Construction of a xylanase-producing strain of Brevibacterium lactofermentum by stable integration of an engineered xysA gene from Streptomyces halstedii JM8.

A xylanolytic strain of Brevibacterium lactofermentum containing the Streptomyces halstedii His-tagged xysA gene was generated. The new strain contains DNA derived from S. halstedii, expresses xylanolytic activity, and was obtained by an integrative process mediated by a conjugative plasmid targeted to a dispensable chromosomal region located downstream from the essential cell division gene ftsZ. The His-tagged Xys1 enzyme was constitutively expressed under the control of the kan promoter from Tn5 and was easily purified by use of Ni-nitrilotriacetic acid-agarose. The new strain is stable for more than 200 generations, lacks any known antibiotic resistance gene, and does not need any selective pressure to maintain the integrated gene. This strategy can be used to integrate any gene into the B. lactofermentum chromosome and to maintain it stably without the use of antibiotics for selection.     

Streptomyces halstedii K122 produces the antifungal compounds bafilomycin B1 and C1

Streptomyces halstedii K122 was previously found to produce antifungal compounds on solid substrates that inhibit radial growth of fungi among Ascomycetes, Basidiomycetes, Deuteromycetes, Oomycetes, and Zygomycetes, and strongly affected hyphal branching and morphology. During growth of S. halstedii K122 in submerged culture, no antifungal activity could be detected. However, cultivation of S. halstedii in thin (1 mm) liquid substrate layers in large surface-area tissue culture flasks caused intense growth and sporulation of S. halstedii K122, and the biologically active compounds could be extracted from the mycelium with methanol. Antifungal compounds were purified using C18 solid phase extraction and silica gel column chromatography, and identified as bafilomycins B1 and C1, using 2D NMR and FAB MS. Production of bafilomycins, which are specific inhibitors of vacuolar ATPases, has not been reported from S. halstedii previously. Minimum inhibitory concentrations (MIC) of bafilomycins B1 and C1, amphotericin B, and nikkomycin Z were determined at pH 5.5 and 7.0 for the target fungi Aspergillus fumigatus, Mucor hiemalis, Penicillium roqueforti, and Paecilomyces variotii. Penicillium roqueforti was the most sensitive species to all the compounds investigated. The MIC values for amphotericin B were 0.5-4 micrograms.mL-1 for the fungi tested, and pH did not affect the toxicity. The MIC values for nikkomycin Z ranged from < 0.5 microgram.mL-1 for Mucor hiemalis to > 500 micrograms.mL-1 for Aspergillus fumigatus, and pH had no influence on toxicity. Bafilomycins B1 and C1 were equally active against the fungal species tested, with MIC values in the range of < 0.5-64 micrograms.mL-1. All fungi were more sensitive to both bafilomycin B1 and C1 at pH 7.0 than at pH 5.5.     

Identification of a promoter sequence in the plasmid pUL340 of Brevibacterium lactofermentum and construction of new cloning vectors for corynebacteria containing two selectable markers

A strong promoter P1 has been found in plasmid pUL340, a cloning vector used to transform corynebacteria. This promoter is also expressed efficiently in Escherichia coli. A gene (cat) for chloramphenicol acetyltransferase from Streptomyces acrimycini and a gene (hyg) for hygromycin phosphotransferase from Streptomyces hygroscopicus were subcloned in different positions of the Brevibacterium lactofermentum plasmid pUL340. Both resistance genes are expressed in B. lactofermentum from their own promoters or from the endogenous promoter in pUL340. These genes provide useful screening markers for selecting transformants of B. lactofermentum together with the kanamycin-resistance gene from the transposon Tn5.     

Overproduction, purification, and biochemical characterization of a xylanase (Xys1) from Streptomyces halstedii JM8.

Streptomyces halstedii JM8, isolated from straw, produces and secretes into the culture supernatant at least two proteins with hydrolytic activity towards xylan. The cloning of a DNA fragment of this microorganism in several Streptomyces strains permitted us to overproduce both proteins. N-terminal sequence analyses, immunoblot assays, and time course overproduction experiments allowed us to ensure that both xylanases were encoded by the same gene and that the smallest form (35 kDa) originated from the large one (45 kDa) by proteolytic cleavage on the C terminus. The production of both forms was studied in different strains carrying the gene in a multicopy plasmid. The best production was obtained with Streptomyces parvulus transformed with the plasmid pJM9, a pIJ702 derivative, which yielded 144 U/ml. Both forms of the xylanase were purified with a fast-performance liquid chromatography system and characterized biochemically. The optimal pH and temperature, for both, were 6.3 and 60 degrees C, respectively, in 7.5-min assays. Both proteins were highly stable in a wide range of pHs (4 to 10) and temperatures (4 to 50 degrees C); nevertheless, after 1-h incubations, both enzymes lost most of their activity at temperatures over 55 to 60 degrees C. Endoxylanolytic activity was demonstrated in both enzymes, but no beta-xylosidase activity was detected.     

Single mutations of residues outside the active center of the xylanase Xys1 Delta from Streptomyces halstedii JM8 affect its activity

Mutagenesis of the xylanase Xys1 of Streptomyces halstedii JM8 has been done by error prone PCR. Mutants with modified hydrolytic activity were isolated, the recombinant variant proteins purified and the catalytic activities of each one determined and compared with the wild type enzyme. Two of the isolated single point mutants, m1 (G133D) and m8 (N148D), showed 22-25% increase in specific activity towards xylan compared to wild type xylanase. Two other mutants, m5a (D175A) and m7 (T160A), showed a significant reduction in specific activity of 40-50% with respect to the wild type enzyme. These residues are mainly located in the beta alpha-loops of the xylanase, the region showing the main structural divergences within family 10 of xylanases. This study shows the usefulness of random mutagenesis to point out some key residues not directly involved in the active center, but in which mutation produces subtle structural rearrangements affecting the enzymatic function.     

链霉菌Streptomyces olivaceoviridis A1 木聚糖酶基因xynA在大肠杆菌及毕赤酵母中的高效表达

从橄榄绿链霉菌StreptomycesolivaceoviridisA1中克隆出木聚糖酶基因xynA ,将带与不带原基因信号肽编码序列的xynA分别以正确的阅读框架克隆到大肠杆菌表达载体pET 2 2b( )上的pellB信号肽编码序列之后 ,得到 2种构建的重组载体 ,在重组大肠杆菌中木聚糖酶得到了表达 ,表达产物具有生物活性。进一步将不带原基因信号肽编码序列的xynA插入到毕赤酵母转移载体pPIC9中 ,转化毕赤酵母得到重组子 ,在重组子中木聚糖酶基因得到了高效分泌表达 ,在摇床培养水平上的表达量达到 2 0 0mg L ,且表达产物具有生物学活性。     

Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model.

The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum. Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pIJ101, pSB24, and pJV1.     

来源于橄榄绿链霉菌A1的高比活木聚糖酶XYNB的高效表达及性质的比较分析

高效表达高比活木聚糖酶是进一步提高木聚糖酶发酵效价、降低其生产成本的有效途径。将橄榄绿链霉菌(Streptomyces olivaceoviridis) A1的高比活木聚糖酶成熟蛋白编码基因xynB克隆到毕赤酵母表达载体pPIC9中,转化毕赤酵母得到重组酵母,在重组酵母中木聚糖酶基因得到了高效分泌表达,且表达产物具有生物学活性。在3L发酵罐中蛋白表达量约14mg/mL, 酶活性(效价)为1200IU/mL。SDSPAGE分析表明,表达的木聚糖酶XYNBa为糖基化蛋白, 分子量为31kD, 经脱糖基化处理得到21kD 的XYNBb, 与橄榄绿链霉菌A1所产原酶XYNB大小一致。通过对XYNB、XYNBa及XYNBb酶学性质的比较发现：三者在比活性、Vmax及热稳定性方面有较大差异。该酶对不同木聚糖的酶解产物的糖份分析表明：酶解产物的主要成分为木二糖、木三糖和木四糖,占总糖含量的95%以上。     

链霉菌zxy19木聚糖酶酶学性质及酶基因克隆

采用平板筛选法,从红树林放线菌中筛选到一株有较强木聚糖酶活的菌株zxy19,其16SrDNA序列与Streptomyces sampsonii的同源性仅为96%.该菌株木聚糖酶活为852.41 IU/mL,酶反应最适pH值为7,最适反应温度为60℃.用针对木聚糖酶基因保守结构域的一对简并引物扩增到该酶基因部分序列,进而通过反向PCR扩增到了完整的酶基因,对该基因序列分析结果表明此木聚糖酶基因属于糖基水解酶家族11的成员,酶蛋白氨基酸序列与已报道序列同源性最高为79%(Streptomyces lividans xylanase B).构建了该酶重组表达质粒pET-28a-xyl 696,经过IPTG诱导实现了该酶蛋白在大肠杆菌BL21(DE3)中的异源表达,且通过镍柱纯化后的表达产物具有生物学活性.     

Transfer of plasmid DNA to Brevibacterium lactofermentum by electrotransformation

The Escherichia coli-Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed. Optimal conditions gave an efficiency of 10(6) transformants per microgram of DNA. Two recalcitrant strains could be electrotransformed when an ampicillin pretreatment step was used. Electrotransformation experiments using DNAase or different structural forms of plasmid DNA showed that the electrotransformation process is quite different from natural transformation involving competence development. Restriction-modification-proficient B. lactofermentum could be efficiently electrotransformed with pBLA DNA isolated from E. coli. This restriction-modification system therefore seems to be overcome by electrotransformation. Thus electrotransformation may efficiently replace the protoplast bacterial transformation method.     

Cloning and nucleotide sequence of celA1, and endo-beta-1,4-glucanase-encoding gene from Streptomyces halstedii JM8.

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.     

Study on thermostability of phospholipase D from Streptomyces sp

Four phospholipases D (PLDs) in the culture supernatants from Streptomyces strains were purified to conduct a comparative study of their thermostabilities. Among the four purified PLDs, the enzyme from Streptomyces halstedii K1 lost its activity at 45 degrees C. PLD from Streptomyces septatus TH-2 was stable at the same temperature. We determined the nucleotide sequence encoding the PLD gene from S. halstedii K1 (K1PLD). The deduced amino acid sequence showed high homology to that of the PLD gene from S. septatus TH-2 (TH-2PLD). By comparison of the optimum temperature and the thermostability among recombinant PLDs, K1PLD, TH-2PLD and T/KPLD that possessed the N-terminus of TH-2PLD and the C-terminus of K1PLD, T/KPLD showed the properties midway between those of K1PLD and TH-2PLD. It was suggested that the 176 amino acids at C-terminus of Streptomyces PLD were important for its thermostability.     

Syntheses of 4-methylumbelliferyl-beta-D-xylobioside and 5-bromo-3-indolyl-beta-D-xylobioside for sensitive detection of xylanase activity on agar plates

4-Methylumbelliferyl-beta-D-xylobioside (MU-X2) and 5-bromo-3-indolyl-beta-D-xylobioside (BI-X2) were synthesized as substrates for the detection of xylanase activity on agar plates. A family F/10 xylanase from Streptomyces olivaceoviridis E-86 (FXYN) was able to be more sensitively detected than RBB-xylan by using MU-X2 as a substrate. A mutant xylanase E128H/FXYN having only 1/1000 of the activity of FXYN was also able to be detected on the MU-X2 plate but was not detected on the RBB-xylan plate. A family G/11 xylanase from Streptomyces lividans 66 (Xyn B) was not detected on the MU-X2 plate, but it was able to be detected on the RBB-xylan plate, suggesting that the MU-X2 substrate is specific to family F/10 xylanases. However, none of the xylanases were detected effectively by using BI-X2 as a substrate.     

<Emphasis Type="Italic">Streptomyces lividans</Emphasis> and <Emphasis Type="Italic">Brevibacterium lactofermentum</Emphasis> as heterologous hosts for the production of X<Subscript>22</Subscript> xylanase from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

The Aspergillus nidulans gene xlnA coding for the fungal xylanase X₂₂ has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum. Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii. B. lactofermentum was also able to produce xylanase X₂₂, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA. These production values are higher than those previously reported for the heterologous expression of the A. nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml). Moreover, the X₂₂ enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale.     

A hydroxylase-like gene product contributes to synthesis of a polyketide spore pigment in Streptomyces halstedii.

A gene, schC, adjacent to the sch gene cluster encoding the biosynthesis of a polyketide spore pigment in Streptomyces halstedii was sequenced. Its deduced product resembled flavin adenine nucleotide-containing hydroxylases involved in the biosynthesis of polycyclic aromatic polyketide antibiotics and in catabolic pathways of aromatic compounds. When schC was disrupted, the normally green spores of S. halstedii became lilac. An schC-like gene was located in an equivalent position next to a large gene cluster (whiE) known to determine spore pigment in Streptomyces coelicolor A3(2).     

Purification and characterization of a family G/11 beta-xylanase from Streptomyces olivaceoviridis E-86

A beta-xylanase (GXYN) was purified from the culture filtrate of Streptomyces olivaceoviridis E-86 by successive chromatography on DE-52, CM-Sepharose and Superose 12. The molecular mass of the xylanase was estimated to be 23 kDa, indicating that the enzyme consists of a catalytic domain only. The enzyme displayed an optimum pH of 6, a temperature optimum of 60 degrees C, a pH stability range from 2 to 11 and thermal stability up to 40 degrees C. The N-terminal amino acid sequence of GXYN was A-T-V-I-T-T-N-Q-T-G-T-N-N-G-I-Y-Y-S-F-W-, and sharing a high degree of similarity with the N-terminal sequence of xylanases B and C from Streptomyces lividans, indicating GXYN belongs to family G/11 of glycoside hydrolases. GXYN was inferior to xylanase B from Streptomyces lividans in the hydrolysis of insoluble xylan because of its lack of a xylan binding domain.