Cloning and Sequencing of the Triacylglycerol Lipase Gene of Aspergillus Oryzae and Its Expression in Escherichia Coli

Home , Ribonuclease, Triacylglycerol lipase

FEMS Microbiology Letters 189 (2000) 159^164 www.fems-microbiology.org

Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli

Jinichi Toida a;*, Mikio Fukuzawa a, Gota Kobayashi b, Kiyoshi Ito c,

Junichi Sekiguchi b Downloaded from https://academic.oup.com/femsle/article/189/2/159/523283 by guest on 27 September 2021

a Food Technology Research Institute of Nagano Prefecture, 205-1 Nishibanba, Kurita, Nagano-shi, Nagano 380-0921, Japan b Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano 386-8567, Japan c National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima-shi, Hiroshima 739-0046, Japan

Received 13 May 2000; received in revised form 12 June 2000; accepted 14 June 2000

Abstract

Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglycerol lipase, respectively). We cloned the triacylglycerol lipase gene (provisionally designated tglA) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes. Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene (tglA) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp. The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi. Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Aspergillus oryzae; Triacylglycerol lipase gene; Lipase gene

1. Introduction activity toward fatty acid ethyl esters or triacylglycerols with short-chain fatty acids. L2 is a 39^41-kDa protein Aspergillus oryzae has been used as koji (solid-state cul- which hydrolyzes monoacylglycerols and diacylglycerols, ture of koji mold) in the manufacture of traditional Japa- but not triacylglycerols. The cloning and sequencing of nese fermented foods: sake, miso (paste made from soy- the genes encoding L1 and L2 (cutL and mdlB, respec- beans, koji and salt), and soy sauce. The most important tively) have been reported by our group [6,7]. L3 is a role of koji is to supply various useful enzymes produced 25^29-kDa protein which exhibits high activity toward by A. oryzae, for example, amylase, protease and lipase. triacylglycerols with medium- or long-chain fatty acids. Although A. oryzae produces only a small amount of li- Since triacylglycerol with long-chain fatty acids is the pases in miso-koji [1], lipases act on lipids and thereby main lipid in soybeans, L3 seems to be the most important make important contributions to the quality and function enzyme in miso fermentation. of miso. Lipases hydrolyze the glycerides of soybeans and In this paper, we describe the cloning and sequencing synthesize esters in miso during fermentation [2], whereby of the genomic DNA and cDNA of the triacylglycerol the £avor and taste are formed. A. oryzae produces at lipase gene from A. oryzae, and its expression in Escheri- least three kinds of extracellular lipolytic enzymes exhibit- chia coli. ing di¡erent substrate speci¢cities (L1, L2 and L3). These three enzymes have been puri¢ed and characterized [3^5]. L1 is a 24-kDa cutinase-like enzyme which exhibits high 2. Materials and methods

2.1. Microorganisms, plasmid and culture conditions * Corresponding author. Tel.: +81 (26) 227 3131; Fax: +81 (26) 227 3130; E-mail: [email protected] A. oryzae RIB128 was used as DNA and RNA sources

FEMSLE 9495 26-7-00 160 J. Toida et al. / FEMS Microbiology Letters 189 (2000) 159^164 for cloning. E. coli JM109 (recA1 v(lac-proAB) endA1 2.4. Synthesis of cDNA gyrA96 thi-1 hsdR17 relA1 supE44 [FP: traD36 proAB laqIq vM15]) was used as a host for transformation. Total RNA from an A. oryzae culture at the L3 pro- E. coli XL1-Blue MRA (v(mcrA)183 v(mcrCB-hsdSMR- duction phase was prepared with Isogen (Nippon Gene) mrr)173 endA1 supE44 thi-1 gyrA96 relA1 lac), which is according to the manufacturer's instructions. First-strand lysogenic for the P2 phage, was used as a host strain for cDNA was synthesized using a RT-PCR kit (Stratagene) recombinant phages. pUC118 (Apr ; Takara Shuzou) was with an oligo(dT) primer and the total RNA as a tem- used for cloning and expression in E. coli. A. oryzae was plate. Double-stranded tglA cDNA was ampli¢ed by cultured at 30³C in a glucose medium consisting of 2% PCR with the ¢rst-strand cDNA as a template and two peptone, 0.5% yeast extract, 0.1% NaNO3, 0.1% KH2PO4, primers, TGLATG-E (5P-CGGAATTCCATGCATGC- 0.05% MgSO4W7H2O and 2% glucose (pH 5.5). For prep- ATCTTGCTATCAAGTCT-3P; the underlining indicates aration of the chromosomal DNA, 2% olive oil instead of theEcoRI site; italics indicate the sequence of positions

glucose was used for the production of L3 and preparation 31^21; Fig. 1) and TGLTTA-B (5P-CGGGATCCT- Downloaded from https://academic.oup.com/femsle/article/189/2/159/523283 by guest on 27 September 2021 of total RNA. E. coli was cultured in LB medium. If TAGTTCGCAGCCGCAACAGCA-3P; the underlining in- necessary, ampicillin was added to a ¢nal concentration dicates the BamHI site; italics indicate the sequence of of 50 Wgml31. positions 954^936; Fig. 1). The PCR product was subcloned into pUC118 after EcoRI and BamHI digestion. 2.2. Construction of a DNA probe by PCR 2.5. Sequencing of genomic DNA and cDNA L3 was cleaved with cyanogen bromide and then the N- terminal amino acid of the cyanogen bromide-cleaved DNA sequencing was performed with a BigDye Termi- fragment was determined as described previously [4]. nator Cycle sequencing kit and an automatic DNA se- PCR was carried out using the chromosomal DNA of quencer (Applied Biosystems, model 310). For genomic A. oryzae as a template and two oligonucleotides, 5P- DNA sequencing, a 7.4-kb EcoRI fragment and a 0.3-kb AA(CT)GCICCI(CT)TIAA(CT)GA(AG)TT(CT)(CT)T-3P or 0.7-kb HindIII fragment within the 7.4-kb EcoRI frag- and 5P-GCIGGIA(AG)(CT)TG(CT)TCIACIGC(AG)TT- ment were subcloned and then sequenced with universal or 3P, as primers. They were designed and synthesized as reverse primers (Takara), or gene-speci¢c oligonucleotides sense and antisense primers corresponding to the N-termi- as primers. For cDNA sequencing, the PCR product was nal amino acid sequence of the mature L3 (NAPLNEFL) subcloned into theEcoRI and BamHI sites of pUC118 and and a part of the cyanogen bromide-cleaved fragment then sequenced. The sequence data were analyzed with (NAVEQLPA), respectively. The ampli¢ed fragments computer programs: GENETYX-MAC (Software Devel- were labeled with a Ready-To-Go DNA labeling kit opment), DNASIS (Hitachi Software Engineering), and (Amersham Pharmacia Biotech) and [K-32P]dCTP was FASTA (Genome Net FASTA server). used as a probe for plaque hybridization. 2.6. Expression of the triacylglycerol lipase (L3) in E. coli 2.3. Construction and screening of a genomic DNA library The plasmid-inserted cDNA encoding the mature L3 (without a signal sequence) was constructed for expression To construct a genomic DNA library, chromosomal in E. coli by IPTG induction. The RT-PCR was performed DNA of A. oryzae RIB128 was prepared as described by with primer TGLAAC-E (5P-CGGAATTCAAACGCTC- Minetoki et al. [8]. The DNA was partially digested with CCCTGAATGAGTTC-3P; the underlining indicates the Sau3AI and then fractionated in a 10^50% sucrose gra- EcoRI site; italics indicate the sequence of positions 91^ dient. DNA fragments of 15^20 kb in length were ligated 111; Fig. 1) and primer TGLTTA-B, and the product was into the BamHI-digested VDASHII vector (Stratagene) subcloned into pUC118 after EcoRI and BamHI digestion, and then packaged in vitro. The library consisted of ap- resulting in the pTGLM plasmid. The transformants of proximately 1U106 independent clones. The recombinant E. coli JM109 (pTGLM) were induced with 2 mM IPTG phage was used for transfection of E. coli XL1-Blue MRA in LB medium at 37³C for 2 h and then collected by (P2), followed by plating on LB medium. The genomic centrifugation. The cells were washed with 50 mM acetate DNA library was screened by plaque hybridization with bu¡er (pH 5.5) and then resuspended in the same bu¡er. a labeled probe at 65³C overnight after transferring it to a The cells in the suspension were disrupted by ultrasonica- Hybond-N nylon membrane (Amersham Pharmacia Bio- tion, and then the resultant homogenate was used to assay tech). Washing was performed three times for 15 min in lipase. Lipase activity was assayed at 30³C for 6 h by the 2USSC/0.1% SDS at 65³C. The inserts isolated from pos- method involving triolein as the substrate, as described itive plaques were subjected to Southern hybridization previously [5]. The amount of total protein was measured after digestion with restriction enzymes. by the method of Lowry et al. [9] with bovine serum al- bumin as the standard.

FEMSLE 9495 26-7-00 J. Toida et al. / FEMS Microbiology Letters 189 (2000) 159^164 161

2.7. SDS^PAGE and zymography cyanogen bromide-cleaved fragment was cloned and sequenced as described above. The resulting fragment was SDS^PAGE of proteins was performed in a 12% poly- 594 bp in size and its sequence was homologous with those acrylamide gel by the method of Laemmli [10]. The trans- of cutinases from fungi. Since the PCR fragment was a formants of E. coli induced with IPTG were collected by part of the triacylglycerol lipase gene, the full-length ge- centrifugation, washed with 50 mM acetate bu¡er (pH nomic DNA was cloned using the fragment as a probe. 5.5), and then suspended in the sample bu¡er. The lysate Southern hybridization of total genomic DNA digested solution was subjected to SDS^PAGE. To detect esterases with EcoRI, BamHI or PstI using the PCR fragment as including lipases, the gel was soaked at room temperature a probe gave one band, suggesting that there is a single in a solution containing 1-naphthylcaprylate instead of 1- copy of the L3 gene in the genome (data not shown). naphthylacetate (Sigma) and Fast Red TR Salt (Aldrich), About 40 000 individual plaques from the genomic DNA and the activity was visualized as a red band [11]. library were screened by plaque hybridization with the 32 P-labeled PCR product described above as a probe, Downloaded from https://academic.oup.com/femsle/article/189/2/159/523283 by guest on 27 September 2021 and six positive clones were selected. Southern blot anal- 3. Results and discussion ysis indicated that the coding region was located in a 7.4-kb region (EcoRI^EcoRI fragment) and therefore 3.1. Isolation of genomic DNA this fragment was cloned into pUC118. Then we sequenced a 1.4-kb region containing the whole L3 gene The N-terminal amino acid sequence of one of the cya- within the 7.4-kb EcoRI fragment. nogen bromide-cleaved fragments (10 kDa) was determined to be SGYSQGXQIVXNAVEQLPAADASKISS- 3.2. Nucleotide sequence of the tglA gene VL (X, unidenti¢ed amino acid). A result of a computer search on the SwissProt data base indicated that this se- The cloned triacylglycerol lipase gene was provisionally quence is homologous with those of cutinases from fungi. designated tglA. The nucleotide sequence of the tglA gene A PCR fragment with two oligonucleotides corresponding comprising 954 nucleotides is shown with the deduced to the N-terminal amino acid sequence of the mature L3 amino acid sequence in Fig. 1. Sequencing of the genomic and a part of the N-terminal amino acid sequence of the DNA and cDNA revealed the presence of three introns of

Fig. 1. Nucleotide sequence of the triacylglycerol lipase gene (tglA)ofA. oryzae. Only the sequence of the non-transcribed DNA strand is numbered from the A (+1) of the translational start codon. The deduced amino acid sequence of the enzyme is shown under the nucleotide sequence, and the amino acid numbers on the right are positions with respect to the N-terminal amino acid of L3. The N-terminal amino acid of the mature L3 protein is Asn at position 31 (indicated by an arrow). An asterisk indicates the stop codon. The protein-coding region is indicated by capital letters. The putative TATA box in the 5P non-coding region, consensus sequences for the 5P and 3P splicing sites or putative internal sequences in introns are underlined. The potential N-glycosylation site is also underlined. The sequence in this paper has been submitted to the DDBJ/EMBL/GenBank DNA data bases under accession number AB039325.

FEMSLE 9495 26-7-00 162 J. Toida et al. / FEMS Microbiology Letters 189 (2000) 159^164 Downloaded from https://academic.oup.com/femsle/article/189/2/159/523283 by guest on 27 September 2021

Fig. 2. Comparison of the deduced amino acid sequences of the tglA gene of A. oryzae (L3) [A. ory Tgl (L3)] with those of the cutinase genes of Botry- tis cinerea (B. cin) [18], Alternaria brassicicola (A. bra) [19], Fusarium solani (F. sol) [20], and A. oryzae (L1) [A. ory Cut (L1)] [6]. The numbers are positions with respect to the N-terminal amino acid of each enzyme. Identical amino acids are shown on a gray background. Overlining above the amino acid sequence indicates the conserved Gly-X-Ser-X-Gly motif in lipase and cutinases. Close squares indicate the serine, aspartic acid and histidine residues which are presumed to form the catalytic triad.

47, 83 and 62 bp, and the sequences of the exon^intron ever, the L3 puri¢ed from the fungal culture was estimated borders in the tglA gene were highly conserved. The 5P and to be 25 kDa in size by SDS^PAGE and 29 kDa by gel 3P splice sites, and putative internal consensus sequence ¢ltration [5]. Since L3 has been detected as a glycoprotein, are GTAAGC, CAG and CTAAC for intron 1; and one potential N-glycosylation site, Asn-Asn-Ser, was GTACGT, CAG and CTGAC for intron 2; and found at positions 226^228, the size di¡erence is consid- GTACGT, TAG and CTGAT for intron 3, respectively ered to be due to the glycosylation of L3. A putative (consensus sequence for 5P splice site, GTPuNGPy; 3P TATA box was present in the 5P non-coding region at splice site, PyAG; putative internal sequence, CTPuAPy) positions 3131 to 3126 (Fig. 1). However, a CAAT box [12]. The tglA gene has an open reading frame encoding and a CT-rich region were not observed. The preference 254 amino acid residues with a calculated molecular mass for A at position 33, which has been reported in many of 25 960 Da (Fig. 1). The amino acid sequences of posi- genes of ¢lamentous fungi [12], was also observed in the tions 31^55 and 167^195 in Fig. 1 are identical to the tglA gene. determined N-terminal sequence of the mature L3 protein A homology search indicated that L3 is homologous to (NAPLNEFLSALLSHLPAIDGTIDAV) [5], and the N- cutinases from fungi. Fig. 2 shows a comparison of the terminal amino acid sequence of a cyanogen bromide- amino acid sequence of L3 with those of cutinases of fun- cleaved fragment described above, respectively. Therefore, gi. The overall amino acid sequence identities of L3 with the signal sequence is concluded to be from Met1 to the cutinases from Botrytis cinerea [18], Alternaria brassi- Arg30, which contains a basic amino acid at position 2 cicola [19], Fusarium solani [20], and A. oryzae (L1) [6] are and a long hydrophobic region at positions 8^23. The 52%, 37%, 35% and 30%, respectively. Lipases and cutin- same cleavage sites after an arginine residue were found ases are known to have the Gly-X-Ser-X-Gly motif and to in L2 of A. oryzae [7], Aspergillus secreted enzymes, ribo- form the catalytic triad consisting of Ser, Asp and His nuclease T1 [13] and polygalacturonase of A. oryzae [14], residues in common. L3 had the Gly-X-Ser-X-Gly se- and xylanases of A. kawachii [15,16] and A. niger [17]. The quence at positions 168^172, and Ser, Asp and His resi- mature L3 protein is predicted to be a polypeptide of 224 dues at 170, 222 and 235, respectively, and thus they were amino acids with a molecular mass of 22 840 Da. How- completely conserved in both L3 and cutinases. In addi-

FEMSLE 9495 26-7-00 J. Toida et al. / FEMS Microbiology Letters 189 (2000) 159^164 163 tion, four cysteine residues, which may contribute to the was con¢rmed to exhibit lipase activity toward triolein. structural stability of L3, were also conserved in these Therefore, we consider that L3 should be classi¢ed as a enzymes. lipase. The amino acid sequence di¡erences of L3 from cutinase, such as in the N-terminal region, may be related 3.3. Expression of the triacylglycerol lipase (L3) in E. coli to its high substrate speci¢city toward triolein.

We attempted to express the triacylglycerol lipase in E. coli using pTGLM constructed by insertion of tglA cDNA Acknowledgements (without a signal sequence) into the EcoRI and BamHI sites of pUC118. The cell homogenates of transformants We wish to thank T. Akeno for DNA sequencing, and harboring plasmids, pUC118 (vector only) and pTGLM K. Iwashita for helpful suggestions. This work was sup- (cDNA without a signal sequence), were assayed for lipase ported in part by a research grant from the Hokuto Foun-

activity. The activity was not detected in E. coli JM109 dation for Bioscience. Downloaded from https://academic.oup.com/femsle/article/189/2/159/523283 by guest on 27 September 2021 (pUC118), while E. coli JM109 (pTGLM) showed 0.95 U (mg total protein)31 activity. Moreover, SDS^PAGE and zymography were carried out to con¢rm the expression of the triacylglycerol lipase (L3), as shown in Fig. 3. SDS^ References PAGE of proteins from E. coli JM109 (pTGLM) gave a [1] Ohnishi, K. (1982) E¡ect of lipase activity on aging and quality of protein band with a molecular mass corresponding to that miso (study on lipids of miso during aging, part I) (in Japanese). of the L3 (calculated 23 kDa, lane 2). Zymography re- Nippon Shokuhin Kogyo Gakkaishi 29, 85^92. vealed a single band of esterase activity (including the li- [2] Ohnishi, K. (1982) Change of lipids during miso fermentation (in pase activity) at the corresponding location (lanes 2 and Japanese). Nippon Jozo Kyokaishi 78, 848^853. 4). These results indicate that the cloned cDNA encodes a [3] Ohnishi, K., Yoshida, Y., Toida, J. and Sekiguchi, J. (1994) Puri¢- cation and characterization of a novel lipolytic enzyme from Asper- functional triacylglycerol lipase. gillus oryzae. J. Ferment. Bioeng. 77, 413^419. L3 is homologous to cutinases. However, the puri¢ed [4] Toida, J., Kondoh, K., Fukuzawa, M., Ohnishi, K. and Sekiguchi, J. L3 is di¡erent from L1 (cutinase) in substrate speci¢city: (1995) Puri¢cation and characterization of a lipase from Aspergillus L3 exhibits high activity toward triacylglycerols with me- oryzae. Biosci. Biotechnol. Biochem. 59, 1199^1203. dium- or long-chain fatty acids and low activity toward [5] Toida, J., Arikawa, Y., Kondoh, K., Fukuzawa, M. and Sekiguchi, J. (1998) Puri¢cation and characterization of triacylglycerol lipase from fatty acid ethyl esters or triacylglycerols with short-chain Aspergillus oryzae. Biosci. Biotechnol. Biochem. 62, 759^763. fatty acids [5]. Moreover, the expressed recombinant L3 [6] Ohnishi, K., Toida, J., Nakazawa, H. and Sekiguchi, J. (1995) Ge- nomic structure and nucleotide sequence of a lipolytic enzyme gene of Aspergillus oryzae. FEMS Microbiol. Lett. 126, 145^150. [7] Tsuchiya, A., Nakazawa, H., Toida, J., Ohnishi, K. and Sekiguchi, J. (1996) Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB)ofAspergillus oryzae. FEMS Microbiol. Lett. 143, 63^67. [8] Minetoki, T., Gomi, K., Kitamoto, K., Kumagai, C. and Tamura, G. (1995) Nucleotide sequence and expression of glucosidase-encoding gene (agdA) from Aspergillus oryzae. Biosci. Biotechnol. Biochem. 59, 1516^1521. [9] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 257^265. [10] Laemmli, U.K. (1970) Cleavage of structural protein during the as- sembly of the head of bacteriology T4. Nature 227, 680^685. [11] Takahashi, S., Ueda, M., Atomi, H., Beer, H.D., Bornscheuer, U.T., Schmid, R.D. and Tanaka, A. (1998) Extracellular production of active Rhizopus oryzae lipase by Saccharomyces cerevisiae. J. Fer- ment. Bioeng. 74, 413^419. [12] Unkles, S.E. (1992) Gene organization in industrial ¢lamentous fungi. In: Applied Molecular Genetics of Filamentous Fungi (Kinghorn, J.R. and Turner, G., Eds.), pp. 28^53. Blackie Academic and Pro- fessional, Glasgow. [13] Fujii, T., Yamaoka, H., Gomi, K., Kitamoto, K. and Kumagai, C. Fig. 3. SDS^PAGE and zymography of proteins from whole cells of E. (1995) Cloning and nucleotide sequence of the ribonuclease T1 gene coli JM109 harboring pUC118 and pTGLM. SDS^PAGE and zymogra- (rntA) from Aspergillus oryzae and its expression in Saccharomyces phy were carried out as described in Section 2. Lane M, molecular mass cerevisiae and Aspergillus oryzae. Biosci. Biotechnol. Biochem. 59, marker proteins (Pharmacia); lanes 1 and 3, pUC118; lanes 2 and 4, 1869^1874. pTGLM. Lanes 1 and 2 on SDS^PAGE correspond to lanes 3 and 4 [14] Kitamoto, N., Kimura, T., Kito, Y., Ohmiya, K. and Tsukagoshi, N. on zymography. The arrow indicates the triacylglycerol lipase (L3). The (1993) Structural features of a polygalacturonase gene cloned from molecular sizes of the marker proteins are shown on the left. Aspergillus oryzae KBN616. FEMS Microbiol. Lett. 111, 37^41.

FEMSLE 9495 26-7-00 164 J. Toida et al. / FEMS Microbiology Letters 189 (2000) 159^164

[15] Ito, K., Ogasawara, H., Sugimoto, T. and Ishikawa, T. (1992) Clon- [18] Van der Vlugt-Bergmans, C.J., Wagemakers, C.A. and van Kan, J.A. ing and sequencing of xynA gene encoding xylanase A of Aspergillus (1997) Cloning and expression of the cutinase A gene of Botrytis kawachii. Biosci. Biotechnol. Biochem. 56, 906^912. cinerea. Mol. Plant Microbe Interact. 10, 21^29. [16] Ito, K., Iwashita, K. and Iwano, K. (1992) Cloning and sequencing of [19] Yao, C. and Koeller, W., GenBank Accession No. U03393. xynC gene encoding xylanase C of Aspergillus kawachii. Biosci. Bio- [20] Soliday, C.L., Dickman, M.B. and Kolattukudy, P.E. (1989) Struc- technol. Biochem. 56, 1338^1340. ture of the cutinase gene and detection of promoter activity in the 5P- [17] Kinoshita, K., Takano, M., Koseki, T., Ito, K. and Iwano, K. (1995) £anking region by fungal transformation. J. Bacteriol. 171, 1942^ Cloning of the xynNB gene encoding xylanase B from Aspergillus 1951. niger and its expression in Aspergillus kawachii. J. Ferment. Bioeng. 79, 422^428. Downloaded from https://academic.oup.com/femsle/article/189/2/159/523283 by guest on 27 September 2021

FEMSLE 9495 26-7-00