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

1. Hibi, M. et al. Appl Microbiol Biotechnol, 97, 2467-2472 (2013))
2. Hibi, M. et al. Commun Biol, 4:16 (2021).)
3. Kishino, S. et al. Proc Natl Acad Sci USA, 110, 17808-17813 (2013))
4. Mo, B. K. H. et al. Biosci Biotechnol Biochem, 85, 1252–1265 (2021))
5. Mohd Fazli, F. A. et al. Biosci Biotechnol Biochem, 83, 774-780 (2019))
6. Saika, A. et al. FASEB Bioadv, 2, 59-71 (2020))
7. Mitsukawa, Y. et al. J Biosci Bioeng, 125, 38-45 (2017))
8. Nojiri, M. et al. Appl Microbiol Biotechnol, 99, 9961-9969 (2015))
9. Horinouchi, N. et al. Microbial Cell Factories, 11, 82, (2012))
10. Watanabe, H. et al. Sci Rep, 11, 23715, (2021))
11. Watanabe, H. et al. J Biosci Bioeng, 129, 552-557 (2020))
12. Sakurama, H. et al. Appl Microbiol Biotechnol, 98, 4021-4032 (2014))
13. Takeuchi, D. M. et al. Biosci Biotechnol Biochem, 86(10) 1467–1475 (2022))
14. Suzuki, T. et al. Biocatal Agric Biotechnol, 30, 101837 (2020))
15. Kimoto, S. et al. J Biosci Bioeng, 134, 213-219 (2022))
16. Saijai, S. et al. Biosci Biotechnol Biochem, 80, 2247-2254 (2016))
17. Meeboon, J. et al. Sci Rep, 12, 7968 (2022)

Research Interests

• Protein design and engineering
• Biocatalysis and biosynthesis
• Synthetic biology and self-organizing materials

Claudia Schmidt-Dannert

Self-assembly and self-organization are key principles of biological systems that offer tremendous opportunities for the bottom-up design of functional biomaterials from simple building blocks such as as proteins, nucleic acids, and lipids. Proteins and peptides provide the greatest versatility for the design and low-cost production of supramolecular materials because of their chemical diversity and ability be manufactured recombinantly by cell factories or, in the future, by cell-free expression systems. Protein nanostructures also play important roles in the spatial organization of enzymes at the subcellular level and direct the formation of inorganic-organic composite materials with properties unmatched by synthetic materials. Inspired by these functions, we are harnessing the self-assembling properties of proteins for the design of protein-based nano-architectures for scaffolding of enzymes for heterogenous biocatalysis and the fabrication of new types of materials. Functionalization of protein-building blocks with additional protein domain and peptide tag fusions allows for control over enzyme attachment and the incorporation of other emergent functions such as biomineralization to create mechanically robust biocomposite materials. In this presentation, I will discuss strategies and examples for the design of such types of materials.

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.

Emma Master received her Ph.D. degree from the University of British Columbia in Canada (2002) and carried out postdoctoral research at the Royal Institute of Technology (KTH) in Sweden (2002-2005). She is an Adjunct Professor at Aalto University and Full Professor at the University of Toronto.

The aim of her research is to create biotechnologies that customize nature’s most abundant structural biopolymers for use in value-added materials, including textiles. After joining the University of Toronto (2005), she was awarded a Finland Distinguished Professor Fellowship (2010) to develop her research program in Finland. In 2015, she was awarded a European Research Council Consolidator grant to further grow her team at Aalto and network within Europe, and in 2020 she was awarded a Future and Emerging Technologies (FET) Open grant to integrate bioscience, computational sciences and materials sciences for the advancement of biotechnologies in circular bio-based economies.

Over the past ten years, she has supervised 20 post-doctoral fellows, 20 Ph.D. students, and 27 Master’s students. ORCID iD https://orcid.org/0000-0002-6837-9817 | Researcher ID O-3554-2014

Research Interests

• Industrial enzyme development and application
• Molecular biology and enzyme engineering
• Enzymatic treatment of lignocellulosic material

Advancing the role of biocatalysts in bio-based materials manufacturing

Thu Vuong¹, Owen Mototsune¹, Xuebin Feng¹, Olan Raji¹, Majid Haddad Momeni² Deepika Dahiya², Emma Master^1,2

1. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
2. Department of Bioproducts and Biosystems, Aalto University; FI-00076 Aalto, Kemistintie 1, Espoo, Finland.

Genomics initiatives have uncovered the critical importance of microbial enzymes to expand-ing the range of products that can be made from plant biomass (i.e., lignocellulose). So far, most applications of such enzymes focus on the deconstruction of lignocellulose to mono-saccharides and monolignols for subsequent fermentation to fuels and target chemicals. While necessary for capturing the full potential of renewable plant biomass, this approach inevitably foregoes the benefit of upgrading the energy and carbon already fixed in struc-tures. In this presentation, I will describe our efforts to discover and develop enzymes and enzyme systems that introduce new chemical and physical functionality to underused ligno-cellulose fractions, leading to lignocellulosic building blocks primed for use in value-added applications. In particular, this presentation will describe our characterization and application of carbohydrate oxidoreductases oxidases [1-3], lytic polysaccharide monooxygenases, car-bohydrate-active transaminases [4], and microbial expansin-related proteins [5].

________________
References

[1] Vuong TV, Master ER. Curr Opin Biotechnol. 2022. 73:51.
[2] Vuong TV, Master ER. Biotechnol Biofuels. 2020.13:51.
[3] Karppi J, Zhao H, Chong SL, Koistinen AE, Tenkanen M, Master E. Front Chem. 2020.8:11.
[4] Aumala V, Mollerup F, Jurak E, Blume F, Karppi J, Koistinen AE, Schuiten E, Voß M, Bornscheuer U, Deska J, Master ER. ChemSusChem. 2019. 12: 848.
[5] Monschein M, Ioannou E, Koitto T, Al Amin LAKM, Varis JJ, Wagner ER, Mikkonen KS, Cosgrove DJ, Master ER. Appl Environ Microbiol. 2023 89(1):e0186322

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 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

Gregg Whited received his Ph.D. in Bacterial Physiology from the University of Texas, Austin (1986). He joined The IFF Company Inc. in 1988. He has been engaged in Metabolic Pathway
Engineering for sustainable large scale commodity chemicals. He is also passionate about engineering and producing industrial enzymes for sustainable applications.

Research interests:

• Industrial enzymes in sustainable applications.
• Metabolic Pathway Engineering for high efficiency catalysis.
• Identifying, expressing, and fermentation production of novel enzyme catalysis.

Removal of cyanuric acid in swimming pools using a cell-free thermostable cyanuric acid

Gregg Whited, Ph.D.

Cyanuric acid (CYA) is used commercially for maintaining active chlorine to
inactivate microbial and viral pathogens in swimming pools and hot tubs. Repeated
CYA addition can cause a lack of available chlorine and adequate disinfection.
Acceptable CYA levels can potentially be restored via cyanuric acid hydrolases
(CAH), enzymes that hydrolyze CYA to biuret under mild conditions. Here we
describe a previously unknown CAH enzyme from Pseudolabrys sp. Root1462
(CAH-PR),mined from public databases by bioinformatic analysis of potential CAH
genes, which we show to be suitable in a cell-free form for industrial applications
based upon favorable enzymatic and physical properties, combined with high-yield expression in aerobic cell culture. The kinetic parameters and modeled structure
were similar to known CAH enzymes, but the new enzyme displayed a surprising
thermal and storage stability. The new CAH enzyme was applied, following addition
of inexpensive sodium sulfite, to hydrolyze CYA to biuret. At the desired endpoint,
hypochlorite addition inactivated remaining enzyme and oxidized biuret to primarily dinitrogen and carbon dioxide gases. The mechanism of biuret oxidation with hypochlorite under conditions relevant to recreational pools is described.

Dr. Pam Ismail is the Founder and Director of the Plant Protein Innovation Center and is a Professor at the Department of Food Science and Nutrition, University of Minnesota. Dr. Ismail has over 25 years of experience in Food Chemistry research focused on analytical chemistry, protein chemistry, and chemistry and fate of bioactive food constituents. Her research focuses on structural characterization and enhancement of functionality, safety, bioavailability, and bioactivity of food proteins, following novel processing and analytical approaches. She is the recipient of a “Distinguished Teaching Award” and an “Outstanding Professor Award.”

Targeted use of Enzymes for Enhanced Plant Protein Functionality

B. Pam Ismail, Ph.D.

By 2025, the global demand for protein ingredients is expected to reach 7 million tons and generate revenues of nearly $70 billion. Specifically, there is a growing interest in novel plant-based protein ingredients to partially replace a market sector that has been dominated by traditional protein ingredients such as milk and soy proteins. However, emerging plant proteins are lagging behind soy protein in functionality. Processor are seeking solutions to overcome formulation challenges. Therefore, efficient, and safe functionalization procedures are needed. This presentation will cover our efforts at the Plant Protein Innovation Center in collaboration with industry to enhance the viability of plant proteins through targeted enzymatic approaches. Enzymatic modification of pulse and hemp proteins to enhance gelation, emulsification and solubility will be covered as examples.

Keita Okuda joined Amano Enzyme Inc. in 2008. He received Ph.D. degrees in molecular biology from Nagoya University (2015) He has been engaged in development of industrial enzymes. He has interest in advanced technologies to create innovative enzyme. He believes that an enzyme will play a central role in the future for sustainable industry. He wants to dedicate to development of innovative enzymes to contribute to implementing the circular economy.

 Research Interests

• Industrial enzyme application.
• Molecular biology of enzyme engineering
• Introduction of advanced technologies into enzyme design

Enzyme Application for Plant-based Food

Keita Okuda

Plant-based food is a major trend in the world to keep the sustainable world. However, one of the technical challenges the market faces is to fill the gap between plant-based food and traditional food. Amano Enzyme Inc. has been working to improve functionalities of plant-based food with a wide range of unique enzymes. We will present the following three topics.

• The solubility of plant protein was significantly improved with Protein Glutaminase (PG). PG is an enzyme to catalyze the deamination, which converts glutamine in protein sequence into glutamate without hydrolyzing protein. As a result, protein hydration is increased as more water is able to interact.

With increasing glutamate residue in protein, the isoelectric point shifts to a lower pH. Then it contributes to prevent the curdling of plant-based milk when added to coffee.

• A novel off-flavor masking system was developed with Cyclodextrin glucanotransferase (CGT). It was observed that cyclodextrin produced with CGT during the process decreased the amounts of volatile compounds (ex. Hexanal, Benzaldehyde, etc.). It was also confirmed that trained sensory panelists detected the reduction of beany flavor of the sample.
• The savory flavor of plant protein is increased by hydrolyzing protein. Our proprietary protease blend, Umamizyme Pulse MA, effectively hydrolyzes plant protein without increasing bitterness. It was demonstrated that a vegetable broth treated with Umamizyme Pulse MA had a strong salty taste, leading to a potential of 20% salt reduction.

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.