Ken Izumori
Research Advisor, International Institute of Rare Sugar Research and Education, Kagawa University; Professor Emeritus, Kagawa University
Background: Ken Izumori graduated from Department of Agricultural Chemistry, Faculty of Agriculture, Kagawa University in 1965, and became assistant professor at the Faculty of Agriculture, Kagawa University in 1968. After serving as an associate professor and professor there, he became a professor at the Rare Sugar Research Center, Kagawa University in 2003. In 2008, he became visiting professor at Kagawa University, as well as professor emeritus at Kagawa University and specially appointed professor at the Faculty of Agriculture, Kagawa University. Since 2016, he has served as research advisor at the International Institute of Rare Sugar Research and Education, Kagawa University.
I coined the term "rare sugar" to describe minor monosaccharides. This word has spread widely, and even an academic society (International Society of Rare Sugars) has been founded. Furthermore, the word was even included in the seventh edition of Kojien (one of the most traditional Japanese dictionaries) in 2018. In my lectures, I always encourage my students to create "new words".
This article series provides an introduction to rare sugars. The authors of this series are experts from industrial, academic, and governmental sectors, mainly members of the International Institute of Rare Sugar Research and Education, Kagawa University. Beginning with an explanation of what rare sugars are, we introduce the basic properties of rare sugars. We then provide an easy-to-understand overview of the current status of the applications of rare sugars in a variety of fields.
This is the first article of the series; the topics are "What is a rare sugar?" and "History of rare sugars".
The source of energy for life on the Earth is sunlight. Fig. 1 illustrates this fact schematically from the human standpoint. Humans only get sunburned when exposed to sunlight and cannot directly use it as energy to live. Plants, on the other hand, use sunlight energy to photosynthesize sugars; plants convert light energy into chemical energy and store it in the form of sugars. Humans and many other organisms consume the sugars accumulated in the plants and use them as a source of energy. In other words, one of the major roles of sugars in the biological world is to store the energy of sunlight and supply it to living organisms on the Earth.
Sugar molecules are classified into three main categories according to size (Fig. 2). A monosaccharide is the smallest sugar unit and can no longer be called a sugar when broken down further. An oligosaccharide is a rather larger sugar composed of several monosaccharides bound together. A polysaccharide is a large sugar consisting of many monosaccharides joined together to form a macromolecule. These sugars have distinct roles in nature. With this understanding of the roles and classifications of sugars, I now proceed to the definition of rare sugars.
The International Society of Rare Sugars, which was established in April 2001, defines rare sugars as "monosaccharides and their derivatives being rarely found in nature". Previously, these monosaccharides were described by several different terms, such as "unnatural monosaccharides" or "monosaccharides rarely found in nature." A dictionary of biochemistry previously referred to them as "non-fermented sugars." Fig. 3 presents a schematic illustration of rare sugars, as defined by the International Society of Rare Sugars. In this diagram, the area of the circle represents the amount of each monosaccharide present on the Earth. D-Glucose, which occupies the largest area, is the most abundant monosaccharide. Small red circles are monosaccharides classified as rare sugars, e.g., D-allose, D-allulose (D-psicose), xylitol, and many others.
Note that this figure does not reflects the amounts of free monosaccharides, but the amounts when polysaccharides and oligosaccharides are broken down to monosaccharide units. In fact, free D-glucose rarely exists in nature, and most D-glucose is found in polysaccharides such as α-1,4-linked starch and β-1,4-linked cellulose. For example, there is little free D-glucose present in soil; even if added to the soil, D-glucose powder will be rapidly degraded by a huge number of microorganisms waiting with their mouths open. The same applies to the other sugars shown as relatively large green ovals in Fig. 3. For example, D-ribose is abundant in nature as a component of nucleic acids, and D-mannose as a component of Konjac mannan.
Terms that indicate the properties of the substance, such as "non-fermented sugar", can help you imagine the characteristics of the substance. However, the definition of rare sugars does not let you imagine the "structural or functional characteristics" of these substances. The concept used instead is "abundance." Key points of the definition are "Being monosaccharides" and "rarely found in nature." As structure and function are not considered in this definition, the International Society of Rare Sugars discussed whether certain sugars, such as xylitol, are to be categorized as rare sugars. Xylitol is produced in large amount by reducing D-xylose, which is abundant in nature, and is used as a sweetener or in other applications. However, the low abundance of xylitol in nature led to the conclusion that it is a rare sugar. This group of monosaccharides once had no established terminology and was only vaguely understood, but the definition of the term "rare sugar" triggered rapid and systematic progress in the research of this field.
"Rare sugar" was a coined term that had not been used widely. However, after the definition of rare sugars was established, research progressed rapidly and the use of this term became more common. In line with this situation, the coined term "rare sugar" was newly included in the seventh edition of Kojien (one of the most traditional Japanese dictionaries) published in 2018 (Fig. 4). In this dictionary, rare sugar is described as "a generic term for monosaccharides and their derivatives rarely found in nature—mass production became possible by the discovery of an enzyme that converts D-fructose to D-psicose." The dictionary also has a section on psicose, which states that psicose is "a kind of rare sugar—used as a synthetic raw material for the production of other rare sugars."
As noted above, one of the important roles of sugars is to store and transport the energy of sunlight in the form of chemical energy, which can be consumed by the organisms on the Earth. Then, do rare sugars also play such an important role?
No one would consider that rare sugars are involved in the photosynthesis shown in Fig. 1. Plants use the energy of sunlight to produce D-glucose (C6H12O6) from water (H2O) absorbed through their roots and carbon dioxide from the atmosphere. The sugars produced by photosynthesis mostly become the energy used for sustaining living organisms. It is unlikely that rare sugars found only in trace amounts in nature have the same energy-carrying role as D-glucose. "Rare sugars," therefore, are characterized as sugars not produced by photosynthesis, not well understood in terms of what role they play, and present only in small amount.
The next chapter describes the possible origin of rare sugars, which are not even a source of energy for living organisms, and how research on such rare sugars has progressed.
Almost all organic substances, including sugars, on the present Earth were originally produced by living organisms, mainly plants. From those organic substances, humans not only obtain energy to live, but also use scientific methods to create many organic substances from the abundant sugars for a variety of purposes. Since they are organic, rare sugars are thought to be produced by living organisms, but the details are not clear. This chapter describes how research into rare sugars, many of which are unknown, has progressed. Let’s begin with the topic of formation of sugars by chemical evolution on the primitive Earth.
On the primitive Earth before the emergence of life (i.e., before there were living organisms to produce organic substances), organic substances are thought to have been formed by chemical evolution. When formalin, a solution of formaldehyde (HCHO; a one carbon atom [C-1] aldehyde) in water, is treated at high temperatures and under alkaline conditions, a complex organic mixture containing sugars (formose) is formed. This process is called the formose reaction and has been demonstrated to produce a variety of sugars, including branched ones, in experiments that replicate conditions on the primitive Earth. Formose was selected as a research topic and a comprehensive study of formose was funded by a Grant-in-Aid for Scientific Research; this project was led by Dr. Ruka Nakashima of the Faculty of Engineering, Tottori University, and carried out by researchers from various disciplines from all over Japan1.
The main objective of this study was to produce bioavailable sugars from the C-1 compound formaldehyde by chemical reaction. The goal was also to elucidate the reaction mechanisms and the composition of the products, as well as to investigate the pathways to the emergence of life from the perspective of chemical evolution. Intensive efforts were made to confirm the chemical reaction conditions and the molecular structure of the products, to determine the sugar composition of the products in studies using enzymes, and to examine whether formose could be used by microorganisms. The research results are summarized as follows. (1) Only mixtures of branched and miscellaneous sugars were produced, suggesting the difficulty of producing a specific, biologically useful sugar (though a specific chemical could be produced in relatively large amounts by controlling reaction conditions). (2) HPLC analysis of the reaction products detected numerous peaks containing unknown substances, indicating that formose is an extremely heterogeneous mixture of sugars. Furthermore, enzymatic quantification of D-glucose, D-fructose, and sugar alcohols in the products revealed that these sugars were only produced in ultra-trace amounts of 1% or less of the original formaldehyde. (3) In the study of formose utilization by microorganisms, it was difficult to isolate a single microorganism capable of degrading formose; therefore, its degradation in soil where many types of microorganisms coexist was studied using a thermometric method. As a result of formose degradation by soil microorganisms, heat generation corresponding to about 25% of that for D-glucose was observed. Although it is unclear which microorganisms degraded which formose components, it is clear that about 25% of the components in formose are degraded by microorganisms2.
Based on these results and findings from other studies, it is likely that trace amounts of D-glucose and D-fructose would have been present in the formose that was chemically produced on the primitive Earth, but they probably did not exist in same form as that on the Earth today. After a long period of chemical evolution, thermodynamically stable monosaccharides such as D-fructose accumulated and may have provided the basis for the emergence of life. It is also assumed that trace amounts of rare sugars as well as D-glucose and D-fructose existed in the formose on the primitive Earth but differed in the way they exist and in composition from rare sugars on the Earth today. Did the production of many kinds of sugars begin after the emergence of life? Or, did the process of chemical evolution produce all monosaccharides (including rare sugars) and enable them to continue to exist to the present? In any case, an attractive hypothesis is that the most useful monosaccharides for life were selected during evolution, and those that fell outside this selection process became rare sugars. The question of how the sugar composition in nature came to be as it is today (why are rare sugars "rare"?) is an important issue that will need to be clarified in the future.
As an aside, I here show an imaginary picture of the Earth during a period of chemical evolution, which was painted by Dr. Arthur Weber of NASA (Fig. 5).
The earth was born 4.6 billion years ago, and life emerged 4 billion years ago. Let us leave, for the moment, the open question of whether or not rare sugars existed at the stage of chemical evolution, before life emerged, and turn the clock forward and summarize the history of rare sugar research up to the present day. Monosaccharides have been the subject of research since the early days of organic chemistry. Around 1890, Emil Fischer and colleagues, through extensive research, determined the molecular structures and names of all the monosaccharides. Their excellent work must have made the researchers at the time feel that the basic research on monosaccharides was completed. Subsequently, the emphasis of monosaccharide research shifted to applied research on the production mechanisms of substances such as alcohols, lactic acid, and acetic acid from D-glucose using microorganisms. Although alcoholic fermentation had long been used in brewing beer and making wine, the role of microorganisms was not recognized. It was Louis Pasteur who demonstrated that alcoholic fermentation proceeds by the action of microorganisms. Pasteur, in the course of his study of alcoholic fermentation, uncovered many basic facts in life sciences. One of these discoveries was that the tartaric acid crystals formed at the bottom of vessels during alcoholic fermentation have different structures that are mirror images of each other. This finding demonstrated the existence of chiral molecules; Pasteur also contributed greatly to the establishment of stereochemistry for organic molecules produced by living organisms.
At the time when the emphasis of monosaccharide study was on the production of substances useful to humans, researchers would have considered rare sugars unattractive and not worth studying. Biological science subsequently developed in fields related to the fundamentals of life, such as transmission and expression of genetic information. The rediscovery of Mendel's laws of inheritance led to competition among researchers to find the biological substance responsible for the genetic laws. As a result, DNA was identified, and DNA’s double-helical structure was revealed. In this major research undertaking, monosaccharides were not in the limelight, much less rare sugars.
Fig. 6 is my personal rough summary of the history of biological sciences research. Basic research on monosaccharides seems to have been completed around 1890 by the work of Emil Fischer and colleagues. After that, research on macromolecules such as proteins with physiological function and genetic DNA advanced, leading to the remarkable achievements that brought us closer to understanding the essential nature of life. And now it is the 21st century, the century of biotechnology. In Fig. 6, the period from the end of the 19th century to around 1990 is described as the "100 years of blank history" because for about 100 years after the main targets of biological research became macromolecular substances, researchers considered the study of monosaccharides to be completed, and little attention was paid to basic research on monosaccharides. Under these circumstances, Kagawa University has taken up the challenge of reviving monosaccharide research by purposely focusing on rare sugars in trace amounts. Fortunately, genetic engineering methods developed during the "100 years of blank history" have been a major force in the revival of monosaccharide research.
The major research focus in the century of biotechnology is macromolecules, represented by proteins that perform biological functions and DNA that carries genetic information. On the other hand, rare sugars are only "monosaccharides and their derivatives being rarely found in nature," as defined at the first meeting of the International Society of Rare Sugars in 2001, the dawn of the 21st century. Research on rare sugars, a group of monosaccharides with low molecular weight, low abundance, and little information on their properties, has begun in earnest in the 21st century, the century of biotechnology.
No research begins without experimental materials. However, few rare sugars are commercially available, and even those that can be purchased are extremely expensive, making it impossible to obtain sufficient quantities needed for experiments. Therefore, research on rare sugars had to start with "making" rare sugars. Although it was not clear whether rare sugars have any physiological functions or useful applications, research on rare sugar production began with the belief that "anything that exists in nature, even in small quantities, must have some meaning and significance." A summary of (1) starting materials, (2) tools, and (3) plans for rare sugar synthesis follows.
(1) As starting materials for rare sugar synthesis, monosaccharides that are abundant in nature and can be purchased at low cost (such as D-glucose, D-fructose, and D-galactose) should be selected.
(2) There are two tool options for rare sugar production: synthesis by chemical reaction or bioreaction using enzymes and microorganisms. As chemical reactions of monosaccharides generally produce a wide variety of byproducts, it is preferable to produce rare sugars through specific reactions using enzymes and microorganisms.
(3) In order to synthesize all types of rare sugars, it is necessary to have an overall plan that identifies the starting materials and reactions necessary to obtain the target products.
Based on the above points, the fundamental strategy was established quickly; however, there was no information on enzymes or microorganisms that produce rare sugars. Therefore, we began our research by using already known enzymatic and microbial reactions. The first rare sugar synthesized by this strategy was D-tagatose (a href="#fig_7">Fig. 7). D-galactose, a constituent of lactose, was used as the starting material to be reduced to galactitol. Next, we explored microorganisms that specifically oxidize the carbon at position 2 with reference to Bertrand's work. As a result, the target microorganisms were obtained relatively easily from the soil and successfully used to convert galactitol to D-tagatose3.
Subsequently, we attempted to produce other rare sugars by using previously reported oxidoreductases and isomerases, but without success. At that time, we found, in the soil behind the cafeteria of the Faculty of Agriculture at Kagawa University, a microorganism that produces a completely unexpected enzyme. This microbial enzyme was D-tagatose 3-epimerase (D-TE), which epimerizes the 3-position of D-tagatose (Fig. 8)4. Fortunately, D-TE had broad substrate specificity and C-3 epimerization activity for all eight ketohexoses to produce the corresponding epimers.
D-TE was a new type of enzyme that catalyzes the epimerization at the 3-position of free ketoses; this type of enzyme had not been previously reported. This discovery of D-TE led to the creation of a blueprint (Izumoring) that would guide the production of all rare sugars.
