Microbiol. Published online ahead of print on March 7th, doi: 10.1128/AEM.00492-14
• Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Schindler, B.D., R.V. Joshi, and C. Vieille. Respiratory glycerol metabolism of Actinobacillus succinogenes 130Z for succinate production. Revised manuscript submitted to J. Ind. Microbiol. Biotechnol.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2014 Citation: Joshi, R.V., B.D. Schindler, and C. Vieille. 2014. Engineering Succinate Production by Actinobacillus succinogenes on Glycerol. In: Proceedings of the 25th Anniversary Meeting of The Association for the Advancement of Industrial Crops (AAIC) and USDAs Agriculture and Food Research Initiative Bioenergy Program: New Crops: Bioenergy, Biomaterials, and Sustainability. In press.

Progress 01/15/12 to 01/14/13

Outputs
OUTPUTS: Actinobacillus succinogenes is a promising bacterial catalyst to develop a succinate production process from glycerol and create a high-value use for crude glycerol, the major byproduct of biodiesel production. Activities. Aim 1. Characterize the parameters of A. succinogenes' growth on glycerol and the metabolic pathways involved. After characterizing the respiratory metabolism of A. succinogenes grown in batch cultures in year one, we optimized A. succinogenes growth in a microaerobic chemostat system, we monitored product formation by the wild-type strain grown at microaerobic steady state, and we are started characterizing our pflB deletion mutant in the same conditions. These chemostat studies should complete Aim 1. Aim 2. Engineer A. succinogenes or the growth conditions for anaerobic growth on glycerol. Microarobic conditions are hard and costly to maintain in industrial settings. Two parallel strategies have been initiated to satisfy the redox requirements of A. succinogenes's anaerobic growth on glycerol. i) Because A. succinogenes expressing the Clostridium butyricum 1,3-propanediol (PDO) pathway genes from a strong promoter on a plasmid grows anaerobically on glycerol but mostly produces PDO, we constructed a cassette to insert the PDO genes into the A. succinogenes chromosome. ii) We are constructing a plasmid that will express the Escherichia coli hydrogen-evolving hydrogenase 3 in A. succinogenes. Work on aim 3 has not started. Aim 4. Optimization of our knockout methodology and engineering of A. succinogenes for increased succinate production from glycerol. After developing a gene deletion technology for A. succinogenes, we are now adapting the E. coli-based recombineering method to A. succinogenes that would allow us to rapidly generate single point mutations in the chromosome. We also started attempts to block the acetate production pathway by deleting the phosphate acetyltransferase and acetate kinase genes. Events. Posters on this project have been presented in June 2012 at the Metabolic Engineering IX conference in Biarritz, France, and at the 112th General Meeting of the American Society for Microbiology in San Francisco, CA. The PD gave an invited talk on this project in November 2012 at Grand Valley State University, Grand Rapids, MI. The PD gave lectures based on this project in BMB101 and MMG801 at MSU and in CMB150 at Grand Valley State University. PARTICIPANTS: Claire Vieille (PD) In charge of overall project direction. Bryan Schindler was a graduate student in the Vieille lab until last spring 2011, when he graduated with his PhD. Bryan characterized the growth and products of A. succinogenes under different respiratory conditions. He also constructed the A. succinogenes pflB deletion mutant described in this report. Nikolas McPherson. Graduate student. Started working in the Vieille lab in summer 2009. While focused on evolving A. succinogenes for fast growth on lignocelluloses hydrolysates. Nik's project also includes the development a recombineering method for Actinobacillus. Rajasi Joshi. Graduate student. Started working in the Vieille lab in summer 2011. Rajasi's project focuses on studying A. succinogenes's growth in a microaerobic chemostat, and engineering the strain for homosuccinate fermentation from glycerol Maeva Bottex is a junior Human Biology major. She worked in the Vieille lab from the end of summer 2010 to spring 2012. During that time Maeva prepared most growth media for A. succinogenes, and did much of the molecular biology work involved in this project (e.g., plasmid extractions, PCRs, clonings, and transformations) under guidance of Bryan Schindler first, then Nikolas McPherson. Xiaofei (Alex) Yang is a senior Microbiology major who started working in the Vieille lab in spring 2011. Rajasi Joshi is training him and supervising his work. Alex is constructing new deletion mutants of A. succinogenes as well as expression plasmids. Jacob Gibson is a freshman who started working in the Vieille lab in fall 2012. He is preparing growth media and helping Nik McPherson develop the recombineering method. Professional development. As part of the project, Nik McPherson attended the Metabolic Engineering IX conference in Biarritz, France, and Rajasi Joshi attended the 112th General Meeting of the American Society for Microbiology in San Francisco, CA. TARGET AUDIENCES: At this point of our project, the target audience is undergraduate and graduate students at Michigan State University. The research activities conducted as part of this project are regularly highlighted in the freshman Frontiers in Biochemistry (BMB 101) course, the senior undergraduate Biochemistry Seminar (BMB 495 course), and the graduate Integrative Microbial Biology (MMG 801) course. We have an agreement with MBI International, a subsidiary of the Michigan State University Foundation. MBI International focuses on developing and commercializing sustainable bio-based technologies, and already has several patents on succinate production by A. succinogenes. We expect that the commercial viability of the strains and processes developed in this project will be demonstrated in larger scale by MBI International, with further technology transfer to commercial partners. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Aim 1. A. succinogenes microaerobic steady-state growth on glycerol was established in 1-L chemostats. Optimum conditions for stable growth and succinate production were in 200-ml minimum medium, pH 6.8, at a 0.02 h-1 dilution rate and 2.5 ml/min gassing with a 95%CO2‒5%O2 gas mixture. Fermentation balances in microaerobic steady-state conditions confirmed the high succinate yields observed in batch cultures. Approximate products yields in mmol/100 mmol glycerol consumed were 78 for succinate, 10 for acetate, and 28 for biomass, 9.5 for CO2 with almost no formate and ethanol produced (two independent chemostats, 33 time points total). In our first chemostat with the pflB deletion mutant (20 time points), products yields in mmol/100 mmol glycerol consumed were 89 for succinate, 14 for acetate, 25 for biomass, and 14 for CO2. A first paper is in preparation on this aim. Aim 2. i) We constructed a cassette that will substitute the pflB gene with the PDO genes in the A. succinogenes chromosome. The construct has not been inserted in the chromosome yet. ii) Construction of the plasmid that will express the Escherichia coli hydrogen-evolving hydrogenase 3 under control of a strong promoter in A. succinogenes is in progress. Aim 4. A second paper is in preparation on the development of a knockout method for A. succinogenes. Attempts to delete the phosphate acetyltransferase and acetate kinase genes have been unsuccessful so far. Next deletion attempts will be performed in the presence of the two genes expressed on a plasmid. To develop a recombineering method for A. succinogenes, we first identified a point mutation in the rpo gene that confers rifampicin resistance. We then used 80-base oligonucleotides containing that mutation to transform A. succinogenes, and demonstrated that A. succinogenes is naturally unable to perform recombineering. Because transient expression of the recT gene is involved in recombineering, we selected three promoters of different strengths in the natural transformation regulon that are induced in carbon depleted medium in the presence of cAMP. We are currently cloning the Vibrio fisherii luciferase operon under control of these promoters to verify promoter inducibility and induction levels before expressing E. coli recT.

Publications

• No publications reported this period

Progress 01/15/11 to 01/14/12

Outputs
OUTPUTS: Because it produces large quantities of succinate and because it grows on glycerol, Actinobacillus succinogenes is a promising bacterial catalyst to develop a succinate production process from glycerol and create a high-value use for crude glycerol, the major byproduct of biodiesel production. Aim 1. We characterized the respiratory growth of A. succinogenes on glycerol with nitrate or dimethyl sulfoxide (DMSO) as the electron acceptor as well as in microaerobic conditions. How the electron acceptor concentration affects growth and succinate production was monitored. Growth and product profiles were monitored during growth in batch cultures with 20 mM starting concentrations of nitrate or DMSO, as well as in batch cultures grown in microaerobic chambers under 1% oxygen. To explain the variations in product balances observed in the different respiratory growth conditions, the in vitro activities of fumarate reductase and other terminal oxidoreductases were tested under the different respiratory conditions. We are now developing a chemostat system to optimize A. succinogenes microaerobic growth on glycerol, and monitor product formation in steady state microaerobic conditions. Aim 2. A. succinogenes' growth on glycerol is slow, requires a terminal electron acceptor, and cultures reach only low final cell densities. All bacterial species known to grow on glycerol by fermentation have been shown to produce 1,3-propanediol (PDO). To determine if we could develop a glycerol-to-succinate fermentation process, we expressed the Clostridium butyricum 1,3-propanediol pathway genes in A. succinogenes and tested the recombinant strain for anaerobic growth on glycerol. Aim 4. We developed a knockout methodology for A. succinogenes and engineered A. succinogenes for increased succinate production from glycerol. Development of a marker excision system. In our initial proposal, we had already constructed a frdAB deletion mutant of A. succinogenes using a delta-frdAB cassette that contained an icd selection marker (encoding Escherichia coli isocitrate dehydrogenase) flanked by two direct FRT sequences for excision by the yeast flippase, Flp. We have demonstrated this year that the Flp/FRT recombination system can work in A. succinogenes and that we can remove the icd marker used in the knockout step. The final markerless delta-frdAB mutant was identified by colony PCR and confirmed by isocitrate dehydrogenase in vitro assays, and the shuttle plasmid expressing the yeast Flp was cured with acridine orange. Engineering of A. succinogenes for increased succinate production from glycerol. An A. succinogenes pflB deletion mutant was built. This mutant strain was tested for succinate production on glycerol in different respiratory conditions. PARTICIPANTS: Claire Vieille (PD) In charge of overall project direction. Bryan Schindler was a graduate student in the Vieille lab until last spring 2011, when he graduated with his PhD. Bryan characterized the growth and products of A. succinogenes under different respiratory conditions. He also constructed the A. succinogenes pflB deletion mutant described in this report. Nikolas McPherson. Graduate student. Started working in the Vieille lab in summer 2009. Initially focused on evolving A. succinogenes for fast growth on lignocelluloses hydrolysates. Nik's project now has evolved to include the development of more genetic tools for Actinobacillus. Rajasi Joshi. Graduate student. Started working in the Vieille lab in summer 2011. Rajasi's project focuses on studying A. succinogenes's growth in a microaerobic chemostat, and engineering the strain for homosuccinate fermentation from glycerol Maeva Bottex is a sophomore Human Biology major. She started working in the Vieille lab at the end of summer 2010. From the start Maeva has been preparing most growth media for A. succinogenes, and doing much of the molecular biology work involved in this project (e.g., plasmid extractions, PCRs, clonings, and transformations) under guidance of Bryan Schindler first, then Nikolas McPherson. Xiaofei (Alex) Yang is a junior Microbiology major who started working in the Vieille lab in spring 2011. Graduate student Rajasi Joshi is training him and supervising his work. Alex is constructing new deletion mutants of A. succinogenes. TARGET AUDIENCES: At this point of our project, the target audience is undergraduate and graduate students at Michigan State University. The research activities conducted as part of this project are regularly highlighted in the freshman Frontiers in Biochemistry (BMB 101) course, the Basic Biotechnology (MMG 445) course, the senior undergraduate Biochemistry Seminar (BMB 495 course), and the graduate Integrative Microbial Biology (MMG 801) course. We have a technology agreement with MBI International, a subsidiary of the Michigan State University Foundation. MBI International focuses on developing and commercializing sustainable bio-based technologies, and already has several patents on succinate production by A. succinogenes that are licensed to a large industrial company. We expect that the commercial viability of the strains and processes developed in this project will be demonstrated in larger scale by MBI International, with further technology transfer to the commercial partner. PROJECT MODIFICATIONS: In Aim 1, we demonstrated that, of the three respiratory conditions tested, microaerobic conditions are the most favorable for succinate production. For this reason, the remainder of our work in Aim 1 will focus on better characterizing A. succinogenes' metabolism in these conditions. The microaerobic conditions that are the most favorable for succinate production will be identified in chemostat cultures using response surface methodology to determine the optimum levels of the process parameters that likely affect succinate yield and productivity. Parameters chosen for optimization will include culture density, dissolved oxygen level, bicarbonate concentration, and glycerol concentration. The response will be measured in succinate yield and succinate production rate. Microaerobic cultures grown on process glycerol produce 30% more acetate and CO2 than cultures grown on pure glycerol. The composition of the process glycerol will be determined by mass spectrometry. Contaminants in the process glycerol will be tested individually for their effects on succinate yields of microaerobic A. succinogenes grown on pure glycerol. Aim 2. Evolution of A. succinogenes for fast respiratory growth on glycerol will be performed in the respiratory conditions that are the most favorable for succinate production: DMSO-respiring and microaerobic A. succinogenes cultures, rather than nitrate-respiring cultures. Aims 3 and 4 are unchanged.

Impacts
Aim 1. Growth by nitrate respiration. Acetate and CO2 were the predominant products of A. succinogenes grown on glycerol-nitrate. The ratio of succinate to acetate (S/A ratio) produced was constant with different initial NaNO3 concentrations. Growth on nitrate was biphasic. A. succinogenes first respired nitrate to nitrite, then respired nitrite. Succinate was produced only in the nitrite reduction phase. Nitrate reduction to ammonia has two redox steps. No reducing equivalents are available for succinate production in this phase. In contrast, nitrite reduction to ammonia involves a single redox step, leaving reducing equivalents available for succinate production. Growth by DMSO respiration. Succinate was the most abundant product, followed by acetate, formate, and CO2 in A. succinogenes cultures grown on glycerol-DMSO. The S/A ratio varied during growth, with the end-point value decreasing with increasing DMSO concentrations. The glycerol metabolism of DMSO-respiring cultures was comparable with pure glycerol and glycerols partially purified after transesterification of vegetable and animal fats. Succinate, formate, and acetate yields were very similar on pure and process glycerols. Because fumarate reductase is highly expressed in DMSO-grown cultures, variation of the S/A ratio as a function of DMSO availability is likely due to the combined effect of enzyme kinetics and the relative redox potentials of DMSO/DMS and fumarate/succinate. Microaerobic growth. Succinate was the predominant product of A. succinogenes grown on glycerol under 1% O2. The S/A ratio increased from 0 to 2.8 during growth. The succinate yield was similar to that of DMSO-respiring cells, but DMSO-respiring cultures produced formate, whereas microaerobic cultures produced CO2. In DMSO-grown cultures, nearly all pyruvate-dissimilating flux likely goes through pyruvate formate lyase (PFL). Under 1% O2, the flux to acetyl-CoA is likely shared between PFL and pyruvate dehydrogenase. Aim 2. Fermentative growth. A. succinogenes expressing the C. butyricum dha operon from plasmid pCV932 fermented glycerol, producing mostly PDO and acetate. This result suggests that the strong promoter in pCV932 forces the majority of glycerol to be reduced to PDO, creating an intracellular redox state unfavorable for succinate production. Aim 4. Construction of knockout mutants. After proving that we could make marker-less knockout mutants of A. succinogenes, we built a delta-pflB strain. Strain delta-pflB grown on glucose produced no formate, confirming that delta-pflB is devoid of PFL activity. Strain delta-pflB grown on glycerol with 10 mM DMSO or under 1% O2 had higher succinate yields and higher S/A ratios than the wild-type strain. Under 1% O2 and with 150 mM glycerol, succinate production by delta-pflB peaked between 26 h and 40 h. During this time, the succinate yield was 85% of the maximum theoretical yield, and only 4.7% of the glycerol consumed was used to produce biomass. These results suggest that cell density and O2 tension could be optimized in a continuous culture to maximize succinate production.

Publications

• No publications reported this period