木糖苷酶

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2003, p. 5957–5967 Vol. 69, No. 10 0099-2240/03/$08.00�0 DOI: 10.1128/AEM.69.10.5957–5967.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved. Cloning, Characterization, and Functional Expression of the Klebsiella oxytoca Xylodextrin Utilization Operon (xynTB) in Escherichia coli† Yilei Qian, L. P. Yomano, J. F. Preston, H. C. Aldrich, and L. O. Ingram* Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611 Received 4 June 2003/Accepted 1 August 2003 Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohy- drates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligo- saccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na�/melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase. Cellulose and hemicellulose (primarily methylglucuronoxy- lan) are the most abundant carbohydrate constituents of woody biomass and agricultural residues (2, 9). High cost as- sociated with the depolymerization of these polymers into mo- nomeric sugars is a primary obstacle preventing their use as a feedstock for chemicals and automotive fuels (31, 46). All native lignocellulosic materials must be pretreated to solubilize hemicellulose constituents and expose cellulose surfaces prior to enzymatic degradation. Although hemicellulose can be de- polymerized by mineral acids, conditions required for com- plete hydrolysis generate toxins that complicate biological uti- lization (20, 29, 33, 49). Conditions which are less severe generate soluble xylodextrins (xylooligosaccharides) that must be further degraded prior to entering pentose metabolism. Xylodextrin utilization has been demonstrated in a variety of bacteria (10, 15, 32, 40, 43, 44, 47). Bacillus stearothermophilus contains a gene cluster involved in the transport and metabo- lism of large soluble products from methylglucuronoxylan (40). To facilitate the bioconversion of xylodextrins into useful chemicals, such as ethanol, genes encoding xylosidase and xy- lanase have been expressed in Saccharomyces cerevisiae (23, 25, 26) but with limited success in xylan fermentation. Ethanolo- genic strains of Escherichia coli KO11 and Klebsiella oxytoca M5A1(pLOI555) expressing bacterial xylosidase and xylanase genes have been shown to metabolize xylan and soluble xylo- dextrins by using a complicated two-step process (6). None of these studies, however, have included heterologous genes en- coding xylobiose uptake systems to facilitate metabolism. Isoprimeverose, a xyloside dimer composed of xylose linked �1,6 to glucose, is transported by a proton symport in Lacto- bacillus pentosus (8, 16, 17). This gene has been cloned and expressed at high levels in L. lactis for detailed investigations of transport. Xylobiose uptake in Streptomyces lividans appears to utilize a different mechanism involving an ATP-dependent transport system (19). Fungi such as Aureobasidium pullulans transport xylobiose by an uncharacterized, energy-dependent permease (30). Previous investigations in this and other laboratories devel- oped ethanologenic strains of E. coli and K. oxytoca M5A1 that metabolize all of the monomeric sugar constituents in ligno- cellulose (37, 38, 45). Subsequent studies characterized genes from M5A1 encoding a cellobiose phosphotransferase system and phosphocellobiase (27) and added these genes to eth- anologenic derivatives of E. coli (34). M5A1 was also found to hydrolyze chromogenic xylosides and to metabolize xylobiose (6), consistent with the presence of an efficient xylosidase ac- tivity and uptake system. In this paper we report the cloning and characterization of the xynTB operon encoding a xylobiose/cation symport and a new xylosidase (glycosyl hydrolase family 43). Functional ex- * Corresponding author. Mailing address: Department of Microbi- ology and Cell Science, P.O. Box 110700, University of Florida, Gainesville, FL 32611. Phone: (352) 392-8176. Fax: (352) 846-0969. E-mail: ingram@ufl.edu. † University of Florida Agricultural Experiment Station publication no. R-09658. 5957 by on June 22, 2010 a e m .a sm .o rg D ow nloaded from pression of these K. oxytoca M5A1 genes in ethanologenic E. coli KO11 enabled the metabolism of soluble �-1,4-linked xy- lodextrins containing up to six xylosyl residues. MATERIALS AND METHODS Bacterial strains and media. Bacterial strains and plasmids used in this study are listed in Table 1. Cultures of K. oxytoca M5A1 and E. coli were grown at 37°C in Luria-Bertani (LB) medium supplemented with sugar as indicated. Ampicillin (100 �g/ml) was used for plasmid selection. Bacterial cell mass was estimated by measuring optical density at 550 nm (OD550) by using a Bausch & Lomb Spec- tronic 70 spectrophotometer (330 mg of dry cell weight per liter at an OD550 of 1.0). Isolation of clones containing K. oxytoca xylosidase gene. A pUC18 library containing 4- to 6-kbp Sau3AI fragments of K. oxytoca chromosomal DNA (27) was transformed into E. coli DH5�. A second K. oxytoca library was prepared from 6- to 9-kbp Sau3AI fragments by using vector pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). Both libraries were screened for xylosidase activity (fluorescent colonies) by using LB plates containing ampicillin and 10 �g of 4-methylumbel- liferyl 7-�-D-xylopyranoside (MUX)/ml. Preparation of GAX1 and GAX2 standards. Aldouronic acid standards, 2�-O- (4-O-methyl-�-D-glucopyranosyluronic acid)-D-xylose (methylglucuronoxylose [GAX1]) and 2�-O-(4-O-methyl-�-D-glucopyranosyluronic acid)-D-xylobiose (methylglucuronoxylobiose [GAX2]), were prepared from 4-O-methylglucuron- oxylan using a modification of methods described by Jones et al. (21). 4-O- methylglucuronoxylan was isolated from sweet gum (Liquidambar styraciflua) by alkaline extraction and was structurally defined by 13C nuclear magnetic reso- nance (NMR) spectroscopy (22). After partial acid hydrolysis in 0.1 N H2SO4 at 122°C (30 min), GAX1 and GAX2 were purified by using gel filtration (BioGel P2 [Bio-Rad, Hercules, Calif.] in 0.05 M formic acid). Products were identified by 1H and 13C NMR (K. Zuobi-Hasona, F. M. St. John, J. D. Rice, and J. F. Preston, unpublished data). Uncoupled NMR spectra were obtained by using a Nicollet NT-300 spectrometer at 25°C in the Fourier transform mode at 300 MHz for 1H and 75.45 MHz for 13C. Structural assignments for peaks were made on the basis of those reported for aldouronic acids (1H/13C 2D-NMR) from sunflower (3) and birchwood (7) hemicellulose. Preparation of soluble xylodextrin. A mixture of xylodextrins was prepared by partial acid hydrolysis of birchwood methylglucuronoxylan (Sigma, St. Louis, Mo.) with trifluoroacetic acid (13). Approximately 38 mg of methylglucuronoxy- lan was mixed with 7.5 ml of 1.6 N trifluoroacetic acid in a 15-ml screw-cap tube. Xylan was dispersed by using an ultrasonic water bath prior to hydrolysis at 100°C for 90 min (manual mixing). After cooling to room temperature, hydro- lysates were neutralized over a 15-min period by adding anion exchange resin (�1.5 g of Amberlite IRA 400 in the -OH form). Neutralized hydrolysates were filtered, lyophilized, and dissolved in 150 �l of distilled water (�125 mg of total carbohydrate per ml). Based on thin-layer chromatography and densitometry, approximately half of the soluble xylodextrins contained six or fewer sugar residues: 7.4% xylose, 11.5% xylobiose (X2), 9.6% xylotriose (X3), 5.7% xylote- trose (X4), 7.4% xylopentose (X5), and 3.9% xylohexose (X6). Additional com- pounds were identified as methylglucuronoxylosides but were not quantified. Fermentation of xylodextrins. Seed cultures of K. oxytoca M5A1- and E. coli KO11-harboring plasmids were grown in 250-ml flasks containing 50 ml of LB broth (5% xylose) for 12 h (37°C, 125 rpm). Sufficient culture was harvested by centrifugation to provide 0.17 mg of dry cell weight (approximately 1 ml of cells at an OD550 of 0.5), washed twice with 1 ml of LB lacking sugar, and resuspended in 50 �l of filter-sterilized LB containing 50% (vol/vol) soluble xylodextrins (hydrolysate). Small samples (10 �l) were removed during incubation at 37°C and were stored frozen. Xylodextrins were separated by thin-layer chromatog- raphy as described previously (50). After visualizing with N-(1-naphthyl)ethyl- enediamine reagent (4), relative amounts were estimated by densitometry using Quantity One Software and a VersaDoc Imaging System Model 1000 (Bio-Rad). Measurement of xylosidase and arabinosidase activities. Cultures of K. oxy- toca M5A1 and E. coli KO11 were grown to half maximal density (OD550 of �2.0) in LB containing 5% total sugar (glucose, xylose, or a combination of both). Sufficient culture was harvested by centrifugation to provide approxi- mately 0.33 mg of dry cell weight, washed with 50 mM sodium phosphate buffer (pH 6.8), and resuspended in 1 ml of the same buffer. Cells were permeablized by mixing with 1 drop of 0.1% sodium dodecyl sulfate (SDS) and 2 drops of chloroform for 10 s using a Vortex mixer. Dilutions of permeabilized cells were assayed at 37°C using either 1 mM p-nitrophenyl �-D-xylopyranoside (pNP-XP) or 1 mM p-nitrophenyl-�-L-arabinofuranoside (pNP-AF) as substrate (1 ml total volume). Reactions were terminated by adding 2 ml of 0.5 M sodium carbonate. Protein concentrations were measured by using the Bradford reagent (Bio-Rad). Activity is expressed as nanomoles of p-nitrophenol released per minute per milligram of protein. Construction of a xynTB expression plasmid. The 3.6-kbp AseI-PstI fragment from pLOI3705 containing the ribosomal binding site and full coding region for xynT and xynB (lacking the CRP and XylR regulatory regions) was gel purified. This DNA fragment was blunt ended, ligated behind the lac promoter (PmeI site) in pNEB193 (New England Biolabs, Beverly, Mass.), and transformed into E. coli TOP10F� (Invitrogen). Both orientations were recovered and were des- ignated pLOI3708 (forward with respect to lac promoter) and pLOI3707 (re- verse). For measurement of xylosidase activity, cultures were grown in LB broth lacking sugar. At an OD550 of 0.7, each culture was divided into two flasks. Isopropyl �-D-thiogalactopyranoside (IPTG; 1 mM) was added to one while the other served as a control. After 2 h of further incubation, cells were harvested by centrifugation and were assayed for �-xylosidase and �-arabinosidase activities. A portion of each culture was also harvested and washed in TE buffer (10 mM Tris-Cl, 1 mM EDTA [pH 8.0]) for protein analysis by SDS-polyacrylamide gel TABLE 1. Description of strains and plasmids used in this study Strains and plasmids Description Reference or source Strains E. coli KO11 �frd, CmR, carrying the Zymomonas mobilis pdc adhB cassette 38 E. coli TOP10F� lacIq lacZ�M15 Invitrogen K. oxytoca M5A1 Wild type 37 Plasmids pCR2.1-TOPO 3.9 Kbp, KmR AmpR, pUC origin, TA cloning vector Invitrogen pNEB193 AmpR cloning vector similar to pUC19 New England Biolabs pLOI3701 pUC18 derivative with �7-kbp DNA fragment, xynT� xynB This study pLOI3702 pUC18 derivative with �6.3-kbp DNA fragment, xynT� xynB This study pLOI3703 pUC18 derivative with �5.6-kbp DNA fragment, xynT� xynB This study pLOI3704 pCR2.1-TOPO derivative with �6.0-kbp DNA fragment, xynT� xynB This study pLOI3705 pCR2.1-TOPO derivative with �6.0-kbp DNA fragment, xynTB operon This study pLOI3706 derivative of pLOI3705 with all Klebsiella DNA removed between the two vector EcoRI sites This study pLOI3707 pNEB193 derivative carrying the 3.6-kbp AseI-PstI fragment with the complete xynTB coding regions (lacking regulatory sites); transcribed opposite to the lac promoter This study pLOI3708 pNEB193 derivative containing the 3.6-kbp AseI-PstI fragment with the complete xynTB coding regions (lacking regulatory sites); transcribed in the same direction as the lac promoter This study pLOI3709 Derivative of pLOI3708, �xynT, xynB (918-bp ClaI internal deletion of xynT) This study 5958 QIAN ET AL. APPL. ENVIRON. MICROBIOL. by on June 22, 2010 a e m .a sm .o rg D ow nloaded from FIG. 1. Genes encoding �-xylosidase and xylobiose uptake. (A) K. oxytoca library clones. AseI and PstI restriction sites were used to subclone the full-length xynT and xynB genes lacking CRP and XylR regulatory sequences (solid bars). The directions of the vector lacZ promoters are also shown here. A single scale bar denotes size for all plasmids. (B) Comparison of gene organization in the xynTB region. Each solid rectangle represents a contiguous set of CRP and XylR regulatory sequences. Individual scale bars are included for each plasmid. (C) Comparison of CRP and XylR regulatory sequences associated with the xynTB operon and respective upstream genes in K. oxytoca M5A1 and K. pneumoniae. Sequences are numbered relative to start codons for the unidentified ORF (K. oxytoca M5A1), xylA2 (K. pneumoniae), and xynT (both organisms). Thin arrows indicate direction of transcription for the respective genes. VOL. 69, 2003 XYLODEXTRIN UTILIZATION OPERON (xynTB) FROM K. OXYTOCA 5959 by on June 22, 2010 a e m .a sm .o rg D ow nloaded from 5960 QIAN ET AL. APPL. ENVIRON. MICROBIOL. by on June 22, 2010 a e m .a sm .o rg D ow nloaded from electrophoresis (PAGE) (10 to 15% acrylamide gradient) using a PhastGel system (Amersham, Piscataway, N.J.). Phylogenetic analyses. Homologs of XynB and XynT were identified by BLASTP search (1). Protein sequences were aligned using ClustalX version 1.81 (42). Phylogenetic trees were constructed using MEGA (v.2.1) (http://www.me- gasoftware.net) (24). Phylogenetic relationships were inferred by using the neighbor-joining algorithm and were tested by bootstrap analysis with 1,000 repetitions. Nucleotide sequence accession number. The sequence for the K. oxytoca xynTB operon and upstream region was submitted to GenBank and was assigned ac- cession number AY297960. RESULTS AND DISCUSSION Isolation of the K. oxytoca xynTB operon. Five unique clones exhibiting �-xylosidase activity were initially isolated as fluo- rescent colonies (MUX positive) from the 4- to 6-kbp K. oxy- toca library. Two were weakly fluorescent and contained the previously characterized K. oxytoca casB gene encoding phos- phocellobiase (27). The three remaining clones (Fig. 1A) con- tained an identical open reading frame (ORF) encoding a 559-amino-acid product (64 kDa) that shared homology with a putative xylosidase (XynB) from Lactococcus lactis (accession number NP_267661). This new K. oxytoca gene was also des- ignated xynB. Based on a SignalP analysis (http://www.cbs.dtu .dk/services/SignalP/) (35), K. oxytoca xynB was predicted to encode a cytoplasmic protein that lacks a signal peptide. All three MUX-positive clones (pLOI3701, pLOI3702, and pLOI3703) also included an upstream incomplete ORF that resembled the carboxy terminus of a transport protein in L. lactis. A second K. oxytoca gene library was constructed to facilitate isolation of the complete sequence for the putative transport protein. One MUX-positive clone was recovered that con- tained both genes (pLOI3705). An additional clone was recov- ered that contained a truncated transport gene (pLOI3704). Using the TMpred program (http://www.ch.embnet.org/software /TMPRED_form.html), the putative transporter gene was pre- dicted to encode a membrane protein (484 amino acids) con- taining at least 10 transmembrane helices. This translated se- quence was very similar to XynT in L. lactis (accession number NP_267662; 83% identity). Based on the proximity of the two K. oxytoca genes and their concordant direction of transcrip- tion, both genes are presumed to form a single transcriptional unit, the xynTB operon (Fig. 1B). A divergently transcribed, incomplete ORF was also identi- fied that began 425 bp upstream from the start codon for K. oxytoca xynT (Fig. 1B). This ORF exhibited no significant ho- mology to other sequences in the database. Comparison of K. oxytoca xynT and xynB to the unannotated sequence for K. pneumoniae (http://genome.wustl.edu) readily identified corre- sponding genes. In K. pneumoniae, however, a xylA homolog denoted xylA2 resides upstream from the xynTB region (Fig. 1B) (28). In L. lactis, this upstream region contains a xylose mutarotase (xylM) required for the efficient metabolism of xylan (14). Based on an in silico analysis of the K. pneumoniae genome (28), potential regulatory sites for catabolite repres- sion (CRP) and for xylose induction by XylR were predicted to be within the ORF-xynTB intergenic region. These sequences were very similar in K. oxytoca M5A1 and K. pneumoniae (Fig. 1C) despite the difference in upstream genes (unidentified ORF and xylA2, respectively). In contrast, the gram-positive FIG. 2. Unrooted phylogenetic trees of XynB (A) and XynT (B) homologs. Trees have been provisionally assigned into different groups based on similarities in primary structure of XynB (groups I to IV) and XynT (groups I to V), respectively, and the functions of neighboring genes. Abbreviations for the organisms and their XynB and XynT homologs are listed in alphabetical order with accession numbers in parentheses: Azir, Azospirillum irakense, XynA (AAF66622); Bha, Bacillus halodurans, XynB (BAB07402); BKK, Bacillus sp. strain KK-1, XylB (AAC27699); BlonD, B. longum DJO10A, Blon1245 (ZP_00121429), Blon1244 (ZP_00121428); BlonN, B. longum NCC2705, XynF (AAN25335), BL0183 (AAN24037), LacS (NP_696148); BpuPLS, B. pumilus strain PLS, XynB (AAC97375); BpuIPO, B. pumilus strain IPO, XynB (S19729); Bsu, B. subtilis, XynB (AAB41091), AbnA (CAA99586), YnaJ (NP_389639), YjmB (NP_389113), YdjD (NP_388497); Bts, Bacillus thermodenitrificans TS-3, Abn (BAB64339); Butfi, B. fibrisolvens, XylB (A49776); Cace, C. acetobutylicum, CAC3452 (AAK81382), CAC1529 (NP_348156), CAP0115 (NP_149278), CAC3451 (NP_350041), CAC0694 (NP_347331), CAC3422 (NP_350012); Ccres, C. crescentus CB15, CC0989 (AAK22973), CC2802 (AAK24766), CC0813 (AAK22798); Ceff, Corynebacterium efficiens YS-314, CE2381 (NP_738991); Chlo, Chloroflexus aurantiacus, Chlo2153 (ZP_00019154); Chut, Cytophaga hutchinsonii Chut0663 (ZP_00117295);