Molecular cloning of the genes for xylan degradation of Bacillus pumilus and their expression in Escherichia coli

Summary The 7.7 Mdal PstI fragment of Bacillus pumilus IPO containing genes for xylan degradation, xylanase, and -xylosidase was inserted at the PstI site of pBR322 and cloned in E. coli C600. The hybrid plasmid thus formed was named pOXN29. The amount of xylanase and -xylosidase expressed in E. coli harboring pOXN29 was about 6% and 20% of the activity produced by the donor, B. pumilus. The reverse orientation of the inserted fragment resulted respectively in 5 times and 50 times increases in xylanase and -xylosidase productivities. Both enzymes expressed in E. coli transformants were shown to be indistinguishable from those of B. pumilus by immunological and chemical criteria. Digestion of pOXN29 with BglII produced two fragments; one was 6.7 Mdal in size and contained the whole pBR322 and the -xylosidase gene, and the other was 3.7 Mdal and coded for xylanase. Analysis of enzymes expressed in the transformant cells indicated that neither enzyme was secreted into the culture medium, periplasm nor membrane bound, although xylanase but not -xylosidase, was secreted into the medium in a B. pumilus culture.     

Expression of the xylan-degrading genes of Bacillus pumilus IPO in Saccharomyces cerevisiae

Xylanase (xynA) and β-xylosidase (xynB) genes of Bacillus pumilus were expressed in Saccharomyces cerevisiae by using the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter of S. cerevisiae. Yeast cells harboring a plasmid pNAX2 containing xynA produced xylanase in the cytoplasm of the cell to an extent as much as 5% of the total soluble protein in the cell extract. Xylanase produced in yeast had an extra methionine at the N-terminus, but had the same specific activity as that produced by B. pumilus IPO. The xylanase in the yeast was not glycosylated and was immunologically identical to that of B. pumilus IPO. Yeast cells harboring a plasmid pYXB containing xynB produced β-xylosidase in the cytoplasm of the cell (3% of the total soluble protein). β-Xylosidase purified from the yeast strain exhibited specific activity nearly equal to the value of enzyme purified from B. pumilus, and had an N-terminal sequence identical to the sequence of the enzyme from B. pumilus.     

Cloning, expression and nucleotide sequences of two xylanase genes from Paenibacillus sp.

Two genes, xynA and xynB, encoding xylanases from Paenibacillus sp. KCTC 8848P were cloned and expressed in Escherichia coli, and their nucleotide sequences were determined. The xylanases of E. coli transformants were released into the extracellular culture fluid in the absence of xylan. The structural gene of xynA 636 bp, encoded a protein of 212 amino acids, while the xynB gene consisted of 951 bp open reading frame for a protein of 317 amino acids. The amino acid sequence of the xynAgene showed 83% similarity to the xylanase of Aeromonas caviae, and belonged to the family 11 glycosyl hydrolases. The deduced amino acid sequence of the xynB gene, however, showed 51% similarity to the xylanase of Rhodothermus marinus, and belonged to the family 10 glycosyl hydrolases.     

Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74 511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49 883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-11 xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome. Received: 21 September 1998 / Received revision: 30 October 1998 / Accepted: 29 November 1998     

Family 10 and 11 xylanase genes from Caldicellulosiruptor sp. strain Rt69B.1

Three family 10 xylanase genes (xynA, xynB, and xynC) and a single family 11 xylanase gene (xynD) were identified from the extreme thermophile Caldicellulosiruptor strain Rt69B.1 through the use of consensus PCR in conjunction with sequencing and polyacrylamide gel electrophoresis. These genes appear to comprise the complete endoxylanase system of Rt69B.1. The xynA gene was found to be homologous to the xynA gene of the closely related Caldicellulosiruptor strain Rt8B.4, and primers designed previously to amplify the Rt8B.4 xynA gene could amplify homologous full-length xynA gene fragments from Rt69B.1. The complete nucleotide sequences of the Rt69B.1 xynB, xynC, and xynD genes were obtained using genomic walking PCR. The full-length xynB and xynC genes are more than 5 kb in length and encode highly modular enzymes that are the largest xylanases reported to date. XynB has an architecture similar to the family 10 xylanases from Thermoanaerobacterium saccharolyticum (XynA) and Clostridium thermocellum (XynX) and may be cell wall associated, while XynC is a bifunctional enzyme with an architecture similar to the bifunctional β-glycanases from Caldicellulosiruptor saccharolyticus. The xynD gene encodes a two-domain family 11 xylanase that is identical in architecture to the XynB family 11 xylanase from the unrelated extreme thermophile Dictyoglomus thermophilum strain Rt46B.1. The sequence similarities between the Rt69B.1 xylanases with respect to their evolution are discussed. Received: May 13, 1998 / Accepted: October 22, 1998     

Cloning and production of Xylanase B from Paenibacillus barcinonensis in Bacillus subtilis hosts

Xylanase B from Paenibacillus barcinonensis was cloned in shuttle vectors for Escherichia coli and Bacillus subtilis, and expressed in Bacillus hosts. Several recombinant strains were constructed, among which B. subtilis MW15/pRBSPOX20 showed the highest production. This recombinant strain consists of a protease double mutant host containing P. barcinonensis xynB gene under the control of a phage SPO2 strong promoter. Maximum production was found when the strain was cultured in nutrient broth supplemented with xylans. Analysis of xylanase B location in B. subtilis MW15/pRBSPOX20 showed that the enzyme remained cell-associated in young cultures, consistent with its intracellular location in its original host, P. barcinonensis, and the lack of a signal peptide. However, when cultures reached the stationary phase, xylanase B was released to the external medium as a result of cell lysis. The amount of enzyme located in the supernatants of old cultures could account for 50% of total xylanase activity. Analysis by SDS–PAGE showed that xylanase B is an abundant protein found in the culture medium in late stationary phase cultures.     

Synthesis and secretion of a heat-stable carboxymethylcellulose from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus

Summary The cellulase gene celA of Clostridium thermocellum coding for the thermostable endoglucanase A was transferred from Escherichia coli to Bacillus subtilis 168 and B. stearothermophilus CU21 using plasmids derived from the Bacillus vector pUB110. When the structural part of the gene was joined to a pUB110 promoter the recombinant plasmids (pSE102, pSE105) were stably maintained and expressed carboxymethylcellulase (CMCase) activity. In B. stearothermophilus CU21 (pSE105) the clostridial CMCase was produced over a wide temperature range up to the maximal growth temperature (68° C). In contrast to E. coli, all of the CMCase synthesized in bacilli was released into the culture medium. About 50% of the extracellular protein secreted by B. subtilis 168 (pSE102) carrying the celA gene consisted of endoglucanase A. These findings demonstrate the feasibility of producing cellulolytic enzymes from thermophilic anaerobes in bacilli.     

Cloning of a gene for maltohexaose producing amylase of an alkalophilic Bacillus and hyper-production of the enzyme in Bacillus subtilis cells

Summary The gene for maltohexaose producing amylase from an alkalophilic bacterium, Bacillus sp. # 707, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a Bacillus subtilis plasmid pUB110, designated the resulting plasmids as pTUE306 and pTUB812, respectively. A common DNA region of approximately 2.5 kb was defined among the inserted DNAs. The enzymatic activity was lost when a part of the common region was deleted. The plasmids were stably maintained and the gene was well expressed in the bacterium, B. subtilis[pTUB812] which produced more than 70 times higher activity in the culture medium than did Bacillus sp. # 707. The major product of hydrolysis of starch by the enzymes of B. subtilis[pTUB812] and E. coli[pTUE306] was maltohexaose. The cloned gene corresponded to one of the genes for five components of malto-oligosaccharide-producing amylases of Bacillus sp. # 707.Abbreviations G1, G2, G3, G4, G5, and G6 glucose, maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose, respectively - kb kilobase pairs - kdal kilodalton - [] designated plasmid-carrier state     

Secretory expression of a glutamic-acid-specific endopeptidase (SPase) from Staphylococcus aureus ATCC12600 in Bacillus subtilis

Summary In order to obtain a large quantity of glutamic-acid-specific endopeptidase of Staphylococcus aureus ATCC12600 (SPase) without cultivating its pathogenic host bacterium, expression plasmids enabling secretion of SPase from Bacillus subtilis were constructed by inserting the SPase gene into B. subtilis-Escherichia coli shuttle vectors. B. subtilis harbouring a simple recombinant plasmid containing the coding and the 5-flanking regions of SPase in the shuttle vector pHY300PLK secreted 22 mg/l of SPase into the medium. As this level was lower than that of the natural strain (45 mg/l), we tried to increase the expression level by constructing a series of hybrid plasmids with the following features: (1) the terminator sequence of the alkaline protease gene from B. subtilis, (2) the promoter and the leader sequences of the -amylase gene or of alkaline protease gene from B. amyloliquefaciens, (3) the vector pHY300PLK and the fused vector of pHY300PLK and pUB110. By using a variety of hybrid plasmids, the resulting transformants secreted SPase at levels of 33–120 mg/l. The recombinant SPase isolated from the medium was indistinguishable from the natural one with respect to its behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting as well as its enzyme activity.Correspondence to: S. Kakudo     

Distribution and Evolution of the Xylanase Genes xynA and xynB and Their Homologues in Strains of Butyrivibrio fibrisolvens

The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B. fibrisolvens.     

The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg

Summary Using the bifunctional cloning vehicle pHP13, which carries the replication functions of the cryptic Bacillus subtilis plasmid pTA1060, the effects of BsuM restriction on the efficiency of shotgun cloning of heterologous Escherichia coli DNA were studied. In a restriction-deficient but modification-proficient mutant of B. subtilis, clones were obtained at a high frequency, comparable to frequencies normally obtained in E. coli (10⁴ clones per g target DNA). Large inserts were relatively abundant (26% of the clones contained inserts in the range of 6 to 15 kb), which resulted in a high average insert length (3.6 kb). In the restriction-proficient B. subtilis strain, the class of large inserts was underrepresented. Transformation of B. subtilis with E. coli-derived individual recombinant plasmids was affected by BsuM restriction in two ways. First, the transforming activities of recombinant plasmids carrying inserts larger than 4 kb, were, in comparison with the vector pHP13, reduced to varying degrees in the restricting host. The levels of the reduction increased with insert length, resulting in a 7800-fold reduction for the largest plasmid used (pC23; insert length 16 kb). Second, more than 80% of the pC23 transformants in the restricting strain contained a deleted plasmid. In the non-restricting strain, the transforming activities of the plasmids were fairly constant as a function of insert length (in the range of 0–16 kb), and no structural instability was observed. It is concluded that for shotgun cloning in B. subtilis, the use of restriction-deficient strains is highly preferable. Evidence is presented that in addition to XhoI other sequences are involved in BsuM restriction. It is postulated that AsuII sites are additional target sites for BsuM restriction.     

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome

Summary A BamHI restriction endonuclease fragment, B7, which is replicated first among all other fragments derived from the Bacillus subtilis chromosome, was cloned in Escherichia coli using as vector a hybrid plasmid pMS102 that can replicate both in E. coli and B. subtilis. Digestion of pMS102 with BamHI produced two fragments and the smaller one was replaced by the B7 fragment.The cloned plasmid pMS102-B7 exhibited some peculiar properties that were not observed with plasmids containing other fragments from the B. subtilis chromosome. (1) E. coli cells harboring this plasmid stuck to each other and to glass. This property was more apparent when cells were grown in poor media. (2) E. coli cells tended to lose the plasmid spontaneously when they were grown without the selective pressure favorable to the plasmid. (3) The frequency of transformation of B. subtilis by pMS102-B7 was less than 1/1,000 of that by the vector plasmid pMS102. The number of copies of pMS102-B7 present in the transformants was also markedly reduced, although the pUB110 origin of replication on the vector was intact and should be functional in B. subtilis. This inhibitory effect of the B7 fragment on plasmid replication was confirmed more directly by developing a semi in vitro replication system using protoplasts.Both in E. coli and B. subtilis the B7 fragment affected replication of its own molecule but not that of the coexisting plasmid with an identical replication system. The implication of the function of the B7 fragment in the initiation of the B. subtilis chromosome will be discussed.     

Sequence specificity of Bacillus subtilis DNA gyrase in vivo

Linearization of pBG0 (a hydrid between Escherichia coli plasmid pBR322 and Staphylococcus aureus plasmid pUB110) was performed by lysis of the oxolinic acid treated Bacillus subtilis protoplasts with sodium dodecyl sulfate. This plasmid DNA linearization was used both for a detailed mapping of DNA gyrase cleavage sites of various strength and for the nucleotide sequence determinations at the points of gyrase-mediated scission by introducing the XhoI linker DNA. A total of 40 plasmids carrying inserted XhoI linker were sequenced by labeling 3' termini of XhoI sites; 38 of them were found to contain a duplication of four base-pairs of the plasmid sequence flanking the linker, which were characteristic of the oxolinic acid-induced DNA cleavage by E. coli DNA gyrase in vitro and in vivo. The relative strength of these sequenced sites was established by comparing their positions to the sites mapped on the appropriate plasmid genome. This allowed us to propose a consensus sequence of B. subtilis DNA gyrase in vivo cleavage site:GNAT GATCATNC% MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqefm0B1jxALjhiov2D% aebbfv3ySLgzGueE0jxyaibaiiYdd9qrFfea0dXdf9vqai-hEir8Ve% ea0de9qq-hbrpepeea0db9q8as0-LqLs-Jirpepeea0-as0Fb9pgea% 0lrP0xe9Fve9Fve9qapdbaqaaeGacaGaaiaabeqaamaabaabcaGcba% GaaeikaiaabsfacaqGPaGaaeiiaiaabccacaqGGaGaaeiiaiaabcca% caqGOaGaae4raiaabMcacaqGGaGaaeiiaiaabccacaqGGaGaaeiiai% aabccacaqGGaGaaeiiaiaabccacaqGGaGaaeiiaiaabccacaqGGaGa% aeiiaiaabccacaqGOaGaaeyqaiaabMcaaaa!4E92!\[{\rm{(T) (G) (A)}}\]where N is any nucleotide. The bases in parentheses were preferred secondarily. The involvement of DNA gyrase in illegitimate recombination events in Bacillus subtilis is discussed.     

Cloning of a Family 11 Xylanase Gene from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang

Bacillus amyloliquefaciens CH51, an isolate from cheonggukjang, Korean fermented soyfood, secretes several enzymes into culture medium. A gene encoding 19 kDa xylanase was cloned by PCR. Sequencing showed that the gene encoded a glycohydrolase family 11 xylanase and named xynA. xynAHis, xynA with additional codons for his-tag, was overexpressed in Escherichia coli BL21(DE3) using pET-26b(+). XynAHis was purified using HisTrap affinity column. K_m and V_max of XynAHis were 0.363 mg/ml and 701.1 μmol/min/mg, respectively with birchwood xylan as a substrate. The optimum pH and temperature were pH 4 and 25 °C, respectively. When xynA was introduced into Bacillus subtilis WB600, active XynA was secreted into culture medium.     

Cloning, sequencing and expression of a gene encoding a 73 kDa xylanase enzyme from the rumen anaerobe Butyrivibrio fibrisolvens H17c

Summary The cloning, expression and nucleotide sequence of a 3 kb DNA segment on pLS206 containing a xylanase gene (xynB) from Butyrivibrio fibrisolvens H17c was investigated. The open reading frame (ORF) of 1905 by encoded a xylanase of 635 amino acid residues (M_r 73156). At least 850 by at the 3 end of the gene could be deleted without loss of xylanase activity. The deduced amino acid sequence was confirmed by purifying the enzyme and subjecting it to N-terminal amino acid sequence analysis. In Escherichia coli C600 (pLS206) cells the xylanase was localized in the cytoplasm. Its optimum pH for activity was between pH 5.4 and 6, and optimum temperature 55° C. The primary structure of the xylanase showed a significant level of identity with a cellobiohydrolase/endoglucanase of Caldocellum saccharolyticum, as well as with the xylanases of the alkaliphilic Bacillus sp. strain C-125, B. fibrisolvens strain 49, and Pseudomonas fluorescens subsp. cellulosa.Abbreviations ORF open reading frame - pNPCase p-nitrophen-yl--d-cellobiosidase - (xynB) gene coding for XynB - XynB xylanase     

Utilization of promoter regions of coliphage lambda in Bacillus subtilis

Summary In vivo studies with galactokinase monitoring system demonstrated that the coliphage lambda P_RP_L promoter regions could be utilized in B. subtilis. These promoter regions were preferentially utilized during the stationary growth phase of B. subtilis. However, these promoter regions were not controlled by the cI₈₅₇ gene at permissive or non-permissive temperature in B. subtilis, although the P _RM promoter was utilized in B. subtilis. S1-nuclease mapping suggests that B. subtilis RNA polymerase recognizes specific sequences in P _R promoter region that is not utilized in E. coli.     

Isolation of two beta-xylosidase genes of Bacillus pumilus and comparison of their gene products

The chromosomal DNA fragments of Bacillus pumilus IPO, a potent xylan-hydrolyzing bacterium, were ligated to a vector plasmid, pBR322, and used to transform Escherichia coli C600 cells. Two hybrid plasmids, pOXD28 and pOXN29, were found to enable the transformants to produce beta-xylosidase. The former was found to contain a 2.6-MDa Bg/II fragment and the latter, a 7.7-MDa PstI fragment, both coding beta-xylosidase, but xylanase is coded only on the latter hybrid plasmid. The DNAs inserted in both plasmids originated from the B. pumilus chromosome, but from different regions, as shown by Southern hybridization and the analysis of restriction fragments. beta-Xylosidases I and II, coded on pOXN29 and pOXD28 respectively, were purified to homogeneous preparations and compared. Both were dimer enzymes consisting of 65000-70000-Da subunits. Specific activity and the Km value of beta-xylosidase I to p-nitrophenyl beta-D-xyloside as substrate were respectively 100 and 1/40 times those of beta-xylosidase II. The mobilities of beta-xylosidases I and II on polyacrylamide gel electrophoresis were also different. beta-Xylosidase I, the gene of which is located near the xylanase gene on pOXN29, can convert xylooligosaccharides to xylose, but beta-xylosidase II had little activity on xylobiose. These results suggest that beta-xylosidase I is the main enzyme for xylan hydrolysis in B. pumilus.     

Cloning,expression and characterization of glycoside hydrolase family 11 endoxylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> ARA

An endoxylanase gene, xynA, was cloned from Bacillus pumilus ARA and expressed in Escherichia coli. The open reading frame of the xynA gene was 687 bp encoding a signal peptide and a mature xylanase with a molecular mass of 23 kDa. The enzyme was categorized as a glycosyl hydrolase family 11 member based on the sequence analysis of the putative catalytic domain. The recombinant XynA (Bpu XynA) was purified to homogeneity by Ni–NTA and ion exchange chromatography on DEAE–Sepharose FF. The enzyme exhibited highest activity at pH 6.6 and 50°C. The purified Bpu XynA was stable for at least 2 h at 45°C, and retained over 50% residual activity after being incubated at 60°C for 1 h. The activity of the xylanase was not significantly affected by metal ions and EDTA. The K _m and K _cat /K _m of Bpu XynA for oat-spelt xylan were 5.53 mg/ml and 10.14 ml/mg s at 50°C and pH 6.6. The main product of hydrolysis by Bpu XynA was xylooligosaccharide. The results revealed that the consumption of grass xylan by B. pumilus ARA depended on the synergistic reactions of Bpu XynA and Bpu arabinosidase, and that a typical GH11 xylanase e.g. Tla XynA had capability to remove the side chain of xylan. The properties Bpu XynA make it promising for application in the production of Bifidobacterium growth-promoting factors and in feed industry.     

Overproduction and secretion of Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium

A gene coding for endo--1,3-1,4-glucanase (lichenase) containing a recombinant plasmid, pLL200K, was transferred from Bacillus circulans into a new shuttle plasmid, pLLS920, by ligating linearized DNAs of pLL200K and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLLS920 produced the endo--1,3-1,4-glucanase. The enzyme was produced during active growth with maximum activity. The B. subtilis (pLLS920) enzyme was 83 times (8522 mU ml^–1) more active than that of the gene donor cells (103 mU ml^–1). The B. megaterium (pLLS920) enzyme was 7 times (735 mU ml^–1) more active than that of the gene donor cells. While E. coli secreted only about 10% of the produced enzyme, B. subtilis excreted the enzyme completely into the medium and B. megaterium by about 98%. The plasmid pLLS920 was stable in B. megaterium (98%), and in B. subtilis (51%) but not in E. coli (29%).