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ScienceDirect Biotechnological production of natural zero-calorie sweeteners Ryan N Philippe1, Marjan De Mey1,2, Jeff Anderson1 and Parayil Kumaran Ajikumar1 The increasing public awareness of adverse health impacts from excessive sugar consumption has created increasing interest in plant-derived, natural low-calorie or zero-calorie sweeteners. Two plant species which contain natural sweeteners, Stevia rebaudiana and Siraitia grosvenorii, have been extensively profiled to identify molecules with high intensity sweetening properties. However, sweetening ability does not necessarily make a product viable for commercial applications. Some criteria for product success are proposed to identify which targets are likely to be accepted by consumers. Limitations of plant-based production are discussed, and a case is put forward for the necessity of biotechnological production methods such as plant cell culture or microbial fermentation to meet needs for commercial-scale production of natural sweeteners. Addresses 1 Manus Biosynthesis, 790 Memorial Drive, Suite 102, Cambridge, MA 02139, USA 2 Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000, Belgium Corresponding author: Ajikumar, Parayil Kumaran ([email protected]
natural sweeteners can address supply scalability limitations associated with plant-based production while increasing sustainability of the overall endeavor. In addition, the development of plant or microbial cellbased production platforms can allow for the rapid modification of pathway enzymes to generate novel sweeteners with fewer or no negative taste attributes. Given the explosive growth in interest in natural zerocalorie sweeteners in the past few years, we will endeavor to provide a solid background on sweetener science from the past twenty years while emphasizing the recent resurgence in research focused on naturally derived zero-calorie sweeteners. A brief exploration of some of the chemistry and biology underlying natural sweeteners will be followed by a short analysis of the potential for biotechnological methods to tackle some sustainability issues with plantbased production methods. Finally, we identify some potential challenges that must be addressed to ensure the successful development of a biotechnologically produced sweetener product.
Discovery of zero-calorie sweeteners Current Opinion in Biotechnology 2014, 26:155–161 This review comes from a themed issue on Food biotechnology Edited by Mattheos AG Koffas and Jan Marienhagen For a complete overview see the Issue and the Editorial Available online 3rd February 2014 0958-1669/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2014.01.004
Introduction Global demand for naturally sourced, zero-calorie sweeteners has increased significantly over the last decade as consumers have become increasingly health conscious. The popular press publishes frequent articles about health impacts of sugars and sugar substitutes, creating public or personal pressures that steer consumers away from sugar to natural low-calorie or zero-calorie alternatives. Perhaps more importantly, demonstrated health issues related to excessive sucrose consumption have helped propel growth of plant-derived natural sweeteners. However, further growth of natural sweeteners is potentially limited by agricultural sustainability, undesirable taste qualities, perceived safety and commercial viability. Biotechnological production platforms for www.sciencedirect.com
Many synthetic and natural sweet-tasting compounds have been identified since the early 1800s. Most of these compounds are much more potent than commonly used sucrose and are generally referred to as high-potency (HP) sweeteners. Sweeteners can be found in many areas of chemical space, with at least 50 structural classes of organic compounds represented [1,2]. Despite centuries-long usage of plants traditionally known for their sweetening potential by certain societies, the original non-sucrose sweeteners developed were artificially synthesized molecules, not natural products. This is in part due to their discovery at a time when taste and smell were key methods employed for the characterization of newly synthesized compounds. The first artificial sweetener to be commercialized is saccharin, discovered in 1878 by Fahlberg and Remsen . This was followed by cyclamate, neohesperidin dihydrochalcone, aspartame, acesulfame K, sucralose, neotame, and advantame . All of these artificial sweeteners are approved for use in various countries, but not all are approved everywhere due to differences in public health administrations. For example, cyclamate is not approved for use in the United States due to health concerns , while it is approved in Canada, Europe, Central and South America, and Asia. Identification of the sweetness receptor first in rats  and then in humans  has enabled high-throughput screening (HTS) of large compound libraries via cell-based Current Opinion in Biotechnology 2014, 26:155–161
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assays, and promises to speed the discovery of even more compounds with sweetening potential. This technology has also enabled the identification of sweetness enhancers named positive allosteric modulators (PAMs), compounds that while not necessarily sweet themselves are capable of enhancing the perception of sweetness from other compounds [6,7]. These technologies have resulted in faster and safer methods for the discovery of novel sweetening compounds.
Natural high-potency sweeteners Given this variability in acceptance of artificial sweeteners, an increasing realization of the effects of excessive sugar consumption, and growing interest for natural products by consumers in general, the demand for natural zero-calorie sweeteners has increased significantly. A wide range of plant derived natural products can elicit sweet responses or can modulate sweetness, including terpenoids, phenylpropanoids, dihydroisocoumarins, flavonoids, steroids, proanthocyanidins, amino acids, and proteins. Numerous excellent reviews are available on this subject [7,8,9], with ongoing work highlighting the potential of biotechnological production of proteins with sweetening or sweetness enhancing ability — thaumatin, mabinlin, monellin, neoculin, brazzein, and miraculin [10–12]. Relatively few sweet-tasting plant-derived natural products have been commercially launched to date, but those numbers are rapidly increasing. These include thaumatin, glycyrrhizic acid, monatin, brazzein, mogroside V, stevioside, and rebaudioside A . The latter two compounds are ent-kaurene glycosides from Stevia rebaudiana. Along with the mogrosides from Siraitia grosvenorii, these glycosylated terpenoid compounds have become increasingly interesting targets for commercial production. The two plant sources that produce the former two molecules are discussed in more detail below.
S. rebaudiana (Bertoni) S. rebaudiana (Bertoni) (stevia) is a sweet herb native to Paraguay and Brazil . The sweet taste of the leaves (Figure 1a) is due to ent-kaurane diterpenoid glycosides, which contain the steviol aglycone core in common and differ in the number and type of sugars attached to C-13 and C-19 (Figure 1b). There are more than a dozen steviol glycosides (SGs) identified in S. rebaudiana, including stevioside and rebaudioside A [15–21]. These latter two form the majority of SGs found in stevia, which all told can accumulate up to 20% of dry leaf weight in some strains, and are approximately 200 and 300 times sweeter than sucrose, respectively [16,17]. More highly branched sugar chains at the C-13 and C-19 positions result in increasing sweetness , while reduction of the C-16 exocyclic double bond greatly reduces sweetness . SGs can be perceived as bitter or ‘metallic’; SGs with fewer glycosylations appear more so [16,17]. Pawar and colleagues have provided a timely review of analytical methods for SGs . Current Opinion in Biotechnology 2014, 26:155–161
While earlier studies reported mutagenic potential for steviol , subsequent studies have shown that steviol is not absorbed by humans . Steviol glycosides have since been demonstrated as safe  and have received Generally Regarded as Safe (GRAS) status from the FDA in the United States. Additional information on the nutritional aspects of stevia is available [21,28].
S. grosvenorii (Swingle) The fruit of S. grosvenorii (Swingle), or Luo Han Guo (LHG) (Figure 1c), has a long history of usage as a sweetener and in traditional Chinese medicine for the treatment of colds, dry cough, sore throat, and minor stomach or intestinal discomfort . The major sweet component of LHG fruit was identified as mogroside V (Figure 1d), with further work leading to the identification of seven additional sweet-tasting mogrol glycosides (MGs) . These MGs have the mogrol triterpenoid aglycone in common and differ in the sugars attached, requiring at least three glycosylations for sweetness . The commercial extract mixture of mogrosides IV, V, and VI, siamenoside I, and 11-oxomogroside-V is 300 times sweeter than sucrose [31,32]. LHG extract and mogroside V have been acknowledged as GRAS by the FDA. As with the SGs, MGs with fewer glycosylations such as mogrosides II and IIIE, found in unripe fruit, can have a bitter or metallic taste [29,30,33], necessitating careful agricultural production to maximize the quality of the plant extract produced.
Criteria for sweetener development While numerous chemical entities possess sweet or sweetness-enhancing properties, very few prove to be suitable for development and application in food and beverage products. Given the cost-effectiveness of sucrose or artificial sweeteners, high commercial potential is required to justify the major investments required to develop a new natural sweetener product. To have high commercial potential, a sweetener must score highly in a variety of metrics, including the availability in scale, taste quality, safety, stability, solubility, cost, and patentability (Figure 2) [13,34]. Very few molecules with sweetening potential can satisfy all these metrics . Moreover, taste quality is absolutely critical. Consumers strongly prefer the taste of sucrose, and HP sweeteners that perfectly mimic the taste of sucrose have not yet been identified. HP sweeteners commonly show (a) low maximal sweetness intensities, (b) undesirable tastes such as bitter, metallic, and licorice-like, (c) slow-onset sweetness that lingers, and (d) an ability to desensitize perception of sweetness . Blending HP sweeteners has been shown to alleviate some of these issues . To be a successful product, a HP sweetener should be soluble enough to sweeten to an equivalent of 10% sucrose with a clean taste that develops quickly and does not linger. It needs to be safe to the consumer and not exhibit toxicity or mutagenicity. It needs to be pH, www.sciencedirect.com
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HO HO OH
HO HO OH
OH O O
HO HO O OH O
O HO HO HO
O HO H
Siamenoside I HO HO HO
HO O O O HO OH OH
Mogroside V Current Opinion in Biotechnology
(a) Stevia rebaudiana (Bertoni) leaves. (b) Steviol glycosides accumulate to high levels in stevia plants; these glycosylated terpenoids have 300 more sweetness than sucrose on a per weight basis. Note the additional glycosylation present in rebaudioside A. (c) Siraitia grosvenorii (Swingle) fruit. (d) Mogrosides can be even more than 300 sweeter than sucrose, and again demonstrate varying properties based on increasing glycosylation of the terpenoid core.
temperature, and light stable for use in food products, and it should not break down into unsafe metabolites or undesirable taste components. Despite bitter taste for some family members [16,17,29,30,33], the terpenoid glycosides found in stevia and LHG are promising candidates for development given their high potency, good temporal profiles, and high stability [22,35].
Sustainable biotechnological production In terms of a fully sustainable production system, HP sweetener supply can be satisfied by biotechnological production while synergistically generating beneficial side-effects. Land can be used to produce high yielding crops such as sugarcane or corn, whose sugar can be converted via plant cell culture or microbial fermentation to natural HP sweeteners, instead of relying on variable potency plant formulations. With many natural HP sweeteners being hundreds of times sweeter than sucrose [8,13], we can effectively multiply sweetening potential www.sciencedirect.com
while minimizing calories. Plant sources of sweeteners are limited in total area that can be cultivated, and in total productivity on a per weight basis. Additionally, it can be difficult or impossible to produce superior but minor products using plant-based methods. For example, while rebaudioside D has enhanced sweetening potential and taste profile compared to stevioside and rebaudioside A , it accumulates at much lower levels than the two latter compounds and thus its total production is limited. The quality of commercial extracts can be diminished if extracts of unripe and ripe fruits or leaves are mixed. Additionally, fluctuations in climate or political stability can affect relatively low-production crops such as stevia or LHG. Finally, the work involved in plant-based production of these sweeteners also limits their overall market; the labor-intensive approaches required for agricultural production of the plant material and its processing to a finished product cap the potential scale of production. This is of critical importance, because sugar is Current Opinion in Biotechnology 2014, 26:155–161
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Biotechnological Production of Natural Sweeteners
- Develop cost-effective plant cell culture method
OR - Metabolic engineering of functional pathway in microbial host - Optimization of production strain and bioprocess
THEN - Fermentation scale-up & economical purification
Discovery of Novel Sweetener
Technical Hurdles to Overcome
Reality Check: Criteria for Product Success
- Taste quality (should match sucrose) - Sweetness intensity - Clean, pure taste - Temporal profile
Compound with Sweetening Activity Identified
- Low desensitization effect - Safe for consumption - Chemically stable compound - High solubility
- Cost-effective (sweetening vs. price)
- Patentability & IP protection - Positive consumer perception
Current Opinion in Biotechnology
(1) Once a promising candidate compound with attractive sweetening properties has been identified, whether by organoleptic assessment of plant material or high-throughput screens of compound libraries using the human sweet taste receptor, there still remains the need for a sober analysis of the candidate’s potential for development into a high-potency sweetener product (2) to justify the expenditure of resources required to bring that product to market. Even having passed the test for potential product success, there remain significant technical hurdles (3) to the successful development of a biotechnological production method for the desired natural sweetener molecule.
traded as a commodity in staggering numbers: the global market for all sweeteners was $77.5 billion in 2012 and is expected to grow to $97.2 billion by 2017. Of this, sugar accounts for 175 000 000 metric tons (MT)/year, or roughly $65 billion, while the markets for HP sweeteners and steviol glycosides account for $1.5 billion and $1.7 billion respectively. Adjusting for sweetness intensity, for the 2012 sucrose production to be replaced by rebaudioside A, 574 000 MT would be needed; this 66-fold increase over 2010 production would require roughly 425 000 more hectares of dedicated farmland (calculated by authors from data available at Food Product Design; URL: www.foodproductdesign.com). For any natural HP sweetener to reach this scale, it is necessary to develop sustainable biotechnological production methods Current Opinion in Biotechnology 2014, 26:155–161
such as plant cell or microbial fermentation for its stable manufacture.
Challenges to successful biotechnological production of natural sweeteners While the benefits of biotechnological production of natural sweeteners are numerous, the successful achievement of the goal is not without technical challenges (Figure 2). Ideally, if it is possible to produce the compound of interest via cell cultures of a critical plant, efforts can be limited to maximizing sweetener yield without requiring detailed knowledge about the biochemistry and biochemical pathway . In the absence of productive cell culture, however, there still remains the potential for metabolic engineering of plant or microbial cells for www.sciencedirect.com
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production of the sweetener. However, metabolic engineering of the plant cell for the production of complex natural products requiring multistep biosynthetic pathways is challenging due to the complexity of plant cells, and lack of genetic tools and adequate methods [37,38]. Moreover, plant cell culture is expensive, time consuming, and low yielding for the production of specialized natural products [39,40]. Therefore, plant cell culture may not be a viable method for the large-scale production of natural product sweeteners. In the past decade, metabolic engineering of microbial systems has demonstrated astonishing progress, and several tools and methods are now in place for manipulating biosynthetic pathways to enable scalable production of specialized natural products [41–43,44,45]. The first challenge to overcome, after having identified a promising chemical candidate (see ‘Criteria for Sweetener Development’ section), is to assemble a complete biochemical pathway capable of biosynthesizing the compound of interest in vivo, in your desired microbial host. The key requirement for microbial engineering is a detailed biochemical and genetic understanding of target compound biosynthesis from the native organism. There is a considerable amount of information available for some targets, while much less can be had for others. For instance, the pioneering work of Brandle and colleagues [46–49] has resulted in the identification of a near-complete biosynthetic pathway for steviol glycosides in S. rebaudiana. There is only a single glycosylating enzyme left to uncover to complete the pathway leading to rebaudioside A production. In contrast, while a transcriptomic profiling approach by Tang et al.  has yielded candidate genes for every unknown step in the pathway to mogrosides, biochemical function has not yet been verified. However, the challenge of completely identifying the biochemical pathway has been greatly reduced with the advent of next-generation sequencing technologies, allowing for the rapid generation of high-quality transcriptomes from practically any plant tissue, especially when coupled with the use of recombinant microbial systems to rapidly assess biochemical function of any candidate genes. Once the enzymes leading to a desired product have been identified and characterized, the challenge moves first to creating an engineered microbial strain capable of producing said compound, then second to optimizing an engineered microbial strain capable of maximum efficient sweetener production from whatever carbon source employed. Engineering multistep secondary metabolite pathways in microbes for scalable production is not a trivial task. However, recent progress in the metabolic engineering of metabolites such as artemisinic acid and hydroxylated taxanes shows great promise [44,45]. Despite these demonstrated successes in expressing plant pathways in microbial hosts, the challenges of specialized www.sciencedirect.com
metabolic enzyme promiscuity and low activity remain . Therefore, approaches combining protein engineering and metabolic engineering for fine tuning of the pathways must be incorporated to maximize production [51,52]. For example, increasing solubility of proteins, decreasing turnover rates, altering substrate or product profiles, and modifying catalytic parameters are potential tools to be employed. To this end, emerging technologies focused on these goals are creating new possibilities for addressing these technical challenges, building a path toward successful microbial production at commercial scale [51,53]. Finally, the economic recovery of the biosynthesized product from completed fermentations will require advances in downstream processing and purification. Here, the identification and engineering of a transport system that efficiently pumps the desired sweetener product out of the microbial cells would be advantageous, as it would allow for filtration-based purification and concentration of product without having to deal with cell debris and cell contents . Such transport systems could also enable higher production titers — by clearing the product from active cells, a metabolic sink is effectively created which will ensure maximum yield and efficiency of sweetener production. Taken together, a great deal of efforts — identifying genes responsible for biosynthesis, metabolic engineering, protein engineering, fermentation and downstream processing — is yet needed to develop sustainable manufacturing processes for naturally derived zero-calorie sweeteners.
Conclusions Growing awareness of the effects of excessive sugar consumption has resulted in changes in consumer habits. Coupled with a mounting interest in naturally derived chemicals and natural products, these changes have resulted in a rapid increase in world markets for naturally occurring, zero-calorie sweeteners. The plants S. rebaudiana and S. grosvenorii are two very promising sources of natural HP sweeteners. The terpenoid glycosides they produce have the desired sweetening potential and flavor profiles for high consumer acceptance while possessing the critical chemical properties to enable successful commercialization. However, limitations and risks inherent in large-scale agricultural production, in addition to the costs associated with extracting and purifying the desired sweeteners from plant material, can together work to limit the potential growth and development of these molecules and the products containing them. In order to achieve the scale of production required to provide a go-to alternative for sucrose, these plant-derived sweeteners need to be produced via biotechnological fermentation of bulk sugars. Current Opinion in Biotechnology 2014, 26:155–161
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While the technological, commercial, and social challenges to the successful development of biotechnology capable of large-scale HP sweetener production are considerable, so are the potential benefits to society. By developing safe, natural, zero-calorie sweeteners with enjoyable taste profiles, we can help mitigate the effects of excessive sugar consumption on a generation of human beings, both in terms of increased quality of life and decreased cost of healthcare. We can generate value from sugar by multiplying its sweetening power, all while providing stable and sustainable farming opportunities and supply markets. And finally, the technologies developed to enable biotechnological production of natural sweeteners such as terpenoid glycosides will have farreaching scientific and industrial implications for the production of other small molecules.
Acknowledgements We thank the National Science Foundation (NSF) for generous support through the SBIR (Small Business Innovation Research) Grant program (award no. 1214339). MDM is supported by the Multidisciplinary Research Partnership Ghent Bio-Economy.
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