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1.
<Emphasis Type="Italic">Bacillus megaterium</Emphasis>—from simple soil bacterium to industrial protein production host 总被引:2,自引:0,他引:2
Vary PS Biedendieck R Fuerch T Meinhardt F Rohde M Deckwer WD Jahn D 《Applied microbiology and biotechnology》2007,76(5):957-967
Bacillus megaterium has been industrially employed for more than 50 years, as it possesses some very useful and unusual enzymes and a high capacity
for the production of exoenzymes. It is also a desirable cloning host for the production of intact proteins, as it does not
possess external alkaline proteases and can stably maintain a variety of plasmid vectors. Genetic tools for this species include
transducing phages and several hundred mutants covering the processes of biosynthesis, catabolism, division, sporulation,
germination, antibiotic resistance, and recombination. The seven plasmids of B. megaterium strain QM B1551 contain several unusual metabolic genes that may be useful in bioremediation. Recently, several recombinant
shuttle vectors carrying different strong inducible promoters and various combinations of affinity tags for simple protein
purification have been constructed. Leader sequences-mediated export of affinity-tagged proteins into the growth medium was
made possible. These plasmids are commercially available. For a broader application of B. megaterium in industry, sporulation and protease-deficient as well as UV-sensitive mutants were constructed. The genome sequence of
two different strains, plasmidless DSM319 and QM B1551 carrying seven natural plasmids, is now available. These sequences
allow for a systems biotechnology optimization of the production host B. megaterium. Altogether, a “toolbox” of hundreds of genetically characterized strains, genetic methods, vectors, hosts, and genomic sequences
make B. megaterium an ideal organism for industrial, environmental, and experimental applications. 相似文献
2.
K. M. J. Van Laere G. Beldman A. G. J. Voragen 《Applied microbiology and biotechnology》1997,47(3):231-235
An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum
activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues
established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl
residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed
no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides.
Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996 相似文献
3.
4.
A. E. Cazemier J. C. Verdoes H. J. M. Op den Camp J. H. P. Hackstein A. J. J. van Ooyen 《Applied microbiology and biotechnology》1999,52(2):232-239
A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA
fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A
had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50–55 °C. The recombinant endoglucanase
Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding
domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262–319 and 448–473,
which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.
Received: 14 January 1999 / Received revision: 29 March 1999 / Accepted: 6 April 1999 相似文献
5.
The effect of extracellular pH and dissolved oxygen on regulation of the pcbAB gene in P.␣chrysogenum was examined, using Northern analysis and a reporter gene fusion. It was found that ambient pH markedly affected levels of
pcbAB mRNA whereas maintenance of dissolved oxygen concentration above 10 % had no detectable effect. The presence of a DNA-binding
protein, which binds upstream of the pcbAB translational start codon, was also related to ambient pH. In all fermentations, pcbAB mRNA was most abundant at around the late exponential/early stationary phase of a culture.
Received: 10 May 1996 / Received revision: 14 October 1996 / Accepted: 25 October 1996 相似文献
6.
An expression system based on the Staphylococcus aureus protein A gene (spa) was developed to allow the production and export of proteins in Lactobacillus. Plasmid shuttle vectors were constructed that carried the eZZ gene, a synthetic gene based on the Protein A gene (spa) but lacking the carboxy-terminal membrane-anchoring region. A gene fusion was created between the eZZ gene and the VD4 region
of a chlamydial major outer-membrane protein gene. Expression studies demonstrated the recognition of the spa regulatory signals by several Lactobacillus, with the recombinant protein being expressed (from 0.1 μg of EZZVD4 fusion protein per ml in L. plantarum up to 10 μg of EZZ protein per ml in L. fermentum) and exported (levels up to 20% in L. fermentum) in several Lactobacillus strains.
Received: 27 August 1996 / Received revision: 27 November 1996 / Accepted: 29 November 1996 相似文献
7.
Z. M. Wang K. M. Devos C. J. Liu R. Q. Wang M. D. Gale 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,96(1):31-36
An RFLP-based map consisting of 160 loci was constructed in an intervarietal cross of foxtail millet [Setaria italica (L.) P. Beauv.], Longgu 25×Pagoda Flower Green. The map comprises nine linkage groups, which were aligned with the nine foxtail
millet chromosomes using trisomic lines, and spans 964 cM. The intraspecific map was compared to an interspecific map, constructed
in a S. italica×S. viridis cross. Both the order of the markers and the genetic distances between the loci were highly conserved. Deviations from the
expected 1 : 2 : 1 Mendelian segregation ratios were observed in both the intra- and inter-specific populations. The segregation
data indicate that chromosome VIII in the Longgu 25×Pagoda Flower Green cross carries a gene that strongly affects gamete
fertility.
Received: 18 December 1996 / Accepted: 4 August 1997 相似文献
8.
K. Scheibner M. Hofrichter A. Herre J. Michels W. Fritsche 《Applied microbiology and biotechnology》1997,47(4):452-457
Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood-
and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene
(TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher
amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [14C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [14C]TNT (100 μM corresponding to 4.75 μCi/l) to 14CO2 within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [14C]TNT significantly.
Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996 相似文献
9.
C. Fernández-González J. A. Gil L. M. Mateos A. Schwarzer A. Schäfer J. Kalinowski A. Pühler J. F. Martín 《Applied microbiology and biotechnology》1996,46(5-6):554-558
The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch
of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B.␣lactofermentum ATCC 13869. The hom- and thrB- disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for
lysine biosynthesis to the production of alanine.
Received: 23 January 1996 / Received last revision: 28 July 1996 / Accepted: 5 August 1996 相似文献
10.
The effect of some culture variables in the production of β-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), β-galactosidase production was partially growth-associated,
as 40%–60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex
medium was used, β-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high
levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific β-galactosidase
activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-l fermenter scale, a threefold increase in volumetric
β-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose
feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented.
Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset
of stationary phase, glucose depletion and biomass concentration.
Received: 18 April 1996 / Received revision: 27 August 1996 / Accepted: 6 September 1996 相似文献
11.
J. M. Crous I. S. Pretorius W. H. van Zyl 《Applied microbiology and biotechnology》1996,46(3):256-260
First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1
P
) and terminator (PGK1
T
) sequences on a multicopy episomal plasmid. The resulting construct PGK1
P
-abf B-PGK1
T
was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively.
Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996 相似文献
12.
Degradation of styrene by white-rot fungi 总被引:2,自引:0,他引:2
A. Braun-Lüllemann A. Majcherczyk A. Hüttermann 《Applied microbiology and biotechnology》1997,47(2):150-155
Degradation of styrene in the gaseous phase was investigated for white-rot fungi Pleurotus ostreatus (two strains), Trametes versicolor, Bjerkandera adusta and Phanerochaete chrysosporium. Fungi were grown in liquid culture and the gas/mycelium contact surface was enhanced with the help of perlite. The influence
of various inducers on styrene degradation was studied. The best inducers for styrene degradation were lignosulphonate for
P. ostreatus and T. versicolor and wood meal for B. adusta and P. chrysosoporium. Under these conditions all fungi were able to degrade styrene almost completely in 48 h at a concentration of 44 μmol/250 ml
total culture volume; one strain of P. ostreatus was able to remove 88 μmol styrene under these conditions. Three transformation products of [14C]styrene in cultures of P. ostreatus were identified: phenyl-1,2-ethanediol, 2-phenylethanol and benzoic acid; 4% of the styrene was metabolised to CO2 in 24 h and no other volatile products were found.
Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996 相似文献
13.
N. Sriubolmas W. Panbangred S. Sriurairatana V. Meevootisom 《Applied microbiology and biotechnology》1997,47(4):373-378
Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG
concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations
(up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results
from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and
inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme
(i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation
of proenzyme (i.e., precursor polypeptide lacking a signal peptide).
Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996 相似文献
14.
R. W. Herzog N. K. Singh D. W. Urry H. Daniell 《Applied microbiology and biotechnology》1997,47(4):368-372
A gene for a synthetic protein-based polymer, G-(VPGVG)119-VPGV, coding for the EG-120mer (elastomer), was cloned into a fungal expression vector to allow constitutive expression of
the polymer controlled by the gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter sequence of Aspergillus nidulans. Stable transformants of A. nidulans showed plasmid integration with varying copy number when analyzed by Southern-blot hybridization. Expression of the synthetic
gene was demonstrated by Northern-blot hybridization. However, the translational efficiency for production of the polymer
polypeptide was low, presumably because of certain codons in the polymer gene (CCG and GUA) that are rarely used by A. nidulans. Partial purification by reversible phase transition followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis
revealed the presence of polymer protein in a transformant that contained multiple copies of the polymer gene. This study
represents the first attempt to express a synthetic gene (with no natural analog) in a fungus.
Received: 23 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996 相似文献
15.
The white-rot fungi Trametes versicolor PRL 572, Trametes versicolor MUCL 28407, Pleurotus ostreatus MUCL 29527, Pleurotus sajor-caju MUCL 29757 and Phanerochaete chrysosporium DSM 1556 were investigated for their ability to degrade the polycyclic aromatic hydrocarbons (PAH) anthracene, benz[a]anthracene and dibenz[a,h]anthracene in soil. The fungi were grown on wheat straw and mixed with artificially contaminated soil. The results of this
study show that, in a heterogeneous soil environment, the fungi have different abilities to degrade PAH, with Trametes showing little or no accumulation of dead-end metabolites and Phanerochaete and Pleurotus showing almost complete conversion of anthracene to 9,10-anthracenedione. In contrast to earlier studies, Phanerochaete showed the ability to degrade the accumulated 9,10-anthracenedione while Pleurotus did not. This proves that, in a heterogeneous soil system, the PAH degradation pattern for white-rot fungi can be quite different
from that in a controlled liquid system.
Received: 20 March 1996 / Received revision: 2 July 1996 / Accepted: 8 July 1996 相似文献
16.
Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for α-amylase activity and low protease
activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect
to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal
peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44 960 Da, in accordance with the
loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley
α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-d-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted
and correctly processed by A. niger using its own signal sequence.
Received: 22 August 1997 / Received revision: 21 November 1997 / Accepted: 29 November 1997 相似文献
17.
The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers. Synthetic genes for a designed analog of a
spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered
sizes as a result of gene rearrangements at the time of transformation. Genes up to 3000 codons in length or longer could
be expressed with no evidence of the prevalent truncated synthesis observed for similar genes in Escherichia coli, though genes longer than 1600 codons were expressed less efficiently than shorter genes. Silk-producing P. pastoris strains were stable without selection for at least 100 doublings.
Received: 4 March 1996 / Received revision: 26 June 1996 / Accepted: 12 August 1996 相似文献
18.
H. Okada T. Sekiya K. Yokoyama H. Tohda H. Kumagai Y. Morikawa 《Applied microbiology and biotechnology》1998,49(3):301-308
A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin,
was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed
S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin.
The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa
and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that
the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no
carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and V
max values toward phosphoric-acid-swollen cellulose as substrate, except that their K
m values were about fourfold higher than that of the native enzyme.
Received: 4 August 1997 / Received revision: 13 October 1997 / Accepted: 31 October 1997 相似文献
19.
N. Kitamoto M. Go T. Shibayama T. Kimura Y. Kito K. Ohmiya N. Tsukagoshi 《Applied microbiology and biotechnology》1996,46(5-6):538-544
Two endo-1,4-β-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family
H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family
C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa,
a pH optimum of 5.0 and temperature optimum of 55 °C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and
temperature optimum of 45 °C.
Received: 3 July 1996 / Accepted: 15 July 1996 相似文献