Appl Microbiol Biotechnol (2014) 98:823–830 DOI 10.1007/s00253-013-5376-x

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Selective production of two diastereomers of disaccharide sugar alcohol, mannosylerythritol by Pseudozyma yeasts Jun Yoshikawa & Tomotake Morita & Tokuma Fukuoka & Masaaki Konishi & Tomohiro Imura & Koji Kakugawa & Dai Kitamoto

Received: 2 October 2013 / Revised: 30 October 2013 / Accepted: 6 November 2013 / Published online: 23 November 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Mannosylerythritol (ME) is the hydrophilic backbone of mannosylerythritol lipids as the most promising biosurfactants produced by different Pseudozyma yeasts, and has been receiving attention as a new sugar alcohol. Different Pseudozyma yeasts were examined for the sugar alcohol production using glucose as the sole carbon source. P. hubeiensis KM-59 highly produced a conventional type of ME, i.e., 4-O-β-D -mannopyranosyl-D -erythritol (4-ME). Interestingly, P. tsukubaensis KM-160 produced a diastereomer of 4-ME, i.e., 1-O-β-D -mannopyranosyl-D -erythritol (1-ME). In shake flask culture with 200 g/l of glucose, strain KM-59 produced 4-ME at a yield of 33.2 g/l (2.2 g/l/day of the productivity), while strain KM-160 produced 1-ME at 30.0 g/l (2.0 g/l/day). Moreover, the two strains were found to produce ME from glycerol; the maximum yields of 4-ME and 1-ME from 200 g/l of glycerol were 16.1 g/l (1.1 g/l/day) and 15.8 g/l (1.1 g/l/day), respectively. The production of 1ME as the new diastereomer was further investigated in fed batch culture using a 5-l jar-fermenter. Compared to the flask culture, strain KM-160 gave three times higher productivity of 1-ME at 38.0 g/l (6.3 g/l/day) from glucose and at 31.1 g/l (3.5 g/l/day) from glycerol, respectively. This is the first report on the selective production of two diastereomers of ME, and should thus facilitate the functional development and J. Yoshikawa : T. Morita : T. Fukuoka : M. Konishi : T. Imura : D. Kitamoto (*) Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan e-mail: [email protected] K. Kakugawa Faculty of Applied Information Science, Hiroshima Institute of Technology, Miyake 2-1-1Saeki Hiroshima 731-5193, Japan

application of the disaccharide sugar alcohol in the food and relative industries. Keywords Sugar alcohol . Mannosylerythritol . Pseudozyma hubeiensis . Pseudozyma tsukubaensis

Introduction Sugar alcohols have attracted considerable interest for the food and pharmaceutical industries. They are generally available for an alternative sweetener of sucrose, a pharmaceutical excipient, and a sugarless coating material due to their low calorie and non-cariogenicity (Dills 1989; Ohmori et al. 2004; van Loveren 2004). The production of monosaccharide sugar alcohol such as sorbitol, arabitol and xylitol has been widely studied, and some of them including optical isomers are provided with microbial and enzymatic reactions (Granström et al. 2004). Carbohydrate often expresses excellent functions by oligomerization. For example, trehalose consists of two D glucose units with a α-1, 1 linkage, and is able to provide much different properties compared with D -glucose (Richards et al. 2002). Thus, oligosaccharide sugar alcohols are supposed to show more attractive properties compared with monosaccharide ones. Candida sp. KSM-1529 was reported to produce a disaccharide sugar alcohol, mannosylerythritol (ME), and the structure was identified as 4-O-β-D -mannopyranosyl-D -erythritol (4-ME; Fig. 1a) (Kobayashi et al. 1987). Erythritol is a commercially produced sugar alcohol with about 60 % to 70 % sweetness of sucrose, and used in various fields such as the low-calorie foods and sugarless confectioneries. It also shows a high negative heat of solution, causing a strong cooling

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a

Materials and methods

CH2OH OH OH O

H

OH

H

OH

HO HO

O

b

CH2

CH3

H3C

O

OR1 O O

O R2 O

CH2OH

n n

O

Microorganisms

H

OH

H

OH

O

CH2

n = 6 – 10 MEL-A : R1 = R2 = Ac MEL-B : R1 = Ac, R2 = H MEL-C : R1 = H, R2 = Ac Fig. 1 Chemical structures of mannosylerythritol (a ) and mannosylerythritol lipids (b)

P. aphidis JCM 10318, P. antarctica JCM 10317, P. antarctica JCM 3941, P. floculosa JCM 10321, P. fusiformata JCM 3931, P. parantarctica JCM 11752, P. prolifica JCM 10319, P. rugulosa JCM 10323 and P. tsukubaensis JCM 10324 were obtained from Japan Collection of Microorganisms (JCM; Saitama, Japan). P. hubeiensis CBS 10079 and P. shanxiensis CBS 10075 were obtained from Centraal bureau voor Schimmelculues (CBS; Utrecht, Netherlands). P. antarctica T-34, P. hubeiensis KM-59 (Konishi et al. 2008) and Pseudzyma sp. KM-160 (Konishi et al. 2007) are our laboratory stocks. Each strain was maintained on YM agar medium (1 % glucose, 0.5 % peptone, 0.3 % yeast extract, 0.3 % malt extract, and 1.5 % agar). P. hubeiensis KM-59 and Pseudzyma sp. KM-160 have been deposited in the National Institute of Technology and Evaluation (NITE), Japan, as FERM P-20987 and FERM P-21481. Screening for microorganisms producing ME

effect like mint (Moon et al. 2010). ME may have unique physicochemical and biochemical properties via the combination of erythritol and mannose. It is thus of interest to apply ME as a new sugar alcohol in the food and related fields. However, except for the above study, there have been no reports on the production and/or characterization of ME. On the other hand, ME is known as the hydrophilic backbone of mannosylerythritol lipids (MELs, Fig. 1b) as the most promising biosurfactants, which exhibit not only excellent surface-active properties but also versatile biochemical actions including skin and hair care effects (Kitamoto et al. 2009; Morita et al. 2009). MELs are efficiently produced from vegetable oils by different Pseudozyma yeasts, and now commercially available from TOYOBO Co., Ltd. (Osaka, Japan) (Morita et al. 2013). Accordingly, Pseudozyma yeasts should be the most suitable candidate for a new ME producer. Based on our previous studies, P. antarctica mainly produces MEL-A, 4-O -β-(2′, 3′-di-O -alka(e)noyl-6′-O -acetylD -mannopyranosyl)- D -erythritol (Fig. 1b), while P. tsukubaensis gives MEL-B having the opposite erythritol configuration, 1-O-β-(2′, 3′-di-O-alka(e)noyl-6′-O-acetyl-D mannopyranosyl)-D -erythritol (Fukuoka et al. 2008). This means that the two diastereomers of ME, that is, 4-ME and 1-O -β-D -mannopyranosyl-D -erythritol (1-ME), would be independently prepared using the above strains. We thus focused our attention on the feasible use of Pseudozyma yeasts, and examined their ability to produce ME using glucose instead of vegetable oils. Here we described for the first time the fermentative production of the disaccharide sugar alcohol using glucose as well as glycerol as the sole carbon source.

The agar culture of the above strains was inoculated in a test tube containing 2 ml of a screening medium (10 % glucose, 0.1 % yeast extract, 0.3 % NaNO3, 0.025 % KH2PO4, and 0.025 % MgSO4) at 25 °C on a reciprocal shaker (180 strokes min−1) for 7 days. ME production was checked by paper chromatography (PPC) as follows. Paper chromatography Sugar alcohols in the culture (10 μl) were analyzed by PPC with 1-propanol/ethyl acetate/water (7:1:2, by vol.) as a solvent system. Visualization was performed by Yoda’s reagent; 5 g/l of KIO4 and 150 g/l of MnSO4/p , p ’-tetramethyl diaminodiphenylmethane saturated by 2 N acetate (1:1, v/v) (Yoda 1953). A standard of ME (4-ME) was prepared from MEL-A produced by P. antarctica T-34 (Fukuoka et al. 2008). Purification of mannosylerythritol The culture (1 ml) was applied to the activated charcoal column (2.4×7.5 cm). Monosaccharide fraction including glucose and erythritol was eluted with 100 ml of H2O at 100 ml/h, and then ME fraction was eluted with 100 ml of 1 % (v/v) ethanol at 100 ml/h. Characterization of mannosylerythritol The chemical structure of the purified ME dissolved in D2O was identified by 1H and 13C nuclear magnetic resonance

Appl Microbiol Biotechnol (2014) 98:823–830

825

(NMR) analysis using Varian INOVA 400 (400 MHz; Varian, California, USA). Specific rotation, [α]D value, of the purified ME was measured by Digital Polarimeter DIP-370 (JASCO, Tokyo, Japan) in aqueous solution.

Morphological and physiological characterization of yeast strains Morphological and physiological characterization was carried out according to the method of Yarrow described in The Yeasts (4th edition, 1998). Cell morphology was examined by microscopy (BX51, Olympus, Japan). The maximum growth temperature was determined in YM broth using metal black broth. The utilization of various carbon sources and

rRNA gene sequencing and molecular phylogenic analysis The genomic DNA of each microorganism was prepared by genomic DNA isolation kit (GenTLE; TAKARA, Japan) after cell destruction by freezing with liquid nitrogen. The D1/D2 domains and ITS1-5.8S-ITS2 regions of the rRNA gene, were sequenced directly from PCR products generated using the primer NL1 (5′-GCATAT CAATAA GCG GAG GAA AAG3′) and NL4 (5′-GGT CCG TGT TTC AAG ACG G-3′) (Begerow et al. 2000), and ITS1 (5′-GAT GAA AAC CTT TTT TCT GAG-3′) and ITS5 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (White et al. 1990), respectively. All of DNA sequences were determined with ABI PRISM 3130x1 Genetic

Erythritol

Glucose

P. aphidis JCM 10318

P. flocculosa JCM 10321

P. fusiformata JCM 3931

P. hubeiensis CBS 10079

P. rugulosa JCM 10323

P. shanxiensis CBS 10075

P. tsukubaensis JCM 10324

Pseudozyma sp. KM-160

P. antarctica JCM 3941 P. hubeiensis KM-59

P. antarctica T-34

Mannosylerythritol (10 g l-1) Mannosylerythritol (10 g l-1)

P. prolifica JCM 10319

Glucose (20 g l-1) Glucose (20 g l-1)

P. antarctica JCM 10317

Erythritol (10 g l-1)

Mannosylerythritol

Erythritol (10 g l-1)

Erythritol

Glucose Mannosylerythritol

P. parantarctica JCM 11752

Fig. 2 Detection of mannosylerythritol produced by the yeast strains of the genus Pseudozyma. The sugar alcohols produced by the Pseudozyma strains were detected by the paper chromatography (PPC). The spots were visualized by spraying 150 g/l of MnSO4/p,p’tetramethyl diaminodiphenylmethane saturated by 2 N acetate (1:1, v/v) after spraying 5 g/l of KIO4

other physiological characteristics were determined with YM plate.

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Analyzer System (Applied Biosystems, California, USA). The D1/D2 and ITS sequences of related taxa were retrieved from GenBank. The BLAST program (Altschul et al. 1997) was used for similarity search in database available on the DDBJ website (http://www.ddbj.nig.ac.jp/). Phylogenetic analysis was performed using the neighbor-joining method with the program MEGA5 (http://www.megasoftware.net/ index.php). The sequences have been deposited with DDBJ (DNA Data Bank of Japan) under the accession number: AB8855769 and AB855770. Production of 4-ME and 1-ME by P. hubeiensis KM-59 and Pseudozyma sp. KM-160 The strains were inoculated in a test tube containing 3 ml of the medium (10 % glucose, 0.1 % yeast extract, 0.3 % NaNO3, 0.025 % KH2PO4, 0.025 % MgSO4) at 25 °C on a reciprocal shaker (180 strokes/min) for 1 day. The seed-culture of the strain KM-59 (2 ml) was inoculated in a 300-ml Erlenmeyer flask containing 30 ml of the medium (10 % or 20 % carbon source, 0.2 % yeast extract, 0.3 % NaNO3, 0.025 % KH2PO4 and 0.025 % MgSO4, pH 6.0) at 25 °C on a rotary shaker at 200 rpm. The seed culture of the strain KM-160 (2 ml) was inoculated in a 300-ml Erlenmeyer flask containing 30 ml of the medium (10 % or 20 % carbon source, 0.6 % yeast extract, 0.3 % NaNO3, 0.025 % KH2PO4, 0.025 % MgSO4, pH 5.5) at 25 °C on a rotary shaker at 200 rpm. The culture of the strain KM-160 (90 ml) was transferred in 1.5 l of the medium in a 5-l jar fermenter at 600 rpm at 25 °C, and a concentrated feed medium (50 % glucose or glycerol, Fig. 3 Phylogenetic relationship of Pseudozyma sp.KM-160 and the species of the genus Pseudozyma and Ustilago, based on the ITS regions. The DDBJ/ GenBank/EMBL accession numbers are indicated in parentheses

3.0 % yeast extract, 1.5 % NaNO3, 0.125 % KH2PO4, 0.125 % MgSO4) was added prior to the short of carbon source. Quantification of ME by high-performance liquid chromatography The quantification of produced ME was performed by highperformance liquid chromatography (HPLC) on a SH1011 column (8 mm×30 cm; Showa Denko, Tokyo, Japan) at 60 °C with a refractive index detector RI-8020 (Tosoh, Tokyo, Japan) using 10 mM H2SO4 as the solvent system at a flow rate of 1 ml/min. The quantification of ME was carried out based on the standard curve using the purified 4-ME and 1ME: 4-ME was prepared from MEL-A of P. antarctica T-34, and 1-ME from MEL-B of P. tsukubaensis JCM 10324, respectively (Fukuoka et al. 2008). All measurements reported here are the means calculated from at least three independent experiments.

Results Screening of ME producers from Pseudozyma yeasts As mentioned above, different Pseudozyma yeasts efficiently produce different MELs from vegetable oils. Thus, ten species (14 strains) of the genus Pseudozyma were examined for their ability to produce ME from the screening medium including glucose as the sole carbon source. The culture broths of these strains were checked by PPC (Fig. 2). Pseudozyma fusiformata (FJ919774)

86

Pseudozyma hubeiensis (DQ008954)

51

Pseudozyma prolifica (AF294700) 52

100

Ustilago maydis (AF135431) Pseudozyma thailandica (AB089354)

Pseudozyma sp. KM160 (AB855769)

84

45

100

Pseudozyma tsukubaensis (AB089372) Pseudozyma flocculosa (AB089364)

54

Pseudozyma jejuensis (EF079966) 73

Pseudozyma shanxiensis (DQ008956)

34

Pseudozyma siamensis (AB117963)

100 65

Ustilago cynodontis (AF038825) Pseudozyma crassa (AB117962) Pseudozyma parantarctica (AB089356)

69

Pseudozyma antarctica (AF294698)

100

Pseudozyma aphidis (AB204896)

96 75

Pseudozyma rugulosa (AF294697) Pseudozyma graminicola (AB180728) Ustilago scitaminea (AF135433)

0.01

Appl Microbiol Biotechnol (2014) 98:823–830

P. hubeiensis KM-59 and Pseudozyma sp. KM-160 clearly displayed a spot corresponding to ME, while P. antarctica JCM10317 and P. hubeiensis CBS 10079 slightly provided a spot of ME. On the other hand, P. antarctica JCM10317, P. antarctica T-34, P. flocculosa JCM10321 and Pseudozyma KM-160 showed a clear spot corresponding to erythritol. Accordingly, the two strains of KM-59 and KM-160 were exclusively used for the production of ME in the following experiments.

827 Table 1 Physiological characterization of Pseudozyma sp. KM-160 and CBS6389 KM-160

CBS6389a

Assimilation of carbon compounds D -Glucose D -Galactose L -Sorbose

+ + +

+ S S

W W + + − − + − + S − − − − W + W −

S S + + S − + + + + − − + + + + + −

+ W − W + W

− + + + + +

+

+

+ W + + −

+ − + ND −

D -Glucosamine D -Ribose D -Xylose

Identification of strain KM-160

L -Arabinose

A taxonomical study of strain KM-160 was then carried out based on the method of Yarrow (1998). In YM broth, after 3 days at 25 °C, the cells of the strain were cylindrical and ellipsoid, 2.0–4.0×5.0–10.0 μm and occur singly, in pairs or in groups. The D1/D2 26S rDNA sequence of the strain was identical to those of Pseudozyma tsukubaensis and Ustilago spermophora. In addition, the ITS sequences of the strain was identical (100 %) with that of P. tsukubaensis (Fig. 3). The physiological pattern of the strain was very similar to that of P. tsukubaensis CBS 6389 (=P. tsukubaensis JCM 10324) as the type strain (Table 1). From these results, Pseudozyma sp. KM160 was identified as P. tsukubaensis.

D -Arabinose L -Rhamnose

Sucrose Maltose α-Methyl-D -glucoside Cellobiose Salicin Melibiose Lactose Raffinose Soluble starch Glycerol Erythritol Ribitol

Structural analysis of ME

D -Mannitol

Each ME produced by the two strains of KM-59 and KM-160 was purified with the activated charcoal column and subjected to detailed structural analysis. The purified ME was then analyzed by 1H and 13C NMR (Table 2). On the NMR spectra, significant differences were observed on the chemical shifts of the erythritol moiety between the two MEs, especially on the methylene protons involved in glycosidic linkage (Fig. 4). The NMR data of ME from strain KM-59 was consistent well with the previous result of ME prepared from MEL-A produced by P. antarctica T-34. Likewise, the data of ME from strain KM160 was well with that of ME from MEL-B produced by P. tsukubaensis JCM 10324 (Fukuoka et al. 2008). The column purified 4-ME showed more than 97 % of the purity on HPLC, and gave white needle-like crystals after recrystallization from 90 % ethanol. The melting point of the crystals was 156.2–158.4 °C and the [α]D value at 22 °C was −37.2 (c = 1.0, H2O). In contrast, the column purified 1-ME did not gave a solid mass but as a colorless syrup. Its [α]D value at 22 °C was −43.2 (c = 1.0, H2O). This indicated that the two MEs have different chirality. Although the absolute configuration has not yet been confirmed by X-ray scattering study, the above results are most likely attribute to a difference in the configuration of erythritol moiety. Consequently, the two MEs produced by strain KM59 and KM-160 were determined as 4-ME and 1-ME, respectively. To our best knowledge, this is the first report on the

2-Keto-D -gluconate D -Glucuronate DL -Lactate Succinate Ethanol Assimilation of nitrogen compounds Nitrate Other tests Growth without vitamins Growth in 50 % glucose Growth at 30 °C Growth at 35 °C Growth at 37 °C

+ positive, L latent, S slowly positive, W weakly positive, − negative, ND not determined a

Boekhout and Fell (1998)

fermentative production of 1-ME as the new diastereomer of ME. Production of 4-ME and 1-ME in shake flask culture To optimize ME production conditions, the effects of the temperature (22 °C, 25 °C, and 28 °C), pH (4.0–8.0), and yeast extract concentration (0.1–0.8 %) on the production

828 Table 2 NMR data of mannosylerythritol produced by P. hubeiensis KM-59 and P. tsukubaensis KM-160 (D2O, 400 MHz)

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Functional groups

P. hubeiensis KM-59 H NMR δ (ppm)

P. tsukubaensis KM-160 C NMR δ (ppm)

H NMR δ (ppm)

C NMR δ (ppm)

1

13

1

13

4.54 d 3.89 dd 3.50–3.52 dd 3.42 t 3.21–3.26 m 3.54–3.58 dd 3.76–3.80 dd

100.6 70.5 73.0 67.0 76.4 61.2

4.55 d 3.89 dd 3.50–3.52 dd 3.42 t 3.21–3.26 m 3.56–3.60 dd 3.77–3.81 dd

100.1 70.4 73.0 67.0 76.4 61.2

3.46–3.50 m 3.64–3.68 m 3.59–3.61 m 3.56–3.59 m 3.63–3.67 dd 3.90–3.94 dd

62.7 70.7 71.9 70.8

3.82–3.86 dd 3.69–3.73 dd 3.59–3.62 m 3.62–3.64 m 3.64–3.68 m 3.46–3.50 m

70.4 71.9 70.7 62.7

D -Mannose

s singlet, d doublet, dd double doublet, t triplet, m multiplet

H-1′ (C-1′) H-2′ (C-2′) H-3′ (C-3′) H-4′ (C-4′) H-5′ (C-5′) H-6′a (C-6′) H-6′b Erythritol H-1a (C-1) H-1b H-2 (C-2) H-3 (C-3) H-4a (C-4) H-4b

glucose for 15 days cultivation (Fig. 5a), strain KM-59 gave the maximum yield of 4-ME at 33.2 g/l (2.2 g/l/day in the productivity), while strain KM-160 gave the maximum yield of 1-ME at 30.0 g/l (2.0 g/l/day). We further investigated the use of glycerol instead of glucose, considering that surplus glycerol derived from biodiesel production has become an alternative feedstock for fermentation (Yazdani and Gonzalez 2007). With 200 g/l of

were investigated. The two strains of KM-59 and KM-160 stably produced ME from glucose at 25 °C. The optimum pH values for stain KM-59 and KM-160 were found to be pH 6.0 and pH 5.5, respectively. Also, the optimum yeast extract concentrations for KM-59 and KM-160 were 0.2 % and 0.6 %, respectively. Figure 5 shows the results of ME production in shake flask culture under the optimized conditions. With 200 g/l of Fig. 4 Partial 1H NMR spectra and chemical structures of ME. a 4-O-β-D -mannopyranosyl-D erythritol, produced by P. hubeiensis KM-59, b 1-O-β-D mannopyranosyl-D -erythritol, produced by P. tsukubaensis KM160

a

1

CH2OH

H-4b

H-4a

H

OH 4’

6’

5’

HO HO 3’

OH O 2’

H

2

OH

3

OH

4

O

CH2

1’

4

CH2OH

b

HO

OH H-4b

OH 4’ 6’ 5’ O

H-4a

HO HO 3’

4.0

3.8

3.6

3.4

3.2 (ppm)

2’

HO

3 2 1

O 1’

CH2

H H

Appl Microbiol Biotechnol (2014) 98:823–830

a

8

15

8

829 Cultivation time (d)

15

40

a

Glucose (g/L)

100

20 10 0 50

100

200

P. hubeiensis KM-59

50

100

200

Glucose (g/L)

50

80

40

60

30

40

20

20

10

0

Pseudozyma sp. KM-160

0

0

b

8

15

8

15

1

b

5

6

Glycerol ME Dry cell weight

30

70

100

10 0 50

100

200

P. hubeiensis KM-59

50

100

200

Glycerol (g/L)

Pseudozyma sp. KM-160

Fig. 5 Production of 4-ME and 1-ME from glucose and glycerol by P. hubeiensis KM-59 and P. tsukubaensis KM-160. The strains were cultivated in the medium containing 50, 100 and 200 g/l of glucose (a) and glycerol (b). The vertical bars show the standard deviation of the mean of three independent experiments

glycerol for 15 days cultivation (Fig. 5b), strain KM-59 gave the maximum yield of 4-ME at 16.1 g/l (1.1 g/l/day), while strain KM-160 gave the maximum yield of 1-ME at 15.8 g/l (1.1 g/l/day). Thus, the two yeasts are likely to efficiently produce ME also from glycerol. Production of 1-ME in fed batch culture As described above, Kobayashi et al. (1987) have already reported that Candida sp. KSM-1529 significantly produces 4-ME from glucose. We thus focused our attention particularly on the improvement of the production of the new diastereomer by KM-160 in fed batch culture. Figure 6 shows the typical time course of 1-ME production by strain KM-160 with a 5-l jar fermenter by feeding of glucose or glycerol as the sole carbon source. After incubation for 2 days, 100 g/l of glucose was completely consumed, and the feed medium was added. During 6 days of cultivation, the total amount of 1-ME reached approximately 38.0 g/l (6.3 g/l/day) from glucose. Likewise, the strain produced 1-ME at 31.1 g/l (3.5 g/l/day) for 9 days cultivation from glycerol. The lag phase in the case of glycerol should be caused by the difference in the metabolic pathway

Glycerol (g/L)

20

60

80

50

60

40 30

40

20 20

10

1-ME, dry cell weight (g/L)

ME (g/L)

2 3 4 Cultivation time (d)

Cultivation time (d)

40

1-ME, dry cell weight (g/L)

60

30 ME (g/L)

Glucose ME Dry cell weight

0

0 0

1

2

3

4

5

6

7

8

9

Cultivation time (d) Fig. 6 Time course of 1-ME production by P. tsukubaensis KM-160 with feeding of glucose (a) and glycerol (b). Strain KM-160 was cultivated in the medium containing 100 g/l of glucose as the sole carbon source at 25 °C, and then the feed medium was directly added to the culture. The 1-ME, residual glucose and glycerol were quantified by HPLC with three independent experiments. Filled circle, 1-ME (g/l); open triangle, dry cell weight (g/l); open square, residual glucose and glycerol (g/l)

generating erythritol between glucose and glycerol. In both cases, the productivity of 1-ME became more than three times higher than that with shake flask culture.

Discussion In the present study, we demonstrated that P. hubeiensis KM59 and P. tsukubaensis KM-160 efficiently produce 4-ME and 1-ME as the diastereomer of 4-ME, respectively (Fig. 4, Table 2). Previously, the fungal strains of Candida sp. KSM1529 (Kobayashi et al. 1987) and Ustilago sp. PRL 627 (Boothroyd et al. 1956) have been reported to produce 4-ME from glucose. To our best knowledge, this is the first report on the efficient production of 1-ME. With 200 g/l of glucose, the maximum yields of 4-ME by strain KM-59 and 1-ME by KM-160 reached 33.2 g/l (2.2 g/l/

830

day) and 30.0 g/l (2.0 g/l/day) in shake flask culture, corresponding to 0.17 and 0.15 g/g of the yield coefficient on a weight basis to glucose, respectively. On the other hand, Candida sp. KSM-1529 produced 4-ME at a yield of 22.0 g/l (3.1 g/l/day) from glucose, corresponding to 0.22 g/ g of the yield coefficient (Kobayashi et al. 1987). In fed batch culture, the production yield and coefficient of 1-ME by strain KM-160 were greatly improved. The productivity of 1-ME from glucose (6.3 g/l/day) became more than three times higher than that (2.0 g/l/day) in the flask culture. This means that the present strains are very likely to have a practical potential for ME production. The present results also demonstrated that the two strains are able to efficiently produce ME from glycerol. Indeed, strain KM-160 produced 1-ME with relatively high yield of 31.1 g/l (3.5 g/l/day) in fed batch culture. The amount of waste glycerol supplied as a by-product in bio-diesel production and oleochemical industry, has been increasing year by year, and the price have been decreasing (Yazdani and Gonzalez 2007). Hence, the present strains should also have a great advantage for converting waste glycerol into useful chemicals. In many bioprocesses, the downstream including product recovery and purification comprises 60 to 70 % of the total production cost (Zeikus et al. 1999). In particular, purification step using column chromatography often causes a significant loss of the recovery rate. In this study, the recovery rate of ME from the culture broth was found to be relatively high; more than 75 % after the charcoal column treatment (data not shown). Thus, the present ME production system may have potential for improving the downstream and production cost. In conclusion, we first attained the selective production of the two diastereomers of ME as a new disaccharide sugar alcohol using Pseudozyma strains. Further studies on the production and characterization of the present MEs would allow us to facilitate a broad range of applications of them in the food and related industries.

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Selective production of two diastereomers of disaccharide sugar alcohol, mannosylerythritol by Pseudozyma yeasts.

Mannosylerythritol (ME) is the hydrophilic backbone of mannosylerythritol lipids as the most promising biosurfactants produced by different Pseudozyma...
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