Agric. Biol. Chem., 55 (5), 1383-1390, 1991 1383 Cloning, Sequencing, and Characterization of the Intracellular Invertase Gene from Zymomonasmobilis Hideshi Yanase,*'1" Hideaki Fukushi,** Naoki Ueda,** Yukitoshi Maeda,** Atsushi Toyoda** and Kenzo Tonomura*** * Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680, Japan ** Department of Agricultural Chemistry, Faculty of Agriculture, University of Osaka Prefecture, Sakai 591, Japan *** Department of Food Technology, Faculty of Engineering, Fukuyama University, Fukuyama 729-02, Japan Received December 13, 1990 The structural gene for the intracellular invertase El of Zymomonasmobilis strain Z6C was cloned in a 2.25-kb DNAfragment on pUSHll, and expressed in Eschevichia coli HB101.The enzyme produced by the E. coli carrying pUSHll was purified about 1,122 fold to homogeneity with a yield of 4%. The molecular weight and substrate specificity of the enzymewere identical with those of the intracellular invertase El from Z. mobilis. The nucleotides of the cloned DNAwere sequenced; they included an open reading frame of 1,536bp, coding for a protein with a molecular weight of 58,728. The N-terminal amino acid sequence predicted was identical with the sequence of the first 20 N-terminal amino acid residues of the protein obtained by Edmandegradation. Comparison of the predicted amino acid sequence of El protein with those of the four other known^-D-fructofuranosidases from Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae indicated a stronger homologyin the N-terminal portion than in the C-terminal portion. Zymomonasmobilis is a Gram-negative its nucleotide sequence was determined and bacterium capable of producing almost the characterized by comparing with genes encod- theoretical yield of ethanol from glucose, and ing for invertase from E. coli,6) B. subtilis,1^ has higher productivity than the yeasts now and S. cerevisiae.9) used industrially. However, this bacterium can ferment only glucose, fructose, and sucrose. In addition, there is little information on the early Materials and Methods metabolism of sucrose, although it has been Plasmids and bacteria used. The bacteria used in this known that sucrose is hydrolyzed to glucose study were E. coli HB101 (F~, hsdS20, recA13, ara-14, and fructose by invertase that is produced proA2, lacYl, galK2, rpsL20, xyl-5, mtl-1, supE44, k~), inducibly,1} and the liberated fructose is and Z. mobilis Z6C, a spontaneous mutant of Z. mobilis converted to sorbitol2'3) and Ievan4'5) in part. subsp. mobilis IFO13756 (=ATCC29191) capable of Preliminary investigation suggested that there producing an extracellular levansucrase (E2) and invertase (E3), constitutively. 1} The plasmids used were pZA22 (Cmr, were three kinds of sucrose-hydrolyzing Tcr, 6.7kb), a shuttle vector between Z. mobilis and E. enzymesin Zymomonas,intracellular invertase coli,10) and pZL7 (lac'Z+, Y+, Cmr, 16.2kb), a lacY+ (that was namedEl), extracellular levansucrase plasmid that can coexist with pUC118or pUC119in E. (E2), and extracellular invertase (E3). There- coli HBIOI.1" fore, we have attempted to clone genes encoding for these enzymes and characterize Cultivation. Z. mobilis was cultivated statically at 30°C in RMmedium (2% glucose, 1% yeast extract, and 0.2% them. This paper describes that the El gene was KH2PO4, pH 6.0) and E. coli in the LB medium (1% cloned from Zymomonasgenomic library, and bacto-tryptone, 0.5% bacto-yeast extract, and 1 %NaCl, To whomreprint request should be addressed. 1384 H. Yanase et al. pH 7.0) at 37°C with shaking. Whennecessary, ampicillin the E. coli HB101carrying pUSHll as follows. The (25 Mg/ml), tetracycline (1 5 fig/ml), and/or chloramphenicol transformant cells harvested from 7 1 of the culture medium (100 /ig/ml for Z. mobilis, 25 /jg/ml for E. coli) were added were washed with 20mMphosphate buffer (pH 7.0), and to the medium. Cells of each organism were harvested by suspended in 500 ml of the same buffer, then disrupted by centrifugation and washed with 20mMphosphate buffer sonication. Thesupernatant solution was fractionated with (pH 7.0). (NH4)2SO4 precipitation between 30 to 50% saturation. The resulting precipitate was dissolved in the above buffer DNAmanipulation. Standard methods were used for the and dialyzed. The enzyme solution was put onto a purification of DNA, plasmid constructions, and transfor- DEAE-Toyopearl 650M column (2.3 x 30 cm) equilibrated mation ofE. coli.12) The plasmids were introduced into Z. with the buffer. The enzyme was recovered from un- mobilis by the transformation method using partial absorbed fractions, and dialyzed against 20mM acetate spheroplasts. 1 3) buffer, pH 5.0. The dialyzed solution was put onto a CM-Sephadex C-50 column (1.2 x 30 cm) equilibrated with Construction of Z. mobilis gene library. Z. mobilis the dialyzed buffer. After the column was washed chromosomal DNAwas isolated by the method of Saito thoroughly with the same buffer, the enzymewas eluted and Miura14) with a partial modification. It was partially with a linear gradient from 0.1 to 1.0m NaCl. Fractions digested with BamHl, and the resulting fragments having showing the enzyme activity were pooled and concentrated a size of about 8kb were ligated into the BamHlsite of in a CENTRICUTU-20 (Kurabo Biomedical P/J). The pZA22, and then transformed E. co// HB101.Recombinant enzyme solution was fractionated with (NH4)2SO4 of 0 to clones were selected for sucrose-hydrolyzing activity in 10%saturation. The resultant precipitate was dissolved in toluenized cells. 20mMacetate buffer, pH 5.0. The El enzyme from Z. mobilis Z6C cells was partially Deletion analysis. To identify the coding region of the purified as follows. Cells harvested from 7 1 of the culture El gene on pZSHl, the 7.85-kb 5amHI-fragment was medium were suspended in 700ml of 20mMphosphate subcloned into pUC118 or pUC119 using E. coli HB101 buffer, pH 7.0, and incubated at 30°C for lOmin with carrying pZL7. E. coli carrying pZL7 could take up shaking to release cell-bound levansucrase (E2) and raffinose by the lactose permease encoded by lacY on invertase (E3) from cells. The washed cells were disrupted pZL7.15) If the gene encoding for the Z. mobilis by sonication, and centrifuged to remove intact cells and sucrose-hydrolyzing enzymewas cloned in the strain, the debris. The supernatant solution was put onto a recombinant clone would be expected to use the fructose DEAE-Toyopearl 650M column (2.3 x 30 cm) equilibrated moiety ofraffinose. This clone, however, could not use the with 20mM phosphate buffer, pH 7.0, and unabsorbed galactose moiety of raffinose or melibiose, because the fractions were pooled. The enzyme was precipitated by strain is galK~. A clone capable of using raffinose was adding (NH4)2SO4 to 50% saturation, and the resultant selected by a red colony formed on a 2,3,5-triphenyl precipitate was dissolved in a small volume of 20mM tetrazolium chloride (TTC)-indicator plate, which con- phosphate buffer, pH 7.0, containing 0.2m NaCl. The tained 2% raffinose, 0.7% KH2PO4, 0.3% K2HPO4, enzymesolution was put onto a SephadexG-200column 0.01% MgSO4, 0.2% proteose peptone, 0.00025% TTC, (2.3 x 100cm) equilibrated with 20mMphosphate buffer, 0.0025% ampicillin, and 1.5% agar.16) pH 7.0, containing 0.2m NaCl, and chromatographed. Fractions having the enzyme activity were pooled and Enzyme and protein assay. The sucrose-hydrolyzing concentrated in a CENTRICUTU-20. activity was assayed at 30°C. The standard assay mixture contained 0.15 m sucrose, 0.1 macetate buffer, pH 5.0, and Molecular weight measurement. The molecular weight in an enzymesolution or toluenized cells in total volumeof a native form was estimated by gel filtration through a 1 ml. After incubated at 30°C, reducing sugar formed in Sephadex G-200 column (1.0x50cm) with 20mM the reaction mixture was measuredby the method of phosphate buffer, pH 7.0 containing 0.2m NaCl. Somogyiand Nelson, and one unit of the activity was Cytochrome c, chymotrypsinogen A, ovalbumin, BSA, defined as the amount of enzymethat produced reducing aldolase, and catalase were used as marker proteins. The sugar equivalent to 1 /miol of glucose per min. The amounts molecular weight of the subunit was measured by the of glucose and fructose liberated were measured with an method of Laemmli.19) Marker proteins used were: F-Kit for Glucose-Fructose (Boehringer Mannheim, a-lactalbumin, soybean trypsin inhibitor, carbonic anhy- GmBH).Protein concentration was assayed by the method drase, ovalbumin, BSA, and phosphorylase b. of Lowry et aL,17) and also by absorption at 280nm. Polyacrylamide gel electrophoresis was done by the Analysis of N-terminal amino acid sequence. The El procedure of Davis.18) enzyme obtained in the final step of purification from E. coli HB101 carrying pUSHll was blotted onto a Purification of the sucrose-hydrolyzing enzymes. The El polyvinylidene difluoride membrane (Millipore), and protein produced by the cloned El gene was purified from degraded sequentially with a protein analyzer, Applied Z. mobilis Intracellular Invertase Gene 1385 Fig. 1. Restriction Maps ofpZSHl and pUSHll. Symbols: H, Z. mobilis chromosomal DNA; =, pZA22; , pUC118. Biosystems model 4701A, equipped with an on-line HPLC apparatus model 120A. DNA sequencing. The DNA sequence of the gene encoding for the ZymomonasEl enzyme was analyzed by the dideoxy method of Sanger et al.20) The entire sequence of the insert contained in pUSHll was analyzed in both directions using an overlapping set of Exonuclease Ill-generated deletions that had been cloned into derivatives of phage Ml3. Results and Discussion Cloning of the gene encoding a sucrose- Fig. 2. Deletion Plasmids Derived from pZSH1. hydrolyzing enzyme The specific activity of sucrose-hydrolyzing enzyme in cell-free extracts prepared from E. coli clones are indicated. Plasmids containing our BamHIlibrary of The plasmids, pUSHl, pUSH5, pUSH6, and pUSHll, Z.