Production of Streptoverticillium cinnamoneum transglutaminase and cinnamic acid by recombinant Streptomyces lividans cultured on biomass-derived carbon sources

Bioresource Technology 104 (2012) 648–651

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Production of Streptoverticillium cinnamoneum transglutaminase and cinnamic acid by recombinant Streptomyces lividans cultured on biomass-derived carbon sources Shuhei Noda a, Takaya Miyazaki a, Tsutomu Tanaka b, Chiaki Ogino a,⇑, Akihiko Kondo a a b

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan

a r t i c l e

i n f o

Article history: Received 20 July 2011 Received in revised form 20 September 2011 Accepted 12 October 2011 Available online 31 October 2011 Keywords: Biomass Streptomyces lividans Cellulase Amylase Xylanase

a b s t r a c t Transglutaminase from Streptoverticillium cinnamoneum (StvcMTG) was produced using recombinant Streptomyces lividans. When grown on glycerol and xylose as sole carbon sources, S. lividans/StvcMTG produced 360 and 530 mg of StvcMTG per liter, respectively. With starch and xylan, the strain produced 230 and 400 mg of StvcMTG per liter, respectively. Recombinant S. lividans/encP, which expresses phenylalanine ammonia lyase from Streptomyces maritimus, produced 160 mg/L of cinnamic acid from cellulose. These results show that S. lividans can assimilate various carbon sources and produce useful compounds in desirable quantities. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction In contrast to yeast and Escherichia coli which are somewhat limited in their ability to utilize biomass and require the provision of substrates through chemical or enzymatic pretreatment of the biomass, Streptomyces lividans is able to grow on a variety of carbon sources. S. lividans possesses genes encoding b-glucosidase (BGL) and b-xylosidase (BXL) and can assimilate cello-oligosaccharide and xylo-oligosaccharide (Hurtubise et al., 1995; Schlösser et al., 1997). It also produces endo-glucanases (EG) and xylanase (EX) and can secrete a-amylase (AMY) into the culture medium (Kluepfel et al., 1986; Tsao et al., 1993). S. lividans has been used for the production of recombinant proteins (Vrancken and Anné, 2009), among them human interleukin-1 b (Lichenstein et al., 1988), glycoprotein from Mycobacterium tuberculosis (Lara et al., 2004), and transglutaminase from Streptoverticillium cinnamoneum (StvcMTG) (Noda et al., 2010). Cinnamic acid was also produced by S. lividans expressing phenylalanine ammonia lyase (Noda et al., 2011). Although optimization of a nitrogen source has significantly increased the production of proteins and cinnamic acid (Noda et al., 2011), the use of carbon sources for protein or cinnamic acid pro-

⇑ Corresponding author. Tel./fax: +81 78 803 6193. E-mail address: [email protected] (C. Ogino). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.10.045

duction has yet to be optimized. Using StvcMTG as a model protein and cinnamic acid as a model compound, carbon sources such as glycerol, starch, xylo-oligosaccharide and cellobiose were utilized for fermentation in the present study. The results suggest that S. lividans is a promising host for the fermentation of various carbon sources.

2. Methods 2.1. Bacterial strain, transformation, and cultivation S. lividans 1326 was used as the host for protein expression. Protoplasts were prepared as described by Hopwood et al. (1995). Mycelium was treated with 1 mg/mL of lysozyme solution (Wako, Osaka, Japan), and plasmids, pUC702-pro-sig-StvcMTG-term encoding transglutaminase from S. cinnamoneum or pUC702p-encP which encodes phenylalanine ammonia lyase from Streptomyces maritimus (Noda et al., 2010, 2011), were introduced by the polyethylene glycol (PEG) method. Selection of transformants was carried out by overlaying soft agar containing 50 lg/ mL of thiostrepton. The selected transformants were named S. lividans/StvcMTG and S. lividans/encP, respectively. For production of StvcMTG and cinnamic acid, a single colony of S. lividans/StvcMTG and S. lividans/encP was inoculated into 5 mL of TSB medium [17 g/L pancreatic digest of casein, 3 g/L papaic digest

S. Noda et al. / Bioresource Technology 104 (2012) 648–651

of soybean meal, 2.5 g/L glucose, 5.0 g/L sodium chloride, and 2.5 g/ L dipotassium phosphate (BD Diagnostic Systems, Sparks, MD, USA)] supplemented with 5 lg/mL of thiostrepton (MP biomedicals, Illkirch-Graffenstaden, France). After 3 days of cultivation in a test tube at 28 °C, the S. lividans/StvcMTG culture was transferred into a baffled flask containing 100 mL of modified TSB medium with 5 lg/mL thiostrepton, 15–50 g/L glycerol, 15 g/L of cornstarch (Nacalai Tesque), 15–100 g/L xylose, or 15 g/L xylan from birch wood (Sigma) as a carbon source, and 50 g/L tryptone as a nitrogen source, respectively, followed by incubation at 28 °C for 4–6 days. The S. lividans/encP culture was transferred to a baffled flask containing 100 mL of TSB medium with 5 lg/mL thiostrepton, 30 g/L cellobiose, xylo-oligosaccharide, or Avicel as a carbon source and 50 g/L tryptone as a nitrogen source, respectively, and incubated at 28 °C for 5 days. 2.2. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) analysis Culture supernatants of S. lividans/StvcMTG were directly mixed with SDS–PAGE buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.002% bromophenol blue, 0.125 M Tris–HCl, pH 6.8) and boiled. The protein samples were fractionated by a 15% SDS– PAGE gel and stained with Coomassie Brilliant Blue R-250 (Nacalai Tesque). The concentration of produced StvcMTG in the supernatant was evaluated with ImageQuant TL (GE Healthcare, Tokyo, Japan) using purified StvcMTG as a standard. The concentration of purified StvcMTG was quantified using a Quick Start Bradford Protein Assay (BioRad Laboratories, Hercules, CA). 2.3. Measurement of degradative enzyme activity b-Glucosidase and b-xylosidase activity was measured in 25 lL of 1 M sodium acetate buffer (pH 7.0) with 100 lL of 10 mM pnitrophenyl-b-D-glucopyranoside (pNPG) or p-nitrophenyl-b-Dxylopyranoside (pNPX) (Nacalai Tesque) as the substrate, respectively. The mixture (containing 375 lL of culture supernatant diluted to 10%) was incubated at 50 °C for 60 min. The reaction was terminated by the addition of 500 lL of 3 M sodium carbonate, and the p-nitrophenol released was determined by measuring absorbance at 400 nm. One unit of enzyme activity was defined as the amount of enzyme that released 1 lmol of p-nitrophenol from the substrate per min. Endo-glucanase, amylase and endo-xylanase activity were measured according to a method established by Miller (1959), with some modification. A 300-lL aliquot of culture supernatant and cell fractions was mixed with 700 lL of a 1% (w/v) solution of carboxy methyl cellulose (CMC) dissolved in 100 mM acetate buffer (pH 5.5) and a 1% (w/v) solution of soluble starch, which was then dissolved in either 100 mM acetate buffer (pH 7.0) or a 1.5% (w/v) solution of xylan, which was then dissolved in 100 mM MES buffer (pH 6.0). The mixture was incubated at 50, 37 and 50 °C for 6, 5 and 6 h, respectively. The amount of reducing sugar released from CMC, starch and xylan was assayed by determining the amount of glucose, for CMC and starch, and the amount of xylose, for xylan and equivalents, using the dinitrosalicylic acid method. One unit of enzyme activity was defined as the amount of enzyme that released 1 lmol of reducing sugar as glucose, for CMC and starch, or xylose, for xylan and equivalents, from the substrate per min. 2.4. Analytical methods The culture supernatant was subjected to centrifugation at 21,880g for 20 min, and the cinnamic acid concentration was determined by high-performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) using a Cholester column (Nacalai Tesque, Kyoto, Japan) and a ultra-violet absorbance detector (Shimadzu

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SPD-20AV). The operating conditions were 30 °C, for the acetonitrile:phosphate buffer (50 mM, pH 2.5) (30:70) mobile phase, with a flow rate of 1.2 mL/min. 3. Results and discussion 3.1. StvcMTG production from glycerol using S. lividans Although glycerol is a by-product of biodiesel production and a potential economical feedstock for fermentation, only a few microorganisms can assimilate glycerol as a carbon source. Using StvcMTG as a model protein, protein production from glycerol was evaluated. The cell growth was also monitored, because it is a key factor for protein productivity (Ogino et al., 2004). Fig. 1(A) shows the StvcMTG concentration in the culture medium with 15, 30 and 50 g/L of glycerol as the carbon source. A StvcMTG concentration of 270 mg/L was achieved from 15 g/L of glycerol, and the maximal level of StvcMTG produced was 360 mg/L after 6 days of cultivation with 30 g/L glycerol. The maximal level was 1.5-fold higher than that produced from 15 g/L of glucose (Noda et al., 2010). However, StvcMTG productivity could not be enhanced by a further increase in the initial glycerol concentration. Fig. 1(B) shows dry cell weights of S. lividans/StvcMTG when using glycerol as the carbon source. The maximal level of StvcMTG and cell growth was achieved using 30 g/L glycerol. During cultivation the pH of the medium increased from 7–8 to 9–10, and the glycerol was consumed within 3–5 days (data not shown). The low StvcMTG productivity in culture using 50 g/L glycerol was attributed to a decrease in the cell growth (Fig. 1). Thus, S. lividans can assimilate glycerol to produce StvcMTG equally as well as glucose can be used to produce it, which shows that glycerol is a waste substance that can be used for the production of useful products. 3.2. StvcMTG production from xylose using S. lividans As opposed to glucose, xylose is a carbon source for a more limited number of microorganisms. Therefore, for an efficient bioprocess using biomass, it is necessary to develop bioconversion systems using xylose. Streptomyces are known to possess a xyl gene cluster and are predicted to produce xylose ABC permease, which stimulates effective xylose assimilation (Schlösser et al., 1997; Bertram et al., 2004; Heo et al., 2008). Fig. 2(A) shows StvcMTG release into culture medium containing 15, 30, 50, 75 and 100 g/L xylose. A maximal amount of StvcMTG (530 mg/L) was observed after 6 days of cultivation with 100 g/L xylose. Fig. 2(B) shows dry cell weight using xylose as the carbon source. After the cultivation using xylose as the carbon source, the pH shifted to 9–10, and xylose was almost consumed within 5 days (data not shown). It appears that xylose uptake is the rate-limiting step in enzyme production. Although a gene encoding the ATP-binding component MsiK (multiple sugar import protein) was identified in S. lividans (Schlösser et al., 1997), the expression level is relatively low, not enough to assimilate 100 g/L xylose immediately. Hence, StvcMTG production rates may be improved by over-expressing a gene encoding MsiK from S. lividans or other Streptomyces xylose ABC permease. 3.3. Cinnamic acid production from oligosaccharides and Avicel Streptomyces possess various kinds of oligosaccharide or biomass degradation enzymes (Morosoli et al., 1986; Kluepfel et al., 1986; Théberge et al., 1992; Tsao et al., 1993; Hurtubise et al., 1995; Yin et al., 1998). Although these enzymes have been characterized using Streptomyces as a host, there are few reports demonstrating direct production from biomass using Streptomyces without additional biomass degradation enzymes.

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Fig. 1. (A) StvcMTG production by S. lividans/StvcMTG in modified TSB medium with 15 g/L glycerol (closed diamonds), 30 g/L glycerol (closed circles) and 50 g/L glycerol (closed triangles). (B) Dry cell weight of S. lividans/StvcMTG in modified TSB medium with 15 g/L glycerol (open diamonds), 30 g/L glycerol (open circles) and 50 g/L glycerol (open triangles). Each data point is the average of three independent experiments, and error bars represent standard deviation.

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Fig. 2. (A) StvcMTG production by S. lividans/StvcMTG in modified TSB medium with 15 g/L xylose (inverted closed triangles), 30 g/L xylose (closed triangles), 50 g/L xylose (closed squares), 75 g/L xylose (closed diamonds), and 100 g/L xylose (closed circles). (B) Dry cell weight of S. lividans/StvcMTG in modified TSB medium with 50 g/L xylose (open squares), 75 g/L xylose (open diamonds), and 100 g/L xylose (open circles). Each data point is the average of three independent experiments, and error bars represent standard deviation.

Since the degradative enzymes, BGL, BXL, EG, AMY and EX are produced by wild-type S. lividans (Table 1), cinnamic acid production was attempted with oligosaccharides as carbon sources. Maximal levels of cinnamic acid (490 mg/L) were produced in modified TSB medium with 30 g/L of cello-oligosaccharide after 5 days of cultivation. After 4 days cultivation, 400 mg/L of cinnamic acid was produced from 30 g/L of xylo-oligosaccharide (Fig. 3). Regardless of the carbon source, S. lividans and S. lividans/encP exhibited maximal BGL and BXL activities in their intracellular fractions during logarithmic growth (Table 1). Maximal levels of StvcMTG reached 160 mg/L after 4 days during cultivation with 30 g/L Avicel. Cinnamic acid productivity was lower compared to that using cellobiose, suggesting that improvements in EG activity might improve cinnamic acid productivity. 3.4. StycMTG production from starch and xylan Starch and xylan, which are major biomass resources, were used as biomass to carry out direct StvcMTG production. The max-

imal level of StvcMTG reached 230 mg/L after 3 days of cultivation with 15 g/L corn starch. The amount of StvcMTG produced using 15 g/L starch was almost the same as when using glucose (Noda et al., 2010). The maximal level of produced StvcMTG reached 400 mg/L after 5 days of cultivation with 15 g/L xylan. Xylanase activity was detected in the culture supernatant of S. lividans (Table 1), indicating that hemicellulolytic compounds are a suitable carbon source.

4. Conclusion Genetically modified S. lividans was able to utilize a number of biomass-derived carbon sources and produced StvcMTG and cinnamic acid. The amount of produced StvcMTG in S. lividans was greater than secretory protein productivity using Bacillus subtilis and E. coli (Ray et al., 2002; Ye et al., 1999), and cinnamic acid productivity using S. lividans higher than that of using E. coli (Vannelli et al., 2007). S. lividans potentially secretes cellulase, amylase and

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S. Noda et al. / Bioresource Technology 104 (2012) 648–651 Table 1 Enzyme activity detected in media during cultivation of wild-type and recombinant S. lividansa. Strain

Amylase activityb (U/L)

Endo-glucanase activityc (U/L)

Endo-xylanase activityd (U/L)

b-glucosidase activitye (U/g-dry cell)

b-xylosidase activityf (U/g-dry cell)

Wild-type S. lividans S. lividans/StvcMTG S. lividans/encP

7.19 ± 1.28 8.38 ± 2.25 –g

1.86 ± 0.0819 –g 2.63 ± 0.359

3.04 ± 1.97 7.64 ± 3.50 –g

0.972 ± 0.0741 –g 0.995 ± 0.0554

0.0477 ± 0.0111 –g 0.149 ± 0.0060

a

Values are means ± standard deviations for three independent experiments. The maximal values of each enzyme activity are shown. Activities in culture supernatants after 2 days cultivation using 30 g/L starch as carbon source. Activities in culture supernatants after 4 days cultivation using 30 g/L Avicel as carbon source. d Activities in culture supernatants after 2 days cultivation using 30 g/L xylan as carbon source. e Activities in the intracellular fraction after 1 day cultivation using 30 g/L cellobiose as carbon source. f Activities in intracellular fractions of wild-type S. lividans and S. lividans/StvcMTG after 2 and 3 days cultivation using 30 g/L xylo-oligosaccharide as carbon source, respectively. g Enzyme activity was not measured. b

c

Cinnamic acid concentration (mg/L)

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Fig. 3. Cinnamic acid production by S. lividans/encP in TSB medium with 30 g/L cellobiose and 50 g/L tryptone (closed circles), 30 g/L xylo-oligosaccharide and 50 g/ L tryptone (closed squares), and 30 g/L Avicel and 50 g/L tryptone (closed diamonds). Each data point is the average of three independent experiments, and error bars represent standard deviation.

xylanase and can be used for the production of useful compounds directly from biomass. Our results indicate that S. lividans is a versatile host for fermentation using biomass as a carbon source. Acknowledgements This work was supported by Special Coordination Funds for Promoting Science and Technology, from the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovation Bioproduction Kobe), MEXT, Japan. References Bertram, R., Schlicht, M., Mahr, K., Nothaft, H., Saier Jr., M.H., Titgemeyer, F., 2004. In silico and transcriptional analysis of carbohydrate uptake systems of Streptomyces coelicolor A3(2). J. Bacteriol. 186, 1362–1373. Heo, G.Y., Kim, W.C., Joo, G.J., Kwak, Y.Y., Shin, J.H., Roh, D.H., Park, H.D., Rhee, I.K., 2008. Deletion of xylR gene enhances expression of xylose isomerase in Streptomyces lividans TK24. J. Microbiol. Biotechnol. 18, 837–844. Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Bruton, C.J., Kieser, H.M., Lydiate, D.J., Smith, C.P., Ward, J.M., Schrempf, H., 1995. Genetic Manipulation of Streptomyces: A Laboratory Manual. The John Innes Foundation, Norwich.

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