Mapping Uncertainty

Friday May 5, 2023
(One day symposium)

McNamara Alumni Center
University of Minnesota
Minneapolis, MN USA.

Map & Directions

Presenters

Romas Kazlauskas, Ph.D.

Professor, Biochemistry, Molecular Biology & Biophysics
Biotechnology Institute
University of Minnesota
rjk@umn.edu

Romas Kazlauskas studied chemistry at the Massachusetts Institute of Technology (Ph.D.) and Harvard University (postdoc with George Whitesides). He worked at General Electric Company (1985-88) and McGill University, Montreal, Canada (1988-2003) and is currently a professor in Biochemistry, Molecular Biology and Biophysics at the University of Minnesota. He has been a visiting professor in Germany, Sweden and South Korea. He is an expert in protein engineering of enzymes for biocatalysis and the author of a forthcoming textbook on protein engineering (www.betterenzyme.com).

 Research Interests

• Engineering new catalytic activity in enzymes
• Rational design of enzyme properties
• Enzyme applications to support sustainability

Shotaro Yamaguchi, PhD.

CTO, Managing Director of Innovation
Amano Enzyme Inc.
shotaro_yamaguchi@amano-enzyme.com

Shotaro Yamaguchi joined Amano in 1984 after receiving a master’s degree in food engineering from the Graduate School of Agriculture, Kyoto University. Since then, he has been engaged in industrial enzymology, fungal genetic engineering, microbial fermentation, and food and medical enzyme applications. He received Ph.D. degree from Kyoto University on lipase in 1991 and spent three years at the Institute of Food Research (UK) from 1999 to 2001. He discovered a novel protein-modifying enzyme, protein glutaminase.

He received the following awards: Encouragement Award from Brewing Society of Japan (2003) and Technology Award from Japan Society for Bioscience, Biotechnology, and Agrochemistry (2010). He is an active Editor for Applied Microbiology and Biotechnology, Headquarters Officer/Auditor for Japan Society for Bioscience, Biotechnology, and Agrochemistry, and Representative for The Society for Biotechnology, Japan.

Todd Hyster, Ph.D.

Prof. Todd Hyster is an Associate Professor of Chemistry and Chemical Biology at Cornell University. He received his B.S. in Chemistry from the University of Minnesota. He did his Ph.D. studies with Tomislav Rovis at Colorado State University. As part of his Ph.D., he was a Marie Curie Fellow with Thomas Ward at the University of Basel. He was an NIH Postdoctoral Fellow with Prof. Frances Arnold at Caltech. He started his independent career at Princeton University in 2015. His group has developed new methods in photoenzymatic catalysis.

Research Interests

• Photoenzymatic Catalysis
• Enzyme engineering via directed evolution
• Selective organic synthesis

Photoenzymatic Catalysis – Using Light to Reveal New Enzyme Functions
Todd K. Hyster

Enzymes are exquisite catalysts for chemical synthesis, capable of providing unparalleled levels of chemo-, regio-, diastereo- and enantioselectivity. Unfortunately, biocatalysts are often limited to the reactivity patterns found in nature. In this talk, I will share my groups efforts to use light to expand the reactivity profile of enzymes. In our studies, we have exploited the photoexcited state of common biological cofactors, such as NADH and FMN to facilitate electron transfer to substrates bound within enzyme active sites. In other studies, we found that enzymes will electronically activate bound substrates for electron transfer. In the presence of common photoredox catalysts, this activation can be used to direct radical formation to enzyme active sites. Using these approaches, we can develop biocatalysts to solve long-standing selectivity challenges in chemical synthesis.

Stefan Lutz, Ph.D.

Sr VP Research
Codexis Inc.
stefan.lutz@codexis.com

Stefan received a B.Sc. in chemistry/chemical engineering from the Zurich University of Applied Sciences, an M.Sc. in Biotechnology from the University of Teesside and a Ph.D. in chemistry from the University of Florida. He was a postdoctoral fellow at Pennsylvania State University. He joined Codexis in 2020 as the Senior Vice President of Research to lead the company’s research team advancing the technology platform, as well as the discovery and engineering of novel enzymes. Prior to his arrival in Redwood City, he was a Professor and Chair of the Chemistry Department at Emory University, having joined the university in 2002 and ascending to Chemistry Department Chair in 2014. Stefan is interested in advanced technologies for creating new, innovative, and economically-sustainable enzyme solutions to benefit society, industry, and the planet.

Research Interests

• Advanced technologies for enzyme design and engineering
• Engineered enzyme applications
• Biocatalysis

Engineering Enzyme Products
Stefan Lutz

Codexis’ CodeEvolver^® directed evolution technology has been applied to improve enzymes for specific functions for well over a decade. Advances in high-throughput gene & protein synthesis (build) and biochemical screening (test) in combination with advanced data analytics (learn) and computational design tools have, and continue to, enable the optimization and drive increasing complexity in developing novel biocatalysts for sustainable manufacturing, the life sciences, and the discovery and optimization of biologics.

Tomoko Matsuda, Ph.D.

Associate Professor
Department of Life Science and Technology
Tokyo Institute of Technology
tmatsuda@bio.titech.ac.jp

Tomoko Matsuda received a doctoral degree in science from Kyoto University (2000). Her doctoral thesis is about the biocatalytic asymmetric reduction of ketone for organic synthesis. She has been engaged in research on biocatalysis since then. She was appointed as an assistant professor at Ryukoku University in Japan (1999-2004) and began the study for biocatalysis using pressurized carbon dioxide. She was appointed as an associate professor in 2004 at the Tokyo Institute of Technology in Japan. She published over 130 scientific articles and received the following awards; the Taisho Pharmaceutical Research Planning Award from the Society of Synthetic Organic Chemistry, Japan (2001), the Morita Scientific Research Encouragement Award from the Japanese Association of University Women (2006), the Shiseido Woman Researcher Science Grant from Shiseido (2011), the Takeda International Contribution Award from Takeda Rika Kogyo (2018).

Research Interests

• Utilization of pressurized CO₂ for biocatalysis
• Green chemistry using biocatalysis

Utilization of Carbon Dioxide as Solvent and Substrate for Biocatalysis
Tomoko Matsuda

As carbon dioxide (CO₂) is an abundant carbon source and is causing global warming, developments in its utilization methods have been awaited. Therefore, pressurized CO₂such as supercritical CO₂ has been applied as a solvent for organic synthesis to develop efficient reactions replacing ordinary organic solvents derived from fossil fuel. However, the application of pressurized CO₂ to biocatalysis has been limited. Therefore, we have been studying on utilization of CO₂ as a solvent for lipase-catalyzed transesterification reactions and as a substrate for biocatalytic carboxylation reactions.

Supercritical and liquid CO₂ has been used for lipase-catalyzed transesterifications to replace conventional organic solvents. In this study, CO₂-expanded liquids, liquids expanded by dissolving pressurized CO₂, were utilized since they can be achieved at a lower pressure than supercritical and liquid CO₂. Then, we found that for lipase-catalyzed transesterifications of bulky substrates, such as 1-(1-adamantyl)ethanol, o-substituted 1-phenylethanol analogs, and substituted 1-tetralol analogs, the conversions were higher for the reaction in CO₂-expanded liquids than those in the corresponding liquids without CO₂ (Figure 1).

Figure 1 Solvent engineering using CO₂ for lipase-catalyzed transesterifications

On the other hand, a two-layer solvent system consisting of an aqueous buffer and the carbon dioxide layer was utilized for the carboxylation reactions since carboxylation enzymes are not stable in pressurized CO₂ without bulk water. Catalyzed by enzymes from a thermophilic microorganism, Thermoplasma acidophilum isocitrate dehydrogenase (TaIDH) and T. acidophilum glucose dehydrogenase (TaGDH), the reductive carboxylation reactions have been successfully conducted using CO₂ as a substrate (Figure 2). These enzymes were also co-immobilized to achieve higher stabilities and activities by forming an enzyme-inorganic hybrid nanocrystal.

Figure 2 Utilization of CO₂v as a substrate of biocatalytic carboxylation

Anne Meyer, Ph.D.

Anne S. Meyer is Professor of Enzyme Technology, Head of the Protein Chemistry & Enzyme Technology Section at Dept. of Biotechnology and Biomedicine, Technical University of Denmark. The Section comprises 8 professor research groups, in total ~75 persons, incl. ~20 PhD students. She is group leader of Enzyme Technology in the Section.

 
Research interests:

• Applied enzyme technology, incl. enzyme enzymatic biorefining of biomass, agro-industrial side streams, starch, pectin, and seaweeds for production of bioactives and functional food compounds.
• Enzymatic synthesis of human milk oligosaccharides.
• Enzymatic degradation of plastic, and enzymatic conversion of CO₂.
• Bioinformatics, enzyme characterization, assays, kinetics, and carbohydrate chemistry

New food processes and ingredients via targeted enzyme catalysis
Anne S. Meyer, Technical University of Denmark, Denmark

One of the major challenges confronting the modern food supply chain is providing safe, nutritious, and preferably functionally healthy food to an expanding global population while utilizing resources sensibly and protecting the environment and the climate. Many agro-industrial co-processing streams are rich in complex plant fibers that should not go to waste, as they may be a valuable source of beneficial, ‘prebiotic’ dietary fibers or a feedstock for functional ingredients production. Corn bran, a residue from large scale corn starch processing, is for example rich in highly substituted feruloylated glucurono-arabinoxylan, and even includes diferuloyl cross links, and is considered recalcitrant to enzymatic modification. We recently discovered a bacterial endo-xylanase (GH30 from Dickeya chrysantemi) that attacks complex corn arabinoxylan to enable gentle solubilization of substituted glucurono-arabinoxylan oligomers¹. The recent news is that the GH30-solubilized corn arabinoxylan molecules modulate the human gut microbiota during simulated colon fermentation in vitro, paving the way for using corn bran streams as a resource to generate new soluble prebiotics². Human milk oligosaccharides (HMOs) are unique, beneficial oligosaccharides in human breast milk. Enzymatic synthesis of HMOs is attractive to create new additives for infant formula and other products. Several glycoside hydrolases can catalyze transglycosylation (incl. transfucosylation) for precise enzymatic synthesis of nature-identical HMO products³. To attain high yields, we are using different types of protein engineering approaches to modify the enzyme to catalyze relevant transglycosylations at high yield⁴. Citrus-pectin residues have turned out to hold fucosylated xyloglucan that can serve as a source of fucose for enzymatic production of fucosylated HMOs via targeted enzymatic transfucosylation⁵. Seaweeds, i.e. marine macroalgae, have for decades been a source of food hydrocolloids. As the demand for hydrocolloids keep increasing, kelp seaweeds are now cultivated in the Northern hemisphere and new enzymes are being discovered for enzymatic refining options for kelp biorefining beyond extraction of hydrocolloids. One line of our research relates to enzymatic modification of alginate from kelp⁶, another concerns enzymatic extraction and modification of fucoidan for medical uses^7,8,9. Lastly, we have recently introduced  new microbial 4-alpha-glucanotransferases to modify starch functionality¹⁰.

1. Munk et al., 2020. ACS Sust Chem Eng 8 (22), 8164-8174.
2. Lin et al. 2023. J Agric Food Chem 71, 385-3897
3. Zeuner and Meyer 2020. Carb Res 493, 108029.
4. Zeuner et al. 2020 J of Fungi 6(4), 295-313
5. Nielsen et al. 2022. Carb Res. 519, 198627
6. Pilgaard et al. 2021. J of Fungi 7, 80-95.
7. Nguyen et al. 2020. Marine Drugs 18(6), 296-313
8. Trang et al. 2022. Frontiers Plant Sci 13, 823668
9. Ohmes et al. 2020 Marine Drugs 18, 481-418
10. Christensen et al. 2023. Intl J Biol Macromol 224, 105-114.

Jun Ogawa, Ph.D.

Professor
Division of Applied Life Sciences,
Graduate School of Agriculture,
Kyoto University
ogawa.jun.8a@kyoto-u.ac.jp

Jun Ogawa studied applied microbiology and completed his doctorate in 1995 at Kyoto University and became an assistant professor at the same university. He was a visiting researcher at French National Institute for Agricultural Research (INRA) (2006-2007) and has appointed as a full professor of the current position in 2009. He has published over 270 papers in applied microbiology such as bioprocess development, microbial metabolism analysis, etc. He was awarded “Agrochemistry Award for the Encouragement of Young Scientists” by Japan Society for Bioscience, Biotechnology, and Agrochemistry (2006), “Oleoscience Award” by the Japan Oil Chemists’ Society (2015 and 2020), “Society Award of Japanese Association for Food Immunology” (2018), “Ching Hou Biotechnology Award” (2020) and “Fellow” (2021) by American Oil Chemists’ Society, and “Chevreul Medal” by the French association for the study of lipids (2021).

Research Interests

• Microbial physiology
• Fermentation technology
• Enzyme technology
• Metabolic engineering
• Microbial consortia studies

 
From function to genes, enzymes, and communities; creating novel biotechnology tools
Jun Ogawa

Information obtained through detail analysis of microbial function leads to finding of unexpected enzymes, metabolisms, and communities useful for bioprocess design. The screening of the novel biotechnological tools required analysis of unrevealed function with difficulties in establishing the methods, however, recent omics technologies make easier to identify the novel genes, enzymes, and communities, expanding their bioprocess application. Here, examples of bioprocess development by applying unique tools found through functional analysis of microbial metabolisms are introduced.

1)  Novel amino acid metabolism involving hydroxylase- and dehydrogenase- catalyzing reactions was found. The hydroxylase library expanded through genomic information analysis and coupled with related enzymes made possible the production of various chiral hydroxy amino acids and chiral amino acid sulfoxides^1,2.

2)  Novel fatty acid reducing metabolism, polyunsaturated fatty acid (PUFA) saturation metabolism, was found in gut microorganisms. The metabolism involving four enzymes of hydratase, dehydrogenase, isomerase, and reductase was applied to the production of various hydroxy, oxo, and enone fatty acids with unique physiological activity useful for health³. Novel desaturases involved in PUFA biosynthesis⁴, and cyclooxygenase⁵ and P450 monooxygenase⁶ generating PUFA-derivatives were found and applied to the production of physiologically active PUFA derivatives.

3)  Novel nucleosidases acting on 2’-O-methylribonucleosides were found and their ribosyl transferring activity was applied for the production of 2’-O-methylribonucleosides⁷. A novel enzyme, allantoinase, in the purine degradation metabolism was found to useful for the production of chiral amides via prochiral cyclic imide hydrolysis⁸. The reversible reactions involved in nucleoside degradation metabolism were applied to produce deoxyribonucleosides⁹.

4)  Phytochemicals in foods and medicines are changed into bioactive molecules by gut microbial metabolism. The analysis of gut microbial metabolism of phytochemicals such as glucosinolates¹⁰, ellagic acid¹¹, baicalin¹², and astragaloside IV¹³ resulted in finding of novel enzymes. Besides, novel aglycon-glycosylating enzymes were found in microorganisms and applied to enhance the applicability of phytochemicals^14,15.

5)  Nitrifying bacteria play an important role in generating nitrate for crop cultivations. Organic nitrogen compounds are converted to nitrate through ammonification and nitrification. Understanding the interactions of the nitrifying microbial consortia is important for controlling the mineralization of organic nitrogen compounds. We established a controllable model consortium for ammonification and nitrification under organic conditions using a co-culture of only three strains selected through metagenomic analysis^16,17. 

References

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