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Enhancement of Cellulose Production by Expression of Sucrose Synthase in Acetobacter Xylinum (Cellulose Synthesis͞sucrose Metabolism)

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Enhancement of Cellulose Production by Expression of Sucrose Synthase in Acetobacter Xylinum (Cellulose Synthesis͞sucrose Metabolism) Proc. Natl. Acad. Sci. USA Vol. 96, pp. 14–18, January 1999 Applied Biological Sciences Enhancement of cellulose production by expression of sucrose synthase in Acetobacter xylinum (cellulose synthesisysucrose metabolism) TOMONORI NAKAI*†,NAOTO TONOUCHI†‡,TERUKO KONISHI*, YUKIKO KOJIMA‡,TAKAYASU TSUCHIDA‡, FUMIHIRO YOSHINAGA‡,FUKUMI SAKAI*, AND TAKAHISA HAYASHI*§ *Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto, 611, Japan; and ‡Bio-Polymer Research Co. Ltd., KSP, Takatsu-ku, Kawasaki, Kanagawa, 213, Japan Communicated by Takayoshi Higuchi, Kyoto University, Kyoto, Japan, November 3, 1998 (received for review May 29, 1998) ABSTRACT Higher plants efficiently conserve energy mung bean gene conserves the activity to synthesize sucrose ATP in cellulose biosynthesis by expression of sucrose syn- from UDP-glucose plus fructose but hardly catalyzes its re- thase, in which the high free energy between glucose and versible reaction (6). The recombinant enzyme was phosphor- fructose in sucrose can be conserved and used for the syn- ylated in vitro at Ser11 byaCa21-dependent protein kinase thesis of UDP-glucose. A mixture of sucrose synthase and from soybean root nodules (7), suggesting that Ser11 is the bacterial cellulose synthase proceeded to form UDP-glucose regulatory target residue in mung bean sucrose synthase (8). from sucrose plus UDP and to synthesize 1,4-b-glucan from The apparent affinity for sucrose was increased in this phos- the sugar nucleotide. The mutant sucrose synthase, which phorylated enzyme and also in an engineered mutant in which mimics phosphorylated sucrose synthase, enhanced the reac- Ser11 (S11E) was replaced by glutamic acid. This shows that the tion efficiency (VmaxyKm) on 1,4-b-glucan synthesis, in which glutamic acid residue at position 11 mimics the phosphorylated the incorporation of glucose from sucrose was increased at low Ser11 residue to bind and cleave sucrose for the synthesis of concentrations of UDP. Because UDP formed after glucosyl UDP-glucose. Therefore, the mutant enzyme would be ex- transfer can be directly recycled with sucrose synthase, UDP- pected to carry out enhanced formation of UDP-glucose from glucose formed appears to show high turnover with cellulose sucrose without any regulation such as phosphorylation. synthase in the coupled reaction. The expression of sucrose Sucrose is potentially the most suitable carbon source for the synthase in Acetobacter xylinum not only changed sucrose production of cellulose in bacterial fermentation, not only metabolism but also enhanced cellulose production, in which because energy can be conserved in the formation of UDP- UDP-glucose was efficiently formed from sucrose. Although glucose with sucrose synthase, but also because this carbon the level of UDP-glucose in the transformant with mutant source is economically attractive throughout the world. There sucrose synthase cDNA was only 1.6-fold higher than that in are potential industrial applications for bacteria that produce plasmid-free cells, the level of UDP was markedly decreased in cellulose during fermentation with sucrose as a carbon source the transformant. The results show that sucrose synthase (9). In Acetobacter xylinum, which has no sucrose synthase, serves to channel carbon directly from sucrose to cellulose and there are at least four enzyme steps in the pathway from recycles UDP, which prevents UDP build-up in cellulose sucrose to UDP-glucose. In addition, A. xylinum strains show biosynthesis. various pathways of UDP-glucose formation. For example, the level of UDP-glucose pyrophosphorylase activity in A. xylinum in strain ATCC23768 differs from that in ATCC23769, al- Cellulose, the most abundant biopolymer on earth, occurs in though cellulose production is similar (10). Herein, we dem- plants and certain algae, fungi, and bacteria. Bacteria have a onstrate the direct synthesis of UDP-glucose from the carbon biosynthetic system different from the system in higher plants, source sucrose during cellulose synthesis both in vitro and in in which sucrose and sucrose synthase participate in cellulose vivo. We then find the major function of sucrose synthase, biosynthesis (1). Higher plants have two systems for the which is the prevention of UDP build-up in the cellulose formation of UDP-glucose, one with sucrose synthase (EC biosynthesis of higher plants. 2.4.1.13) and the other with UDP-glucose pyrophosphorylase The 59 upstream region (about 3.1 kb) of cellulose synthase (EC 2.7.7.9), although bacteria contain only the latter. Sucrose operon (bcs operon) has been isolated by cloning from A. synthase catalyzes the freely reversible reaction: UDP-glucose 1 º 1 xylinum BPR 2001 (11). The expression level of the upstream fructose sucrose UDP. The amount of the enzyme is region was determined by using sucrose synthase cDNA as a much higher in nonphotosynthetic tissues (2, 3), a major sink reporter gene in the shuttle vector pSA19. The expression in plants where sucrose is the source of carbon that is trans- occurred with the 1.1-kb upstream sequence from the ATG of located and cleaved by the enzyme to produce UDP-glucose bcs operon in A. xylinum G7 (12) and Acetobacter aceti 10–852 for synthesis of cellulose. Therefore, the reaction of the (13), although the upstream sequence did not cause any enzyme proceeds to the cleavage of sucrose for the synthesis expression as a promoter in E. coli TB1 (New England of UDP-glucose in the plant tissues (4). In developing cotton Biolabs). This shows that the upstream region functions as a fibers, the sucrose synthase is localized in arrays in parallel with specific promoter for the Acetobacter genus (11). However, the helical pattern of cellulose deposition, participating in the much more sucrose synthase was produced with the lac biosynthesis of cellulose (1). promoter than with the upstream region of bcs operon. Nev- Mung bean (Vigna radiata, Wilczek) sucrose synthase is a ertheless, the production of recombinant protein with the tetramer composed of identical subunits of 95 kDa (5). A ribosome-binding site of lac promoter was reduced to about recombinant protein from Escherichia coli encoded by the half in A. xylinum. Therefore, for the expression of sucrose synthase, we used the lac promoter and the ribosome-binding The publication costs of this article were defrayed in part by page charge site of bcs operon. payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. †T.N. and N.T. contributed equally to this work. © 1999 by The National Academy of Sciences 0027-8424y99y9614-5$2.00y0 §To whom reprint requests should be addressed. e-mail: taka@ PNAS is available online at www.pnas.org. kuwri.kyoto-u.ac.jp. 14 Downloaded by guest on September 23, 2021 Applied Biological Sciences: Nakai et al. Proc. Natl. Acad. Sci. USA 96 (1999) 15 MATERIALS AND METHODS tion, the supernatant was adjusted to 65% saturation with ammonium sulfate and the precipitating proteins were col- Bacterial Strains and Culture Conditions. E. coli lected and dissolved in the same buffer. The suspension was BL21(DE3) was used as the host to produce wild-type and centrifuged and the resultant supernatant was applied to a mutant recombinant sucrose synthases from mung bean by Sepharose CL-6B column (2.5 3 30 cm). The active fractions using the pET-21d expression vector under the control of a T7 (Vo) were pooled and further applied to a Superdex 200 promoter (Novagen). Transformants of strain BL21(DE3) column (2.6 3 60 cm) that had been equilibrated with the same containing various plasmids were cultivated in LB medium at m y buffer containing 0.1 M NaCl. The peak fractions were pH 7.0 and 37°C in a shaker flask; ampicillin (50 g ml) was combined, dialyzed against the Tris buffer, and applied to a included when the bacteria carried plasmids conferring drug Mono Q column (0.5 3 5 cm) that had been equilibrated with resistance. The cells of A. xylinum subsp. sucrofermentans BPR the same buffer. The active fractions containing sucrose 2001 were grown at 30°C for 24 h in a shaking flask in a liquid synthase activity were collected and pooled. The final prepa- medium (300 ml) containing 2% glucose, 0.5% yeast extract, ration of each recombinant sucrose synthase appeared to be 0.5% bactopeptone, 0.3% K2HPO4 (pH 6.0), and 0.1% cellu- homogeneous at 95 kDa by SDSyPAGE. The specific activities lase preparation (14). A. xylinum subsp. nonacetoxidans LD-2 of the purified wild-type and mutant enzymes were 1.46 and was cultured at 28°C for 72 h statically in a roux flask y y 1.35 units mg of protein, respectively, for the formation of containing sucrose corn steep liquor medium consisting of 4% sucrose. One unit of sucrose synthase activity was defined as sucrose and 4% corn steep liquor (15). This strain incorporates 1 mmol of sucrose formed per min at 30°C. sucrose, which may be hydrolyzed by the activity of intracel- Assay for Sucrose Synthase. For determination of the lular invertase. Its cell suspension was inoculated into the y incorporation of glucose from sucrose into UDP-glucose, the production culture to a concentration of 10% (vol vol). A reaction mixture contained 40 mM [14C]sucrose (2.5 mCiy y standard culture was used in 75 ml of the sucrose corn steep mmol, Amersham), 10 mM UDP, 50 mM TriszHCl (pH 7.5), liquor medium in a 300-ml flask and a small-scale culture with and the enzyme preparation (usually 20–30 mg of protein) in 14 m y 2 ml of the medium containing [ C-glucose]sucrose (26 Ci a total volume of 20 ml. For determination of the incorporation 5 mmol;1 Ci 37 GBq; New England Nuclear) as a carbon of glucose from UDP-glucose into sucrose, the reaction mix- source.
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