Journal of Applied Microbiology ISSN 1364-5072

REVIEW ARTICLE

Production of arabitol by yeasts: current status and future prospects M. Kordowska-Wiater Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences in Lublin, Lublin, Poland

Keywords arabitol, bioconversion, biotransformation, waste materials, yeasts. Correspondence Monika Kordowska-Wiater, Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences in Lublin, Skromna 8, 20-704 Lublin, Poland. E-mail: [email protected] 2014/2333: received 21 November 2014, revised 28 February 2015 and accepted 12 March 2015 doi:10.1111/jam.12807

Summary Arabitol belongs to the pentitol family and is used in the food industry as a sweetener and in the production of human therapeutics as an anticariogenic agent and an adipose tissue reducer. It can also be utilized as a substrate for chemical products such as arabinoic and xylonic acids, propylene, ethylene glycol, xylitol and others. It is included on the list of 12 building block C3-C6 compounds, designated for further biotechnological research. This polyol can be produced by yeasts in the processes of bioconversion or biotransformation of waste materials from agriculture, the forest industry (L-arabinose, glucose) and the biodiesel industry (glycerol). The present review discusses research on native yeasts from the genera Candida, Pichia, Debaryomyces and Zygosaccharomyces as well as genetically modified strains of Saccharomyces cerevisiae which are able to utilize biomass hydrolysates to effectively produce L- or D-arabitol. The metabolic pathways of these yeasts leading from sugars and glycerol to arabitol are presented. Although the number of reports concerning microbial production of arabitol is rather limited, the research on this topic has been growing for the last several years, with researchers looking for new micro-organisms, substrates and technologies.

Introduction The polyol arabitol (arabinitol) can be obtained in two forms, as L-arabitol or D-arabitol. This sugar alcohol with a molecular weight of 152 (both forms) belongs to the pentitol family together with xylitol and ribitol. It is sweet, colourless, crystalline and soluble in water. Arabitol, together with other pentitols, is used in the food and human therapeutics industries. Having a sweetness similar to sucrose and a much lower calorie content (due to their slow absorption or lack of absorption by the human digestive tract), sugar alcohols can be used as low-calorie sweeteners (e.g. arabitol has a calorie content of only 0
2 kcal g 1). Sugar alcohol sweeteners have the additional benefit of not sustaining oral bacteria growth, which means that their consumption, unlike that of sugars, does not lead to dental cavities. L-arabitol has been shown to be able to significantly reduce the adipose tissue in the body and prevent the deposition of fat in the digestive tract. Its efficacy is similar to that of soluble

dietary fibres (Mingguo et al. 2011; Kumdam et al. 2013). D-arabitol can be used as a substrate for some chemical products, e.g. arabinoic and xylonic acids, propylene, ethylene glycol, xylitol, enantiopure compounds, immunosuppressive glycolipids, herbicides and anti-pathogenic medicines (Koganti et al. 2011; Koganti and Ju 2013; Yoshikawa et al. 2014; Zhang et al. 2014). Polyols such as xylitol and arabitol are on the list of 12 building block chemicals (C3-C6 compounds) derived directly from the sugars in biomass, that have been earmarked as top targets for further research and development within industrial biotechnology (Erickson et al. 2012). Currently, arabitol is produced on an industrial scale by the chemical reduction in lactones of arabinonic and lyxonic acids, a reaction that requires an expensive catalyst and a constant temperature of 100°C (Kumdam et al. 2013). Moreover, extensive separation steps have to be followed in this process to remove the by-products of the reaction (Zhang et al. 2014). Instead of chemical production, however, arabitol can be synthesized using a

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biotechnological method known as bioconversion or biotransformation, which is believed to be environmentally friendly and more cost-effective, as it does not require the use of pure sugar substrates and chemical catalysts. Bioconversion involves the application of live microbial cells, which carry out multiple enzyme-catalysed reactions. The products obtained from such reactions are usually much easier to remove by down-stream separation (Zhang et al. 2014). There are some waste substrates that can be used for arabitol production by micro-organisms. The main substrates for L-arabitol production are hemicelluloses obtained from renewable lignocellulosic feedstocks. Hemicelluloses are especially valuable because of their complex structure containing hexoses (glucose, mannose) and pentoses (xylose, arabinose). The relative proportion of the individual sugars depends on the raw material used; for instance, hardwoods and agricultural materials are rich in pentose sugars, which can be utilized as substrates in biotechnological production of polyols by pentose-assimilating yeasts (Hahn-H€agerdal et al. 2007). There are some native yeast strains possessing the ability to biotransform L-arabinose to L-arabitol, as well as genetically modified strains of Saccharomyces cerevisiae endowed with a new favourable trait allowing them to assimilate L-arabinose. However, microbial production of L-arabitol from a mixture of hemicellulosic sugars entails high costs of preparing cultivation media (e.g. pretreatment of lignocellulosic materials, hydrolysis and detoxication) and purification of the final product from the mixture of polyols. The best starting material for the production of D-arabitol is glucose, a monosaccharide that can be cheaply obtained due to its abundance in nature. Glucose is found in plant material in many parts of the world and is a rich source of carbon produced enzymatically from starch, sucrose, cellulose and hemicelluloses. There are some native yeasts capable of transforming glucose to Darabitol (Zakaria 2001; Saha et al. 2007; Zhu et al. 2010). Another waste substrate that has been proposed for the production of arabitol is glycerol. Persistent and high global glycerol stocks are the result of the large quantities of crude glycerol produced in the manufacture of soap, biodiesel and in other oleochemical industries (Nicol et al. 2012). Especially, after the prices of this compound had plummeted as a consequence of the recent rapid development of the diesel industry, glycerol started to represent a potentially inexpensive substrate that could lower the costs of arabitol production. Biotechnological application of glycerol seems to be an interesting option. There are some reports on the bioconversion of glycerol to D-arabitol by selected yeasts (Koganti and Ju 2013; Yoshikawa et al. 2014). Yeast strains play an important role in the production of arabitol by bioconversion. This review presents native and engineered yeasts able to utilize waste substrates to 304

produce L or D-arabitol. The metabolism, effectiveness and perspectives for research and industrial application of these micro-organisms are discussed. L-arabitol

production by yeasts

Yeasts produce polyhydroxy alcohols as an integral part of their normal growth process. However, the production and yields of polyols are influenced by growth conditions. The most efficient native yeast strains able to produce Larabitol from L-arabinose are briefly presented in Table 1 in the chronological order of their first published application in this process. The first investigations concerning Larabitol-producing yeasts were reported in the 1970s, during studies on the treatment and application of lignocellulosic materials. Barnett (1976) found that among the 400 yeasts included in his study, about 100 of the strains grew on L-arabinose aerobically. Candida tropicalis and Pachysolen tannophilus were the first species reported to be able to utilize L-arabinose under aerobic and fermentative conditions and produce arabitol aerobically at concentrations of 2
7% (w/v) and 1
8% (w/v), respectively, from 5% (w/v) of this pentose (Gong et al. 1983). Later investigations showed that Pichia stipitis and Candida shehatae also assimilated L-arabinose under microaerobic conditions (Du Preez et al. 1986; Delgenes et al. 1989). A more detailed study was performed by McMillan and Boynton (1994), who showed that L-arabinose was metabolized by the yeasts C. shehatae, C. tropicalis, P. stipitis, P. tannophilus and Torulopsis sonorensis to arabitol as the main product. The best producer of arabitol was C. tropicalis cultured in a medium containing a yeast nitrogen base and arabinose at pH 4
5 (Table 1). Those authors also observed that all of the investigated yeasts assimilated sugars (glucose, xylose and arabinose) in the mixture sequentially, and arabinose was the last sugar to be assimilated, suggesting that the metabolic requirement to produce assimilatory pathway enzymes is greater for the assimilation of L-arabinose than for D-xylose (McMillan and Boynton 1994). Saha and Bothast (1996) examined 49 L-arabinose-utilizing yeasts from the genera Candida, Pichia, Debaryomyces, Kluyveromyces and Saccharomycopsis. Among them, they selected for further study Candida entomaea NRRL Y-7785 and Pichia guilliermondii NRRL Y-2075 on the basis of their superior rates of substrate conversion to L-arabitol (Table 1). The selected yeasts were cultivated in CCY medium supplemented with 50 g l 1 of L-arabinose at pH 5
6 and 4
6, respectively, at different temperatures (22–40°C) and a rotation speed of 200 rev min 1. The effect of the initial pH of the medium was also investigated. It was found that C. entomaea, at pH 5, gave a yield of 0
7 g g 1 and P. guilliermondii, at pH 4,

Journal of Applied Microbiology 119, 303--314 © 2015 The Society for Applied Microbiology

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Microbial production of arabitol

Table 1 Yeast strains able to produce L-arabitol from L-arabinose Strain

Strain source

Yield (g g 1)

Conditions

Candida tropicalis NRRL Y-11860 Pichia stipitis NRRL Y-7124 Candida entomaea NRRL Y-7785 Pichia guilliermondii NRRL Y-2075 Candida auringiensis NRRL Y-11848 Candida succiphila NRRL Y-11998 Debaryomyces hansenii P. guilliermondii PYCC 3012 P. guilliermondii DSM 70052 Candida parapsilosis DSM 70125 Candida shehatae ATCC 22984 Candida sp. NY7122

Culture collection

1
02

29°C; 200 rev min

Culture collection

0
57

Culture collection

Rhodotorula mucilaginosa PTD3 Debaryomyces nepalensis NCYC 3413

By-products

References

1

nd

McMillan and Boynton (1994)

29°C; 200 rev min

1

nd

McMillan and Boynton (1994)

0
66

34°C; 200 rev min

1

nd

Saha and Bothast (1996)

Culture collection

0
63

34°C; 200 rev min

1

nd

Saha and Bothast (1996)

Culture collection

0
73

25°C; 140 rev min

1

nd

Dien et al. (1996)

Culture collection

0
81

25°C; 140 rev min

1

Ethanol

Dien et al. (1996)

Sugar cane Culture collection

0
1 0
47

30°C; 150 rev min 30°C; 200 rev min

1

Xylitol Xylitol

Girio et al. (2000) Fonseca et al. (2007a)

Culture collection

0
43

28°C; 150 rev min

1

nd

Kordowska-Wiater et al. (2008)

Culture collection

0
78

28°C; 150 rev min

1

nd

Kordowska-Wiater et al. (2008)

Culture collection

0
5

28°C; 150 rev min

1

nd

Kordowska-Wiater et al. (2008)

Soil

0
53

37°C; 120 rev min

1

Watanabe et al. (2012)

Stems

0
2

30°C; 175 rev min

1

Ethanol, glycerol nd

Bura et al. (2012)

Rotten apple

0
26

30°C; 180 rev min

1

Ethanol

Kumdam et al. (2013)

1

nd, not detected.

produced L-arabitol at a yield of 0
71 g g 1 in the above medium. The two species were cultivated in mixed sugars (glucose, D-xylose, L-arabinose) and they utilized them separately and sequentially and were not able to produce ethanol from xylose and arabinose. The researchers concluded that both yeasts were promising candidates for the production of L-arabitol from L-arabinose and L-arabinose-rich hemicellulosic materials such as corn fibre hydrolysate (Saha and Bothast 1996). Dien et al. (1996) carried out a screening of 116 yeast strains from the ARS Culture Collection (NRRL), mainly Candida (50 strains), Pichia (43 strains), Debaryomyces (9) and Ambrosiozyma (5), for L-arabinose fermentation to ethanol, but concentrations of arabitol were also detected. One hundred and one strains converted arabinose to arabitol at 30°C, and the most efficient of them was Candida auringiensis, which produced 11–37 g l 1 of arabitol using 32–75% of the initial 100 g l 1 of the sugar. Candida succiphila produced about 8 g l 1 of arabitol from 32 to 35% of the pentose used. During cultivation in optimal conditions (Table 1), C. auringiensis NRRL Y-11848 produced 73 g l 1 of L-arabitol using up all the sugar in the medium and C. succiphila produced

81 g l 1 of the pentitol and 3
9 g l 1 of ethanol in batch cultures (Dien et al. 1996). Girio et al. (2000) studied the ability of Debaryomyces hansenii isolated from sugar cane to assimilate pentoses and hexoses from hemicelluloses. In their experiments, the maximal concentration of arabitol produced from 1 L-arabinose was only 2
86 g l per 35 g l 1 of the substrate after 120 h of cultivation, so the determined yield was also low. This yeast consumed arabinose in a mixture containing a low concentration of glucose, simultaneously producing mainly biomass, but arabitol started to accumulate before glucose depletion, giving a yield of 0
19 g g 1. In a mixture of arabinose with a small amount of xylose, arabitol was the main product, giving a yield of 1
05 g g 1 sugar consumed (Girio et al. 2000). Fonseca et al. (2007a) investigated two yeasts able to metabolize L-arabinose: Candida arabinofermentans PYCC 5603T and P. guilliermondii PYCC 3012. They observed that P. guilliermondii secreted considerable amounts of arabitol during cultivation in an L-arabinose containing medium under oxygen limited conditions. Yields, in the range of 0
21–0
58 g g 1, depended on the concentration of the pentose in the broth. On the other hand,

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C. arabinofermentans produced arabitol only in a medium containing a high concentration of the carbon source. Those researchers proved that an increased accumulation of arabitol was the major consequence of oxygen limitation during the experiment with variable oxygenation conditions (Fonseca et al. 2007a). Kordowska-Wiater et al. (2008) reported that among the six strains of yeasts able to produce arabitol from L-arabinose, Candida parapsilosis was the most efficient one, giving 10–14 g l 1 from 20 g l 1 of L-arabinose in different cultivation conditions with a yield of 0
51– 0
78 g g 1. Quite effective were also C. shehatae ATCC 22984 and P. guilliermondii DSM 70052, which secreted 8
57 and 6
41 g l 1 of arabitol respectively (Table 1). These authors confirmed the influence of conditions such as aeration and temperature on the efficiency of bioconversion (Kordowska-Wiater et al. 2008). More precisely, statistical optimization of L-arabinose biotransformation to arabitol by C. parapsilosis using response surface methodology showed that optimal conditions for obtaining 14
3 g l 1 of arabitol were as follows: temperature 28°C, rotational speed 150 rev min 1 and substrate concentration 32
5 g l 1 (Kordowska-Wiater et al. 2013). Watanabe et al. (2012) screened over 1600 yeast strains and selected one, closely related to Candida subhashii, designated NY7122, which was able to produce L-arabitol and ethanol from L-arabinose. The strain secreted 5
63 and 10
69 g l 1 of L-arabitol from 20 g l 1 of L-arabinose in cultures growing at 30 and 37°C, respectively. When pH of the medium was lowered from 6
2 to 5
0, the concentration of L-arabitol increased. In a medium containing mixed sugars (glucose, D-xylose, L-arabinose), this strain behaved similar to other yeasts, utilizing the sugars sequentially (Watanabe et al. 2012). Kumdam et al. (2013) studied the ability of Debaryomyces nepalensis, an osmotolerant yeast species to produce arabitol and ethanol from hexoses and pentoses in batch

cultures at 30°C and 180 rev min 1. They determined that arabitol was produced from L-arabinose as the sole carbon source at a concentration of 22
7 g l 1 and a yield of 0
26 g g 1. This strain also secreted arabitol during cultivation in a medium containing glucose as the sole carbon source, but at a lower yield 0
09 g g 1. Ethanol was a second product, but only a very small amount of that (2
43 g l 1) was detected in the broth and the yield obtained was 0
03 g g 1 (Kumdam et al. 2013). Bura et al. (2012) investigated the endophytic yeast Rhodotorula mucilaginosa strain PTD3, isolated from the stems of hybrid poplar and observed that it was found to be capable of producing xylitol from xylose; ethanol from glucose, galactose and mannose; and also arabitol from arabinose in batch cultures. This yeast produced arabitol from L-arabinose slowly with a yield of 0
2 g g 1, which represented 29% of the theoretical yield after 100 h of incubation. In mixed sugar fermentation, a sequential pattern of utilization was observed (Bura et al. 2012). Glucose fermentation for D-arabitol production A number of osmophilic or osmotolerant yeasts such as Zygosaccharomyces rouxii, D. hansenii, Candida albicans, Candida pelliculosa, Candida famata and Pichia miso can produce D-arabitol from glucose, but usually the incubation time is too long and yields too small to be industrially acceptable (Saha et al. 2007). Selected yeasts producing D-arabitol from glucose are presented in Table 2 in the chronological order of their reported use in bioconversion. One of the first such reports was presented in 1971; by Kiyomoto in the US patent for D-arabitol production which described a process in which Pichia ohmeri produced 43 g l 1 of D-arabitol in 96 h with a yield 0
43 g g 1 and a productivity 0
448 g (l 9 h) 1. Escalante et al. (1990) reported their results on arabitol production from glucose by Hansenula polymorpha DSM

Table 2 Yeast strains able to produce D-arabitol from glucose Strain

Strain source

Yield [g g 1]

Conditions

Other products

References

Pichia ohmeri No. 230 Hansenula polymorpha DSM 70277 Metschnikowia reukaufii AJ14787 Zygosaccharomyces rouxii NRRL Y-27624 Kodamaea ohmeri NH-9

Culture collection Culture collection

0
43 0
14

30°C; 125 rev min 1 45°C, 1000 rev min 1

– nd

Kiyomoto (1971) Escalante et al. (1990)

Natural source

0
52

33°C; 350 rev min

1

nd

Nozaki et al. (2003)

Honeybee hive

0
48

30°C; 350 rev min

1

Saha et al. (2007)

Honeybee hive

0
41

37°C; 220 rev min

1

Debaryomyces nepalensis NCYC 3413 Pichia anomala TIB-x229

Rotten apple

0
02

30°C; 180 rev min

1

Glycerol, ethanol Glycerol, ethanol Ethanol

Natural environ

0
22

30°C; 250 rev min

1

Ribitol

Zhu et al. (2010) Kumar and Gummadi (2011) Zhang et al. (2014)

nd, not detected.

306

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M. Kordowska-Wiater

70277. This strain produced 18
8 g l 1 of the polyol after 60 h of fed-batch culture at 45°C, pH 4
8 and a stirring rate 1000 rev min 1. Zakaria (2001) reported that Candida famata R28 grew very well in a medium containing D-glucose as the sole carbon source and was able to produce D-arabitol as the sole product of metabolism, but he only performed a qualitative analysis of the product. Another yeast potentially able to produce D-arabitol is Metschnikowia reukaufii reported by Nozaki et al. (2003). This yeast, isolated from a natural source, could efficiently secrete arabitol synthesized from glucose; under optimal conditions, 206 g l 1 of the polyol was produced in 100 h. This strain was also able to produce D-arabitol from other types of sugars. Saha et al. (2007) isolated yeasts from honeybee hives and selected a strain, identified as Z. rouxii, which produced 83
4  1
1 g D-arabitol from 175  1
1 g glucose per litre in 240 h in a batch culture using 5% inoculum. The researchers optimized the process using the one variable at a time approach and found the optimal temperature and pH to be 30°C and 5
0, and the optimal agitation speed to be in the range of 300–450 rev min 1. Besides glucose, the investigated strain could assimilate a variety of monosaccharides, e.g. fructose, galactose, mannose and xylose, and to produce D-arabitol, but it did not utilize L-arabinose (Saha et al. 2007). Zhu et al. (2010) also isolated yeast strains from honeybee-hive and flower samples collected in China and selected 18 strains that could produce more than 10 g l 1 of D-arabitol from glucose. Of these, three strains produced over 60 g l 1 of D-arabitol. The most efficient was a strain genetically identified as Kodamaea ohmeri NH-9, which produced 62
9  0
57 g l 1 from 200 g l 1 glucose. The authors found that under the optimal conditions of 37°C, 220 rev min 1 and pH 6
5 in YGA medium the concentration of D-arabitol increased to 81
2  0
67 g l 1 after 72 h of cultivation and the volumetric productivity was 1
128 g (l 9 h) 1. Repeatedbatch fermentation during 10 cycles improved the efficiency only negligibly to 82
8 g l 1 of D-arabitol and the productivity to 1
38 g (l 9 h) 1, but cultivation time was shortened to 60 h (Zhu et al. 2010). Kumar and Gummadi (2011) isolated a yeast strain, genetically identified as Debaryomyces nepalensis NCYC 3413, which produced arabitol from glucose as the sole carbon source. In their study, arabitol, together with glycerol, was a by-product of ethanolic fermentation. It was produced at the concentrations of 6
3 and 5
0 g l 1 in cultures containing initial concentrations of 400 and 200 g l 1 of glucose, respectively, hence, the yields were rather poor (Kumar and Gummadi 2011). In the latest report, Zhang et al. (2014) described a recently isolated yeast Pichia anomala TIB-x229 (CGMCC

Microbial production of arabitol

No. 5482), which was able to convert glucose to D-arabitol as the main product and ribitol as a by-product in a flask culture. It secreted about 22
5 g l 1 of D-arabitol from 100 g l 1 of glucose after 30 h of incubation and 39 g l 1 of this polyol from 180 g l 1 of glucose after 70 h, giving a yield of about 0
22 g g 1 (Zhang et al. 2014). Glycerol fermentations for D-arabitol production Studies on the use of glycerol as a substrate for arabitol production have been conducted for the last 5 years as a response to increasing biodiesel production. Koganti et al. (2011) screened 214 yeast strains, mainly from the genera Debaryomyces, Geotrichum, Metchnikowia, Candida and Dipodascus, among which, Debaryomyces and Metchnikowia were found to have the largest numbers of strains which produced noticeable amounts of arabitol and very low quantities of other polyols from glycerol after 3 days of batch cultivation. The most valuable strain was D. hansenii NRRL Y-7483, which consumed glycerol and produced arabitol with a yield of about 20% under optimized dissolved oxygen conditions. Those researchers reported that the optimal temperature of cultivation was 30°C and the optimal glycerol concentration was over 90 g l 1. The yield was the highest (50%) when the initial concentration of glycerol was 150 g l 1, but its consumption by the yeast decreased under these conditions, and only 30 g l 1 of the substrate was utilized. (Koganti et al. 2011). After another series of optimization experiments, the researchers found that D. hansenii NRRL Y-7483 could produce arabitol in a bioreactor with a yield of 0
55 g g 1 in 5 days. The efficacy of the process was affected by the N/P ratio in the medium, the level of dissolved oxygen (DO), and pH. The optimal parameters were established as follows: N/P ratio 9, DO value 5%, and pH 3
5, but it was observed that it was beneficial to start the bioconversion from a high DO value (about 100%) and a neutral pH (6
7) and allow the two parameters to drop naturally to the control values. Under the optimized conditions and medium composition, arabitol was produced from glycerol with 0
33 g (l 9 h) 1 volumetric productivity and 0
02 g (g 9 h) 1 specific productivity, which was two to three times higher than in flask cultures (Koganti and Ju 2013). Yoshikawa et al. (2014) screened 10 strains from the genera Candida, Debaryomyces, Metchnikowia and Hansenula coming from the NBRC collection. Among them, four strains produced D-arabitol in the range of 9
75– 41
7 g l 1from 300 g l 1 of raw glycerol (Table 3), and the best producer was Candida quercitrusa NBRC 1022. The researchers also isolated 2300 microbial strains from environmental samples, among which 110 were predicted to produce D-arabitol from raw glycerol. The most

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Table 3 Yeast strains producing D-arabitol from glycerol Concentration (g l 1) or yield (g g 1)

Other products

References

1

nd

Koganti et al. (2011)

30°C; 400 rev min

1

nd

Koganti and Ju (2013)

28°C; 150 rev min

1

nd

Yoshikawa et al. (2014)

28°C; 150 rev min 28°C; 150 rev min

1

nd nd

Yoshikawa et al. (2014) Yoshikawa et al. (2014)

1

28°C; 150 rev min

1

nd

Yoshikawa et al. (2014)

1

28°C; 150 rev min

1

nd

Yoshikawa et al. (2014)

Strain

Strain source

Debaryomyces hansenii NRRL Y-7483 Debaryomyces hansenii NRRL Y-7483 Candida quercitrusa NBRC 1022 Candida quercitrusa 17-2A Debaryomyces hansenii var. fabryi NBRC 0015 D. hansenii var. hansenii NBRC 0083 Hansenula anomala NBRC 10213

Culture collection

0
20 g g

1

30°C; 200 rev min

Culture collection

0
55 g g

1

Culture collection Plant parts Culture collection

41
7 g l 1; 0
41 g g 1 40
7 g l 1 9
75 g l 1

Culture collection

26
5 g l

Culture collection

15
6 g l

Conditions

1

nd, not detected.

efficient strain, designated 17-2A, after a morphological, physiological and molecular analysis, was identified as Candida quercitrusa. Bioreactor experiments showed that C. quercitrusa NBRC 1022 secreted 67
1 g l 1 of D-arabitol in a medium initially containing 250 g l 1 of glycerol, giving a yield of 0
41 g g 1 after 7 days (Yoshikawa et al. 2014). Metabolic pathways of arabitol-producing yeasts Metabolism of L-arabinose to arabitol and other products Sugar transport across the cell membrane is the first step in pentose metabolism. There is very little information about L-arabinose transport in yeasts utilizing natural arabinose. Generally, many yeasts display an active sugar/ proton symport, beside a facilitated diffusion system which shows a lower affinity and an 80–90% higher capacity. The presence of an L-arabinose/proton symporter was reported in the xylose-fermenting yeast C. shehatae (Fonseca et al. 2007b). In the initial step of arabinose metabolism in yeasts, this pentose is converted by L-arabinose reductase (aldose reductase) (AR; EC 1.1.1.21) into the polyol, L-arabitol, which is subsequently oxidized to L-xylulose by L-arabitol-4-dehydrogenase (LAD; EC 1.1.1.12). In the next step, L-xylulose is converted into xylitol by L-xylulose reductase (LXR; EC 1.1.1.10). That pentitol is an intermediate compound in the catabolic pathways of arabinose and xylose. In fungi, L-arabinose and D-xylose reductases prefer NADPH as a cofactor, whereas the polyol dehydrogenases are dependent on NAD. Because under low oxygen conditions regeneration of NAD is limited, arabitol is accumulated. Despite the scanty biochemical data concerning L-arabinose utilization by yeasts, it has been observed that the catabolic pathways of arabinose and xylose partially overlap, 308

because in the first step D-xylose is transformed into xylitol by D-xylose reductase (XR), which is dependent on NADPH (preferentially) or NADH as cofactors. The dual cofactor specificity of aldose reductase (XR) may help some yeast cells to avoid excessive xylitol accumulation under limited aeration. In a further step, xylitol is converted into D-xylulose by the strictly NAD dependent enzyme, xylitol dehydrogenase (XDH; EC 1.1.1.9), and this pentose is shunted into the pentose phosphate pathway (PPP) after it has been phosphorylated by D-xylulose kinase (XK; EC 2.7.1.17). Many types of yeast are known to be able to assimilate L-arabinose as the sole carbon and energy source. They produce biomass aerobically and, under limited aeration, they secret L-arabitol into the medium. Only two species (Candida arabinofermentans and Ambrosiozyma monospora) have been reported to produce trace amounts of ethanol from L-arabinose (Richard et al. 2003; Fonseca et al. 2007a,b). Metabolic pathways for conversion of glucose and glycerol to D-arabitol

Glucose is transported into the cell by two systems: a facilitated diffusion mechanism, based on a glucose gradient, or an active sugar-proton symport, which allows glucose to accumulate in the cell by the proton motive force and is tightly regulated by the concentration of the sugar in the environment (Fonseca et al. 2007b). Which transport system is used depends on the yeast species and its transporters and facilitators. D-arabitol is produced from glucose by two alternative pathways. In the first step, this hexose is phosphorylated to glucose-6-phosphate, which is then converted in PPP. Some organisms can convert glucose-6-phosphate to D-ribulose-5-phosphate, dephosphorylate this latter compound, and then reduce D-ribulose to D-arabitol by NADP-dependent D-arabitol

Journal of Applied Microbiology 119, 303--314 © 2015 The Society for Applied Microbiology

M. Kordowska-Wiater

Microbial production of arabitol

dehydrogenase (EC 1.1.1.11) (D-ribulose forming pathway). Other strains can convert glucose-6-phosphate to D-xylulose-5-phosphate, and after dephosphorylation reduce D-xylulose to D-arabitol by NAD-dependent D-arabitol dehydrogenase (D-xylulose forming pathway) (Saha et al. 2007; Zhang et al. 2014). In yeast cells, arabitol synthesis from glycerol is expected to follow similar routes to those leading from glucose via glucose-6-phosphate. First, glycerol assimilated by simple diffusion or active transport is phosphorylated by glycerol kinase (EC 2.7.1.30) (e.g., in Candida utilis or S. cerevisiae) to glycerol-3–phosphate and then to dihydroxyacetone phosphate (DHAP) by glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). An alternative pathway in yeasts lacking glycerol kinase involves NAD-dependent glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29). Dihydroxyacetone phosphate, formed in the above routes, is converted to glyceraldehyde-3–phosphate and subsequently via the gluconeogenesis pathway to glucose-6-phosphate. This compound is next converted in PPP to arabitol via ribulose-5-phosphate and ribulose (Koganti et al. 2011; Nicol et al. 2012).

Although some trials with yeast fusants have been performed since 1980s, they mainly concentrated on ethanol fermentation from D-xylose or mixed sugars coming from lignocellulosic hydrolysates. Lucca et al. (1999) obtained 13 intergeneric fusants between S. cerevisiae P-158 and Torulaspora delbrueckii CBS 813 able to grow in media containing high concentrations of glucose. Two selected hybrids, PB1 and PB2, were able to produce arabitol, beside ethanol and glycerol. The maximum concentrations of arabitol were 4 and 5 g l 1 for PB1 and PB2, respectively. Unfortunately, they mainly produced ethanol, and glycerol was released intra and extracellularly as the main by-product. One parental strain, T. delbrueckii, accumulated arabitol more efficiently giving about 20 g l 1 of this product. The intra/extracellular arabitol ratio was 3 : 1 in the hybrids and the parental strain. The authors concluded that both arabitol and glycerol were produced as a result of a high osmolarlity of the medium used (Lucca et al. 1999). Consequently, hybrid PB2 was re-examined in media containing 100–800 g l 1 of glucose, and the production of polyols was still observed. In fermentor culture, this hybrid produced 70 g l 1 of arabitol in a medium with 400 g l 1 of glucose with a yield of 0
27 g g 1, but about 120 g l 1 of the sugar remained unassimilated (Table 4). The PB2 hybrid produced more arabitol and glycerol than either parental strain and also showed the highest sugar uptake in aerobic conditions (Lucca et al. 2002).

Genetically modified yeasts for arabitol production Fusant yeasts There are few reports on yeast hybrids obtained by protoplast fusion able to utilize sugars to produce arabitol.

Table 4 Genetically modified yeasts able to produce arabitol

Strain Saccharomyces cerevisiae and T. delbruecki hybrid PB2 S. pombe and L. edodes hybrid S. pombe and L. edodes hybrid S. cerevisiae V30 and Pichia stipitis karyoductant S. cerevisiae AH22 S. cerevisiae BWY02.XA S. cerevisiae TMB 3063 S. cerevisiae TMB 3664 S. cerevisiae TMB 3130 S. cerevisiae 424A(LNH-ST) S. cerevisiae 424A(LNH-ST)pLXRNAD-LAD

Kind of modification

Other products or byproducts

Substrate

Yield (g g 1)

Conditions

Intergeneric fusant

Glucose

0
27

30°C; 500 rev min

Intergeneric fusant Intergeneric fusant Interg. karyoductant

Glucose

0
59

Arabinose

Recombinant Recombinant Recombinant Recombinant Recombinant Recombinant Recombinant

1

References

Glycerol, ethanol

Lucca et al. (2002)

30°C

Ethanol

0
76

30°C

nd

L-arabinose

0
52–0
58

28°C; 150 rev min

1

nd

Cheng-Chang et al. (2005) Cheng-Chang et al. (2005) Kordowska-Wiater et al. (2012)

L-arabinose

0
62 1
14 0
68 0
48 0
95 0
82 0
33

30°C; 30°C; 30°C; 30°C; 30°C; 30°C; 30°C;

1

L-ribulose

1

nd Ethanol nd Acetate nd Ethanol, xylitol

L-arabinose L-arabinose L-arabinose L-arabinose L-arabinose L-arabinose

 0
18  0
17  0
05  0
05  0
003

200 200 200 200 200 200 200

rev rev rev rev rev rev rev

min min min min min min min

1 1 1 1 1

Sedlak and Ho (2001) Karhumaa et al. (2006) Karhumaa et al. (2006) Bettiga et al. (2009) Sanchez et al. (2010) Bera et al. (2010) Bera et al. (2010)

nd, not detected.

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An interesting experiment was carried out by ChengChang et al. (2005), who constructed an intergeneric yeast-like fusant formed from protoplasts of Schizosaccharomyces pombe CCRC 21461 and Lentinula edodes (shiitake mushrooms). During stationary cultivation on glucose, this hybrid produced ethanol, and arabitol as a by-product. The production of arabitol reached 8
56 g l 1 from almost 200 g l 1 of glucose after 9 days of incubation (volumetric productivity 0
11 g (l 9 h) 1). However, arabitol was produced only after the concentration of ethanol reached a plateau. The authors suggested that the production of arabitol might be related to the maintenance of isotonic pressure or the protection of the fusant against autolysis. This hybrid also produced arabitol, as the main product, from xylose at a concentration of 30
24 g l 1 at the end of 8 days’ incubation; low concentrations of xylitol were also present in the reaction mixture during the whole cultivation time. Probably, the enzyme xylitol dehydrogenase was induced to oxidize xylitol to arabitol. This fusant was also able to produce arabitol as the main product from a mixture of xylose/ glucose and from xylan, giving concentrations of 35
12 and 63
78 g l 1, respectively (yields 0
8 and 1
07 g g 1). The S. pombe–L. edodes hybrid also produced arabitol from arabinose as the sole carbon source in a stationary culture with a yield of 0
76 g g 1 (Table 4). The authors concluded that the efficacy of the fusant obtained was satisfactory (Cheng-Chang et al. 2005). Kordowska-Wiater and Targo nski (2001) reported a special kind of fusion between protoplasts of S. cerevisiae V30 and nuclei of P. stipitis CCY 39501, as a result of which they obtained karyoductants able to assimilate Larabinose and secrete arabitol. Seven isolates were obtained, and one of them, named SP-K7, which was capable of producing large quantities of arabitol, was the subject of an optimization study (Kordowska-Wiater and Targo nski 2001; Kordowska-Wiater et al. 2012). The production of arabitol was optimized using the RSM method. Three variables (culture conditions) were tested: the concentration of the pentose, temperature and rotational speed responsible for oxygenation. In a batch culture under optimal conditions (L-arabinose concentration 32
5 g l 1, 28°C and 150 rev min 1), the karyoductant secreted 16
3–18
9 g l 1 of arabitol with a yield of 0
52– 0
58 g g 1, depending on the working volume of the culture, which confirmed the significance of oxygenation in the production process (Kordowska-Wiater et al. 2012). Construction of recombinant yeasts for arabitol production There are only a few reports on the application of engineered S. cerevisiae for utilization of L-arabinose. A 310

summary of the results of those studies is given in Table 4. The experiments were performed using bacterial or fungal genes responsible for L-arabinose metabolism. Sedlak and Ho (2001) constructed S. cerevisiae containing the E. coli araBAD operon using strong promoters from genes encoding the yeast’s glycolytic enzymes. The expression of these cloned genes in the yeast, achieved using shuttle plasmids: pYaraA, pYaraB, pYaraD and pYaraDBA, was demonstrated by the presence of the active enzymes encoded by these cloned genes and by the presence of corresponding mRNAs. Saccharomyces cerevisiae bearing pYaraA with the active gene for L-arabinose isomerase (AI, EC 5.3.1.4) produced L-arabitol in concentrations of 13–15 g l 1 as the main product and L-ribulose as a by-product. Yeasts bearing pYaraDBA with three genes for AI, ribulokinase (EC 2.7.1.16) and L-ribulosephosphate epimerase (EC 5.1.3.4) produced L-arabitol only. The authors concluded that the polyol inhibited the activity of L-arabinose isomerase and that this was one of the reasons why no ethanol was produced from fermentation of L-arabinose. They also confirmed the expression of all studied enzymes in yeast cells (Sedlak and Ho 2001). Together with the increasing knowledge about pentose metabolism in yeast cells, researchers started to construct yeast cells bearing fungal genes of the pentose pathway. Richard et al. (2003), for example, reported obtaining S. cerevisiae H2561 containing all the genes of the L-arabinose pathway (XYL1, lad1, lxr1, XYL2 and XKS1). XYL1, XYL2 and XKS1 of yeast origin were integrated into chromosomes; lad1 and lxr1 of fungal origin were located on two different plasmids. The authors detected activities of all the enzymes of this pathway and confirmed that the construct could grow on L-arabinose and produce very small amounts of ethanol (0
1 g l 1) during 70-h anaerobic cultivation. Unfortunately, they did not analyse the concentration of arabitol, but it might be suspected that this polyol was present in the fermentation broth (Richard et al. 2003). Karhumaa et al. (2006) constructed various recombinants of S. cerevisiae containing bacterial (E. coli, B. subtilis) genes of the L-arabinose pathway and/or yeast (P. stipitis) genes of xylose metabolism, which were able to utilize arabinose and/or xylose. A laboratory strain BWY02.XA, bearing plasmids YIpXR/XDH/XK, YEpURAaraA, YEparaB and YEparaD, slowly consumed xylose and arabinose in anaerobic conditions and secreted arabitol with a yield of about 1 g g 1 of arabinose. This construct grew on arabinose at aerobic conditions and also utilized mixture of 50 g l 1 xylose and 50 g l 1 arabinose simultaneously. These researchers constructed industrial strains S. cerevisiae TMB 3061 and TMB 3063 by transforming PCR fragments containing genes AraA and AraD

Journal of Applied Microbiology 119, 303--314 © 2015 The Society for Applied Microbiology

M. Kordowska-Wiater

flanked by rDNA sequences into strain TMB 3060. Saccharomyces cerevisiae TMB 3060, on the other hand, was obtained by chromosomal integration of plasmid YIpAraB into a xylose-fermenting industrial strain TMB 3400. Unlike the parental strain TMB 3400, the new strains TMB 3061 and TMB 3063 grew on arabinose and were also able to utilize a mixture of arabinose and xylose. Strain TMB 3063 with additional copies of the L-arabinose isomerase gene produced arabitol from arabinose during cultivation in a mixture of glucose, xylose and arabinose, with a yield 0
68  0
17 g g 1 (Table 4). It was also observed that this strain produced ethanol from pentoses (Karhumaa et al. 2006). Sanchez et al. (2010) continued the investigation of the engineered industrial strains of S. cerevisiae obtained by Karhumaa et al. (2006). Those authors used evolutionary engineering to improve mixed-pentose utilization by strain TMB 3061 in continuous culture. The evolved strain TMB 3130 displayed an increased rate of consumption of xylose and arabinose under aerobic and anaerobic conditions and reached the highest final biomass concentration in both xylose- and arabinose-containing medium. It was able to convert L-arabinose to arabitol almost stoichiometrically during the pentose phase of anaerobic co-fermentative culture, which suggested the strain was unable to ferment arabinose to ethanol despite the presence of appropriate genes (Sanchez et al. 2010). Bettiga et al. (2008, 2009) also constructed S. cerevisiae strains able to utilize pentoses, mainly for ethanol production. First, they transformed the starting strain TMB 3042, previously engineered for efficient pentose fermentation, which was characterized by overexpression of PPP and XK and deletion of GRE3, encoding unspecific aldose reductase. They used the bacterial arabinose fermenting pathway (genes AraA, AraB, AraD) to isolate a strain (TMB 3073) able to grow on arabinose. Subsequently, they obtained strain TMB 3075, containing XR/XDH genes from P. stipitis, and strain TMB 3076, with XI gene from Piromyces sp., by transforming strain TMB 3074 using plasmids pY7 and YEplacHXT-XIp, respectively. The two strains exhibited similar aerobic growth in the arabinose-containing medium, but during anaerobic fermentation of glucose, xylose and arabinose, they presented different patterns with regard to arabinose utilization and arabitol production. The transformant bearing genes XR/XDH proved to be more efficient, as it converted almost 90% of arabinose consumed to arabitol giving a concentration of 3
51 g l 1 and a yield of 0
87 g g 1 (Bettiga et al. 2008). In another experiment, Bettiga et al. (2009) transformed strain TMB 3043 with mutated XYL1, XYL2 and XDH genes of fungal origin, all inserted in the integrative plasmid YIpOB9, to obtain a new strain named TMB 3662. In a next step, they

Microbial production of arabitol

introduced into this strain, a multicopy plasmid harbouring synthetic, codon optimized genes sLAD1, sALX1 coding for L-arabitol dehydrogenase and L-xylulose dehydrogenase, respectively. In this way, they obtained strain TMB 3664, encoding a complete L-arabinose pathway, which utilized L-arabinose and D-xylose as sole carbon sources for biomass production and secreted arabitol from consumed 20% of arabinose with a yield of 0
48 g g 1 during anaerobic mixed sugar batch fermentation after xylose depletion. The authors confirmed that this AR expressing strain did not only convert arabinose to arabitol, but channelled it further into the metabolism. Strain TMB 3664 was also examined in a medium containing glucose and L-arabinose during anaerobic fermentation. It was shown that, after glucose depletion, L-arabinose was metabolized to L-arabitol with a yield of 0
39 g g 1, wherein the yield of ethanol was calculated to be 0
42 g g 1 of consumed sugars and 0
35 g g 1 of consumed L-arabinose (Bettiga et al. 2009). In neither of their experiments did Bettiga et al. (2008, 2009) study the ability of the transformants obtained to utilize arabinose as the sole carbon source and produce L-arabitol as the main product of bioconversion. Bera et al. (2010) also obtained a recombinant S. cerevisiae 424A (LNH-ST), containing fungal genes of arabinose metabolism (LAD, LXR) in addition to multiple copies of three xylose-metabolizing genes (XR, XDH, XK), which was able to ferment hemicellulosic sugars. While a control strain slowly assimilated L-arabinose to produce arabitol as the only product, at a yield of 82
5  4
7%, the recombinant, bearing plasmid pLXRNAD-LAD, produced arabitol, ethanol and xylitol from this pentose at metabolic yields of 33
7  0
3%, 42
6  2
3% and 2
2  1
2%, respectively, assimilating 90% of the substrate within 96 h. The consumption of L-arabinose by the 424A (LNH-ST)/pLXRNADP-LAD strain was slightly slower than in the first parental strain, but the amount of arabitol was similar. Moreover, the recombinant 424A (LNH-ST)/pLXRNAD-LAD showed the ability to produce arabitol and ethanol from a mixture of L-arabinose and D-xylose, with yields 35
5  1
0% and 46
0  2
0%, respectively (Bera et al. 2010). The effect of culture conditions on arabitol production The results obtained by the different scientists suggest that there are several factors which limit arabitol production by yeasts in batch cultures, both in shake flasks as well as in bioreactors. Among the environmental factors affecting substrate assimilation and product secretion are incubation temperature, medium composition (the kind of substrate and concentrations of C and N sources) and pH, as well as oxygen supply connected with the working

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volume of culture and the speed of shaker rotation or stirrer agitation. This last factor is key for bioconversion (biotransformation) because it affects the balance of redox cofactors, which in turn determines the kind and concentration of the product secreted into the medium. The basic factors are presented in Tables 1–4 together with the yields reached by the respective yeasts. Data show that optimal temperatures were in the range of 28–45°C and the rotational speed of the shaker or stirrer agitation speed in bioreactor was between 125–250 rev min 1 and 350–1000 rev min 1, respectively. The concentrations of C sources in the media were as follows: 20–100 g l 1 for L-arabinose, 20–600 g l 1 for glucose and 100–350 g l 1 for glycerol; pH was between 3
6–7
0 (mainly 5
5–6
0) depending on the kind of micro-organism studied. In some reports, inoculum doses were also presented. The cultures were inoculated with 2–8% (generally 5%) of cell suspension. Optimization studies were usually conducted by the conventional ‘one variable at a time’ method with only two authors using central composite design (CCD) and response surface methodology (RSM) for statistical optimization of the reaction parameters (Zhu et al. 2010; Kordowska-Wiater et al. 2012, 2013). Zhu et al. (2010) predicted the optimal nutritional conditions and determined, using CCD, the optimal concentration of glucose, yeast extract and (NH4)2SO4 for D-arabitol production by K. ohmerii. Kordowska-Wiater et al. (2012, 2013), in a preliminary study, used the Plackett-Burman design for seven variables to limit the number of factors, selected from among medium components and culture conditions, to those most significant for the process (L-arabinose concentration, temperature and rotational speed of shaker). Next, CCD was used to estimate response surfaces for the three variables affecting arabitol production by the karyoductant SP-K7 and C. parapsilosis. Verification experiments confirmed the usefulness of the model for both yeast strains. Isolation and purification of arabitol from cultivation broth The method of purification and isolation of D- or L-arabitol from cultivation medium depends on the carbon source used for their production. Arabitol present in fermentation broth has to be purified because it can only be crystallized from a solution when its purity exceeds 65%. When L-arabitol is the sole product of yeast metabolism, downstream processes are relatively simple, but when a mixture of sugar alcohols is determined in the medium, purification is necessary. Mingguo et al. (2011) suggested the use, for this purpose, of bacteria from the genus Bacillus able to consume xylitol, sorbitol, mannitol, but not L-arabitol. According to this method, fermentation 312

broth should be inoculated with selected bacteria, cultivated under optimal conditions and then centrifuged to remove micro-organisms. The supernatant should be decolorized by shaking with activated carbon at 80°C and applied to an ion-exchange resin column to remove metal ions; this last step is verified by a conductivity of