Progress 09/01/05 to 08/31/08

Outputs
OUTPUTS: Activities: Mannitol is produced enzymatically from fructose by mannitol dehydrogenase (MtDH) with NAD(P)H as the cofactor. In theory, glucose can be stoichiometrically converted into mannitol in a single electrochemical reactor with immobilized thermostable MtDH and xylose (glucose) isomerase (XI). While thermostable XIs are commercially available, all known MtDHs are mesophilic. We searched the genomes of hyperthermophiles for MtDH sequences. We identified a single putative MtDH in the Thermotoga maritima genome (Genbank # TM0298). TM0298 shares 55% similarity with two known mesophilic MtDHs. We cloned the gene encoding TM0298, and demonstrated that TM0298 is indeed a hyperthermostable MtDH. We characterized the stability of T. maritima MtDH (TmMtDH), identified the conditions for optimum activity, and studied its substrate specificity. We also studied parameters that can affect the mannitol yield in a reactor (i.e., effect of salts, pH, and mannitol as potential TmMtDH and XI inhibitors). Finally, we tested that TmMtDH can be used together with Thermotoga neapolitana XI (TNXI) to produce mannitol directly from glucose. We tested this concept both in the absence and presence of an NADH-regenerating system. Sorbitol is produced from fructose by sorbitol dehydrogenase (SDH), but it can also be produced directly from glucose by aldose reductase (AR). Because no gene was identified in the genomes of hyperthermophiles that potentially encoded an SDH or an AR, we cloned and expressed in E. coli the Candida boidinii AR (CbAR). We optimized CbAR expression in E. coli, we characterized CbAR's thermostability, and we determined its kinetic parameters on glucose and xylose (its natural substrate). Based on the models of xylose and glucose in CbAR's substrate binding site, we have introduced single and double mutations in CbAR to increase CbAR's specificity for glucose. Mentored a minority undergraduate student from the 2008 Michigan State University Summer Undergraduate Research Academy (SURA). In her ten-week program, the student, Allisha Ali, introduced a point mutation in CbAR, verified the sequence of the mutated gene, purified the mutant enzyme, and determined its kinetic parameters on glucose. Events: Poster: Molecular design of oxidoreductases for the biosynthesis of carbohydrate-based industrial polyols. SH Song, B Hassler, RM Worden, JG Zeikus, and C Vieille. USDA NRI annual PD meeting "Genes to Products - Agricultural Plant, Microbe, & Biobased Product Research," 11-14 March, 2007, Bethesda, MD. Poster: Molecular design of oxidoreductases for the biosynthesis of carbohydrate-based industrial polyols. SH Song, B Hassler, RM Worden, JG Zeikus, and C Vieille. USDA NRI annual PD meeting "Genes to Products - Agricultural Plant, Microbe, & Biobased Product Research," 16-18 April, 2008, Bethesda, MD. Products: Vieille, C., Song, S.H., and Zeikus, J.G. 2007. U.S. Patent application no. 60/837,039. Thermotoga maritima mannitol dehydrogenase. Kinetic parameters of TmMtDH and TNXI at 60C were used to build a mathematical model of reaction kinetics at the surface of a bioelectrochemical reactor electrode (manuscript in preparation). PARTICIPANTS: J. Gregory Zeikus (PD) In charge of overall project direction Claire Vieille (co-PD) Dr. Vieille assists Dr. Zeikus in directing the overall project. She also supervises the specific technical aspects of the project, she trains and supervises the students/postdocs currently working on the project, writes the manuscripts resulting from the lab research, and maintains a close collaboration with Dr. Mark Worden (MSU department of Chemical Engineering) to develop enzyme-based bioelectrochemical reactors. When time permits, Dr. Vieille participates in the bench work involved in this project. She initially cloned and started characterizing the Thermotoga maritima mannitol dehydrogenase (TmMtDH) prior to the arrival of Seung Hoon Song (Postdoc) in the lab. Seung Hoon Song (Postdoc) Dr. Song has been working on this project full time for the last twenty months. He finished characterizing TmMtDH (manuscript submitted), and he characterized the activity and stability properties of Candida Boidinii aldose reductase (CbAR). He is now using directed evolution to increase CbAR's thermostability and site-directed mutagenesis to increase CbAR's activity on glucose. During his first year on the project, Dr. Song received a Korean fellowship that was complemented by this USDA CSREES grant. Dr. Song's salary is now completely supported by this grant. Nitasha Ahluwalia (Undergraduate student) Nitasha worked on this project for over two years with hourly support from this grant. Initially trained by Dr. Vieille, she helped her purify TmMtDH, and perform TmMtDH stability and kinetic assays. She later on worked under Dr. Song's supervision. Nitasha graduated from MSU in May, 2008. Allisha Ali (Undergraduate student) Allisha Ali worked on this project for ten weeks during the summer of 2008. She was trained and mentored by Dr. Song. During her ten weeks in the lab, she completed the construction of one point mutation in CbAR and the characterization of the effect this mutation has on CbAR activity on glucose. A sophomore minority student, Allisha had no prior experience with laboratory work, nor did she have any knowledge of molecular biology and enzymology. At the end of her internship, Allisha understood and could clearly present the goals, the methods, and the results of her work to an audience of her peers and professors. TARGET AUDIENCES: This project is used as a research example in a Multidisciplinary Bioprocessing Laboratory (MBL) course for senior undergraduates and beginning graduate students. One interdisciplinary team of students works in the Zeikus/Vieille lab for the first half of the semester, then in the Worden lab, to first learn to work with enzymes then use them in enzyme-based bioelectrochemical reactors. The MBL course is integrated with existing courses in departments relating to bioprocessing, culminating in the development of a new Multidisciplinary Graduate Training Program in Technologies for a Biobased Economy. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: TmMtDH is expressed in E. coli at high level, it is soluble, and it is active with NADH and NADPH. TmMtDH is most active on fructose as its substrate, confirming that it is the first known thermostable MtDH. TmMtDH is most active at 90C-100C. It optimally reduces fructose at pH 6.0, and optimally oxidizes mannitol at pH 8.3. TmMtDH has 3-fold higher affinity for NADH than for NADPH. At 80C, it has a Vmax of 59 umol/mg.min and a Km of 51 mM for fructose at pH 6.0, and a Vmax of 38 umol/mg.min and a Km of 5.5 mM for mannitol at pH 8.3. In an electroenzymatic reactor using TmMtDH and TNXI immobilized on a gold electrode, 300 mM glucose produced 130 mM mannitol without traces of fructose. The initial pH of 6.0 had shifted to 9.0 at the end of the experiment, explaining why the conversion stopped early. We have since shown that up to 500 mM mannitol does not inhibit TNXI activity, but that mannitol behaves as a competitive inhibitor of TmMtDH with a Ki of 99 mM. We have also shown that up to 500 mM NaCl does not inhibit TNXI and TmMtDH activities. We expressed CbAR at high levels and in soluble form in 25C E. coli cultures. We established an easy purification method for CbAR. CbAR reduces glucose optimally at pH 6.5-7.0. At pH 6.7, it is most active at 50C-55C. CbAR is much more active in xylose (Km=0.054 mM and Vmax=39 umol/mg.min) than in glucose reduction (Km=0.45 M and Vmax=7.9 umol/mg.min). It is inactive with NADH; but its Km for NADPH is 0.04 mM. A mesophilic enzyme, CbAR is not highly stable. Its half-life is only 144 min at 37C and below 1 min at 50C. Among the seven mutations introduced in CbAR to increase its affinity for glucose, N129K increased Vmax to 10.35 umol/mg.min and decreased Km to 433 mM, yielding a 25% catalytic efficiency increase. In contrast, mutation L226M decreased Km to 363 mM, but it decreased Vmax as well, yielding a 30% catalytic efficiency decrease on glucose. Several other CbAR mutants were hardly expressed in E. coli. Their activity on glucose remains to be tested. Change in conditions: Chemical industries seek to replace current chemical synthetic approaches for the productions of mannitol and sorbitol with biological syntheses to lower production costs. We cloned and characterized the first thermostable MtDH that, used in combination with a thermostable XI, should allow the biological, stoichiometric production of mannitol from glucose. Our research benefits the engineering field of electroenzymology: What we learn from how our reactors convert glucose to mannitol is knowledge that can be applied to other future electrobiocatalytic processes. This project has been the opportunity for the PD to train one postdoc and one undergraduate student in molecular biology and protein biochemistry techniques. The postdoc gained mentoring experience by supervising a second, minority undergraduate student. The postdoc is also gaining additional professional training by participating in an interdisciplinary collaboration with chemical engineers working on electrobiocatalytic processes.

Publications

• Song, S.H., N. Ahluwalia, Leduc, Y., Delbaere, L., and C. Vieille. 2008. Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase. Appl. Microbiol. Biotechnol. DOI 10.1007/s00253-008-1633-9.

Progress 09/01/06 to 08/31/07

Outputs
OUTPUTS: Activities: Mannitol is produced enzymatically from fructose by mannitol dehydrogenase (MtDH) using NAD(P)H as the cofactor. It is theoretically possible to convert glucose stoichiometrically into mannitol in a single electrochemical reactor system containing both immobilized thermostable MtDH and glucose isomerase. While thermostable glucose isomerases are commercially available, all known MtDHs are mesophilic enzymes. For this reason, we searched for MtDH sequences in the genomes of hyperthermophilic organisms. We identified a single putative MtDH in the Thermotoga maritima genome (Genbank # TM0298). TM0298 shares about 32% identity and 55% similarity with two known mesophilic MtDHs. We cloned the T. maritima gene encoding TM0298. We demonstrated that TM0298 is indeed a hyperthermostable MtDH. We characterized the stability of T. maritima MtDH (TmMtDH), identified the conditions for optimum activity, studied its substrate specificity, and tested that TmMtDH can be used together with Thermotoga neapolitana glucose isomerase in an NADH-recycling electroenzymatic bioreactor to produce mannitol directly from glucose. We studied parameters that might affect the mannitol yield in the reactor (i.e., effect of salts and of mannitol as a potential inhibitors of TmMtDH and glucose isomerase activities). Sorbitol can be produced from fructose by sorbitol dehydrogenase (SDH), but it can also be produced directly from glucose by aldose reductase (AR). Because no gene was identified in the genomes of hyperthermophiles that potentially encoded an SDH or an AR, we cloned and expressed in Escherichia coli the Candida boidinii AR (CbAR). We optimized CbAR expression in E. coli, we characterized CbAR's thermostability, and we determined its kinetic parameters on glucose and on xylose (its natural substrate). Products: Vieille, C., Song, S.H., and Zeikus, J.G. 2007. U.S. Patent application no. 60/837,039. Thermotoga maritima mannitol dehydrogenase. PARTICIPANTS: Dr. J. Gregory Zeikus (PD) and Dr. Claire Vieille (co-PD) are in charge of overall project direction. Dr. Vieille also supervises the specific technical aspects of the project, she trains and supervises the students/postdocs currently working on the project, writes the manuscripts resulting from the lab research, and maintains a close collaboration with Dr. Mark Worden (MSU department of Chemical Engineering) to develop enzyme-based bioelectrochemical reactors. When time permits, Dr. Vieille participates in the bench work involved in this project. She initially cloned and started characterizing the Thermotoga maritima mannitol dehydrogenase (TmMtDH) prior to the arrival of Seung Hoon Song (Postdoc) in the lab. Dr. Seung Hoon Song (Postdoc) has been working on this project full time for the last twenty months. He finished characterizing TmMtDH (manuscript submitted), and he characterized the activity and stability properties of Candida Boidinii aldose reductase (CbAR). He is now using directed evolution to increase CbAR's thermostability. During his first year on the project, Dr. Song received a Korean fellowship that was complemented by this USDA CSREES grant. Dr. Song's salary is now completely supported by this grant. Nitasha Ahluwalia (undergraduate student) has been working on this project for over two years with hourly support from this grant. Initially trained by Dr. Vieille, she helped her purify TmMtDH, and perform TmMtDH stability and kinetic assays. She now is working under Dr. Song's supervision. TARGET AUDIENCES: This project is used as a research example in a Multidisciplinary Bioprocessing Laboratory (MBL) course for senior undergraduates and beginning graduate students. One interdisciplinary team of students works in the Zeikus/Vieille lab for the first half of the semester, then in the Worden lab, to first learn to work with enzymes then use them in enzyme-based bioelectrochemical reactors. The MBL course is integrated with existing courses in departments relating to bioprocessing, culminating in the development of a new Multidisciplinary Graduate Training Program in Technologies for a Biobased Economy.

Impacts
Change in knowledge: TmMtDH is expressed with a C-terminal His-tag (in plasmid pET24a) in E. coli at high level, it is soluble, and it is active with NADH and NADPH. TmMtDH is most active on fructose as its substrate, confirming that it is the first known thermostable MtDH. It also shows 28%, 17%, 5%, and 1% activity on D-tagatose, D-xylulose, L-sorbose, and D-arabinose, respectively. TmMtDH is most active between 90C and 100C. It optimally reduces fructose at pH 6.0, and optimally oxidizes mannitol at pH 8.3. TmMtDH has 3-fold higher affinity for NADH than for NADPH. The enzyme has a Vmax of 59 umol/mg.min and a Km of 51 mM for fructose at 80C and pH 6.0, and a Vmax of 38 umol/mg.min and a Km of 5.5 mM for mannitol at 80C and pH 8.3. In an electroenzymatic reactor using TmMtDH and T. neapolitana xylose isomerase (TNXI) immobilized on a gold electrode, 300 mM glucose produced 130 mM mannitol without traces of fructose. The initial pH of 6.0 had shifted to 9.0 at the end of the experiment, probably explaining why the conversion stopped early. We have since shown that up to 500 mM mannitol does not inhibit TNXI activity. We have also shown that up to 500 mM NaCl does not inhibit TNXI and TmMtDH activities. We succeeded in expressing CbAR at high levels and in soluble form in 25C E. coli cultures. We established an easy purification method for CbAR. CbAR reduces glucose optimally at pH 6.5-7.0. At pH 6.7, it shows maximum activity between 50C and 55C. As expected from its function as xylose reductase in Candida boidinii, CbAR is much more active in xylose (Km=0.054 mM and Vmax=39 umol/mg.min) than in glucose reduction (Km=0.45 M and Vmax=7.9 umol/mg.min). CbAR showed no activity with NADH; in contrast, its Km for NADPH is 0.04 mM. Originating from a mesophilic organism, CbAR is not high stable. Its half-life is only 144 min at 37C, 80 min at 40C, and below 1 min at 50C. Directed evolution experiments are in progress to increase CbAR thermostability and to increase its activity on glucose. Change in conditions: Chemical industries are looking to replace traditional chemical synthetic approaches for the productions of mannitol and sorbitol with biological syntheses to lower production costs and allow polyols to be called natural products. We have cloned and characterized the first thermostable MtDH that, used in combination with a thermostable xylose isomerase, should allow the biological, stoichiometric production of mannitol from glucose. Our research benefits the engineering field of electroenzymatic reactors: What we learn from how our electroenzymatic reactors behave in glucose conversion to mannitol will be knowledge that can be applied to other future electrobiocatalytic processes. This project has been the opportunity for the PD to train one postdoctoral fellow and one undergraduate student in a vast array of molecular biology and protein biochemistry techniques. The postdoctoral fellow is also gaining additional, interdisciplinary professional training from being involved in an interdisciplinary collaboration with chemical engineers specialized in electrobiocatalytic processes.

Publications

• Puttick, P., C. Vieille, S.H. Song, M.N. Fodje, P. Grochulski and L.T.J. Delbaere. 2007. Crystallization, preliminary X-ray diffraction and structure analysis of Thermotoga maritima mannitol dehydrogenase. Acta Crystallog. F 63:350-352.

Progress 09/01/05 to 09/01/06

Outputs
Mannitol is produced enzymatically from fructose by mannitol dehydrogenase (MtDH) using NAD(P)H as the cofactor. It is theoretically possible to convert glucose stoichiometrically into mannitol in a single electrochemical reactor system containing both immobilized thermostable MtDH and glucose isomerase. While thermostable glucose isomerases are commercially available, all known MtDHs are mesophilic enzymes. For this reason, we searched for MtDH sequences in the genomes of hyperthermophilic organisms. We identified a single putative MtDH in the Thermotoga maritima genome (Genbank # TM0298). TM0298 shares about 32% identity and 55% similarity with two known mesophilic MtDHs. We cloned the T. maritima gene encoding TM0298. TM0298 is expressed with a C-terminal His-tag (in plasmid pET24a) in Escherichia coli at high level, it is soluble, and it has MtDH activity with NADH and NADPH. TM0298 is most active on fructose as its substrate, confirming that TM0298 is the first known thermostable MtDH, but it also shows 28%, 17%, 5%, and 1% activity on D-tagatose, D-xylulose, L-sorbose, and D-arabinose, respectively. T. maritima MtDH is maximally active at 90C or above. It optimally reduces fructose at pH 6.0, and it optimally oxidizes mannitol at pH 8.3. T. maritima MtDH has higher affinity for NADH (Km of 0.06 mM) than for NADPH (Km of 0.21 mM). The enzyme has a Vmax of 12 umol/mg.min and a Km of 39.5 mM for fructose at 80C and pH 6.0, and a Vmax of 3.1 umol/mg.min and a Km of 1.4 mM for mannitol at 80C and pH 8.3. Collaborative experiments already showed that T. maritima MtDH can be combined with T. neapolitana glucose isomerase to produce mannitol directly from glucose. Further experiments include directed evolution to increase T. maritima's activity on fructose at 60C. We used T. maritima MtDH to develop a plate screening assay based on the oxidation of NADH by phenazine methosulfate, which in turn reduces nitroblue tetrazolium into an insoluble blue formazan dye. This assay can be used for any thermostable NAD(P)-dependent oxidoreductase. Sorbitol can be produced from fructose by sorbitol dehydrogenase (SDH), but it can also be produced directly from glucose by aldose reductase (AR). Because no gene was identified in the genomes of hyperthermophiles that potentially encoded an SDH or an AR, we cloned and expressed in E. coli the Candida boidinii AR. We succeeded in expressing this fungal AR at high levels and in soluble form in E. coli cultures grown at 30C. We purified the enzyme by two steps of ion exchange chromatography and are in the process of determining its kinetic parameters on glucose and on xylose (its natural substrate). We will later mutagenize this NADP-dependent AR to increase its thermostability and its activity on glucose using screening methods derived from that mentioned above. INVENTIONS Zeikus, J.G., Vieille, C., and S.H. Song. 2006. MSU Invention disclosure no. 07-020F. Thermotoga maritima mannitol dehydrogenase.

Impacts
Chemical industries are looking to replace traditional chemical synthetic approaches for the productions of mannitol and sorbitol with biological syntheses to lower production costs and to allow polyols to be called natural products. We have cloned and characterized the first thermostable mannitol dehdyrogenase that, used in combination with a thermostable glucose isomerase, would allow the stoichiometric production of mannitol from glucose.

Publications

• No publications reported this period