High-level production of erythritol by mutants of osmophilic Moniliella sp.
Introduction
Erythritol is a tetraose alcohol found in several natural sources, such as lichen, hemp leaves, and mushrooms. It can also be found in fermented foods, such as wine, soy sauce, and sake [1]. Although the sweetness of erythritol is about 70–80% that of sucrose, it is low-calorie: the in vivo heat consumption is less than 0.3 kcal/g, 10% that of sucrose [2]. Erythritol has a high negative heat when dissolved in solution, providing a strong cooling effect [3]. Structurally, erythritol is a polyol and shares the health properties of other polyols, such as being safe for tooth enamel and for diabetics [4]. Because it is rapidly absorbed in the intestines, over 90% of consumed erythritol is excreted from the body without being metabolized. It has also been shown to be insulin independent and non-carcinogenic. Erythritol is highly heat resistant and not susceptible to browning reactions with amino acids, both good properties for processing [2].
Erythritol is synthesized from dialdehyde starch by a chemical reaction at high temperatures in the presence of a nickel catalyst [5]. However, because of its low efficiency, this process has not been industrialized. There are many known erythritol-producing microorganisms, most of which are yeasts that can tolerate high osmotic pressures, such as Pichia, Candida, Torulopsis, Trigonopsis, Moniliella, Aureobasidium, and Trichosporon spp. [6], [7], [8], [9]. It has also been reported that Leuconostoc oenos produces erythritol, but only under anaerobic conditions [10]. Erythritol is the first sugar alcohol to be produced commercially by fermentation [2]. The compound is industrially produced by the yeast, Aureobasidium sp., which is isolated from the soil of a sugarcane plant in Okinawa. A mutant of this strain can give erythritol yields of up to 43.8% after 4 days cultivation in a jar fermentor [8]. Candida magnoliae is another important yeast species with a high erythritol yield compared to most yeast species, and it has a preference for fructose over glucose as carbon source [11]. Recently, it was reported that Yarrowia lipolytica Wratislavia K1 produced erythritol at 170 g/L after 7 days when grown in 30% glycerol at pH 3 [12]. Erythritol production from Pseudozyma tsukubaensis KN75, a yeast strain that is capable of growth at high osmolarity and has the highest erythritol yield (61%), has been scaled up from a laboratory scale to plant scales [13].
A biosynthetic mechanism of erythritol production within microbes has been proposed: 1 mol glucose is converted to 2 mol carbon dioxide and 1 mol erythrose-4-phosphate in the pentose phosphate pathway, and then 1 mol erythrose-4-phosphate is converted to 1 mol erythritol by the serial actions of a dephosphorylase and a dehydrogenase [1]. Supplementing the reactions with Cu2+ reduced the production of fumarate, which relieves the inhibition of erythrose reductase and a high yield of erythritol from Torula corallina [14]. Moreover, proteomic information has been used to predict the carbon metabolism of erythritol-synthesizing microorganisms, such as C. magnoliae, to aid in the design of better microbial systems for the production of erythritol [15], [16].
As reported in the previous paper, we screened 658 strains grown on various sources, such as pollen, honey, and high sugar foods, and were able to isolate 6 high erythritol-producing microorganisms [17]. One of these, Moniliella sp. 440, produces erythritol with a 38.8% yield on media containing 30% glucose. In this study, we mutated this strain and obtained a mutant with significantly higher erythritol productivity. We also investigated the optimal culture conditions for erythritol production in the mutant strains.
Section snippets
Materials
Yeast extract was purchased from DIFCO (Detroit, MI, USA). Corn steep liquor was obtained from Fon Nen Co., Ltd. (TaoYuan, Taiwan). All carbohydrates used for substrates and other chemicals used were reagent grade.
Microorganism and cultivation
Moniliella sp. 440 was isolated from honey obtained from the Institute of Bee Breeding in Taiwan [17]. The standard cultivation was done by transferring single colonies of the strain from plates to 10 mL of media consisting of 30–50% glucose and 1.0% yeast extract (pH 5.5) in 50-ml
Isolation of high erythritol-producing mutants
Isolates from six generations of iterative NTG mutagenesis, named N12115-6, N21105-6, N31074-3, N42208-2, N53199-9, and N61188-12, were selected based on their high levels of erythritol production when grown in media containing 40% glucose and 1% yeast extract (Table 1). It is clear that erythritol production increased progressively with each successive round of mutagenesis and erythritol productions from these mutants reached 151.0–237.8 g/L (productivity were between 1.26 and 1.98 g/L/h), a
Discussion
In this study, we mutated the wild type strain, Moniliella sp. 440, through iterative rounds of NTG treatment and screening and six generations of mutants were selected and characterized for their ability to convert glucose to erythritol. It was clear that erythritol production of the mutants increased progressively with each round of mutagenesis, with the terminal mutant, N61188-12, having significantly higher erythritol production (237.8 g/L) than the starting strain (Table 1).
In comparing the
Acknowledgments
The authors thank to Dr. Sung-Yuan Hsieh for help in generating the photomicrographs. This work was supported by grant 89-EC-2A-17-0263 from the Ministry of Economic Affairs of the Republic of China.
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Systematic metabolic engineering of Yarrowia lipolytica for the enhanced production of erythritol
2024, Bioresource TechnologyErythritol production from sugar based feedstocks by Moniliella pollinis using lysate of recycled cells as nutrients source
2022, Process BiochemistryCitation Excerpt :Reutilization of ethanol was observed during fed batch fermentation with both sugarcane juice and molasses. M. pollinis produced 0.38 g/g erythritol from sugarcane juice during fed batch fermentation, which is equivalent to the yield reported on pure glucose by Burschapers et al. [32] in 20 L fed batch culture and only 10 % less than Moniliella sp. mutant N61188−12 [33]. Park et al. [34] demonstrated that intermittent feeding of glucose and CSL resulted in an increase in erythritol productivity.
By-products of sugar factories and wineries as feedstocks for erythritol generation
2021, Food and Bioproducts ProcessingCitation Excerpt :In the case of molasses, the aim was to use a broth with an initial sugar concentration of 300 g/L because of two reasons. In the first place, numerous research works indicate that erythritol-producing fungi, such as Moniliella, Pseudozyma, Torula or Starmerella magnoliae, tolerate glucose concentrations about 300 g/L, attaining erythritol yields of 0.23–0.60 g/g in model solutions (Burschäpers et al., 2002; Cho et al., 1999a, 1999b; Hajny et al., 1964; Jeya et al., 2009; Koh et al., 2003; Lin et al., 2010, 2001; Oh et al., 2001; Ryu et al., 2000). Secondly, if there is no substrate inhibition or excessive osmotic pressure, elevated initial sugar concentrations normally result in higher product concentrations, which can be translated into lower purification costs (Moon et al., 2010; Troostembergh et al., 2001).
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2019, Biotechnological Production of Bioactive CompoundsSugar alcohols
2018, Encyclopedia of Food Chemistry