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1.
I. Faus C. del Moral N. Adroer J. L. del Río C. Patiño H. Sisniega C. Casas J. Bladé V. Rubio 《Applied microbiology and biotechnology》1998,49(4):393-398
A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated,
and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the
expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet.
Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997 相似文献
2.
N. Kitamoto S. Yoshino M. Ito T. Kimura K. Ohmiya N. Tsukagoshi 《Applied microbiology and biotechnology》1998,50(5):558-563
A xylanase gene, xynF1, was cloned and characterized from a shoyu koji mould Aspergillus oryzae KBN616. The xynF1 gene was found to be comprised of 1484 bp with ten introns. The deduced amino acid sequence encodes a protein consisting
of 327 amino acids (35,402 Da) which is very similar to the fungal family F xylanases such as Aspergillus nidulans XlnC, Aspergillus kawachii XynA and Penicillium chrysogenum XylP. The intron/exon organization of xynF1 is very similar to that of the fungal family F xylanase genes. Plasmid pXPR64, which contains 64 copies of the xynF1 promoter region (PxynF1) in the same direction, was constructed and introduced into A. oryzae. This led to reduced expression of both xylanase and β-xylosidase genes in the transformants.
Received: 18 May 1998 / Received revision: 7 July 1998 / Accepted: 9 July 1998 相似文献
3.
The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative
DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate.
Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 1998 相似文献
4.
M. J. Pettinari G. J. Vazquez N. Krüger P. S. Vary A. Steinbüchel B. S. Méndez 《Applied microbiology and biotechnology》1998,49(6):737-742
A Bacillus megaterium genomic fragment, which encoded an activator homologous to σ54 regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B.␣megaterium screened for β-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a
protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties
by recombinant E. coli strains.
Received: 20 October 1997 / Received revision: 18 February 1998 / Accepted: 23 February 1998 相似文献
5.
High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium 总被引:1,自引:0,他引:1
G. Miksch R. Neitzel K. Friehs E. Flaschel 《Applied microbiology and biotechnology》1999,51(5):627-632
The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome
of Klebsiella planticola with high transposition frequency. A Bacillus hybrid β-glucanase located on an RSF1010-derived plasmid was mobilized from E.coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments
it was shown that the production of β-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter
used for the expression of the kil gene the secretion of β-glucanase into the medium started at the transition from the exponential to the stationary phase,
as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins.
Received: 22 July 1998 / Received revision: 25 November 1998 / Accepted: 29 November 1998 相似文献
6.
A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was
efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several
differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus.
Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999 相似文献
7.
Shaw-Reid CA McCormick MM Sinskey AJ Stephanopoulos G 《Applied microbiology and biotechnology》1999,51(3):325-333
The N-succinyl-ll-diaminopimelate desuccinylase gene (dapE) in the four-step succinylase branch of the l-lysine biosynthetic pathway of Corynebacterium glutamicum was disrupted via marker-exchange mutagenesis to create a mutant strain that uses only the one-step meso-diaminopimelate dehydrogenase branch to overproduce lysine. This mutant strain grew and utilized glucose from minimal medium
at the same rate as the parental strain. In addition, the dapE
−
strain produced lysine at the same rate as its parent strain. Transformation of the parental and dapE
−
strains with the amplified meso-diaminopimelate dehydrogenase gene (ddh) on a plasmid did not affect lysine production in either strain, despite an eightfold amplification of the activity of the
enzyme. These results indicate that the four-step succinylase pathway is dispensable for lysine overproduction in shake-flask
culture. In addition, the one-step meso-diaminopimelate dehydrogenase pathway does not limit lysine flux in Corynebacterium under these conditions.
Received: 20 May 1998 / Received revision: 12 August 1998 / Accepted: 3 September 1998 相似文献
8.
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
9.
Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene 总被引:2,自引:0,他引:2
The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant
Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage
continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability,
and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell
yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen
on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture.
The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high
yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress
and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity
over that of a single-stage continuous culture.
Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998 相似文献
10.
Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface 总被引:2,自引:2,他引:0
Murai T Ueda M Shibasaki Y Kamasawa N Osumi M Imanaka T Tanaka A 《Applied microbiology and biotechnology》1999,51(1):65-70
The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization
of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated.
The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal
half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase
activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying
glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only
glucoamylase.
Received: 16 June 1998 / Received last revision: 21 August 1998 / Accepted: 3 September 1998 相似文献
11.
C. J. Park J. H. Lee S.-S. Hong H.-S. Lee S. C. Kim 《Applied microbiology and biotechnology》1998,50(1):71-76
To produce a large quantity of the angiotensin-converting-enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino
acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers
of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units
in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers
by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl β-d-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly
as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were
purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was
obtained from 1 l E. coli culture harboring pETYG9, which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1
in molecular mass, amino acid sequence and ACE-inhibiting activity.
Received: 6 January 1998 / Received revision: 23 February 1998 / Accepted: 24 February 1998 相似文献
12.
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
13.
Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the
Escherichia coliβ-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in
both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the
gratuitous lac inducer isopropyl β-d-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the plac/uidA fusion, was tested for β-glucuronidase activity. We found that the expression of the plac/uidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression
in the clp -deficient mutant.
Received: 1 April 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996 相似文献
14.
Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease
domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular
presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal
sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments
including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin
produced was shown to be biologically active.
Received: 2 December 1996 / Received revision: 17 March 1997 / Accepted: 27 April 1997 相似文献
15.
Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects 总被引:4,自引:0,他引:4
R. J. Gouka P. J. Punt C. A. M. J. J. van den Hondel 《Applied microbiology and biotechnology》1997,47(1):1-11
Filamentous fungi are widely used for the production of homologous and heterologous proteins but, compared to homologous proteins,
the levels of production of heterologous proteins are usually low. During the last 5 years, the levels of production of heterologous
proteins have been drastically improved by fusing the corresponding gene to the 3' end of a homologous gene, encoding a well-secreted
protein such as glucoamylase. Nevertheless, little research has been carried out to determine the limitations that hamper
heterologous protein production. Recently we have carried out a detailed analysis of the levels of production of several proteins
and glucoamylase fusion proteins in defined recombinant Aspergillus awamori strains. In this review we will focus on the use of filamentous fungi for the production of heterologous, especially non-fungal,
proteins. In particular, the effect of gene-fusion strategies will be reviewed. Furthermore, the remaining limitations in
heterologous protein production and suggestions for improvement strategies for overproduction of these protein will be discussed.
Received: 5 July 1996 / Accepted: 6 September 1996 相似文献
16.
Domingues L Onnela ML Teixeira JA Lima N Penttilä M 《Applied microbiology and biotechnology》2000,54(1):97-103
One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being
the flocculating yeast production system. Also, lactose is available in large amounts as a waste product from cheese production
processes. The construction of flocculent and non-flocculent brewer's yeast strains secreting β-galactosidase and growing
on lactose is presented. A plasmid was constructed coding for an extracellular β-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper. This selective marker allows for the transformation of wild-type yeasts. This work
represents an important step towards the study of heterologous protein secretion by flocculent cells.
Received: 13 January 2000 / Accepted: 23 January 2000 相似文献
17.
B. Díez E. Mellado M. Rodríguez E. Bernasconi J. L. Barredo 《Applied microbiology and biotechnology》1999,52(2):196-207
The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its use in gene expression. The nucleotide sequence of a 2816-bp genomic fragment
was determined, showing an open reading frame of 1600 bp interrupted by two introns, of 160 bp and 57 bp respectively, with
fungal consensus splice-site junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate
dehydrogenase enzymes, especially to those from the fungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chrysogenum strains with different penicillin productivity. The use of the gdhA promoter for homologous and heterologous gene expression in fungi and Escherichia coli was analyzed. Heterologous gene expression was ascertained by the construction of gene fusions with the lacZ gene from E. coli and the bleomycin-resistance determinant (ble
R) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-biosynthetic genes pcbC and penDE from P. chrysogenum and the cephalosporin biosynthetic genes cefEF and cefG from Acremonium chrysogenum.
Received: 2 November 1998 / Received revision: 15 January 1999 / Accepted: 5 March 1999 相似文献
18.
19.
N. Michel-Reydellet N. Desnoues M. de Zamaroczy C. Elmerich P. A. Kaminski 《Molecular & general genetics : MGG》1998,258(6):671-677
This work reports the characterisation of the Azorhizobium caulinodans amtB gene, the deduced protein sequence of which shares similarity to those of several ammonium transporters. amtB is located downstream from glnK, a glnB-like gene. It is cotranscribed with glnK from an NtrC- and σ54-dependent promoter. glnK and amtB insertion mutant strains have been isolated. Methylammonium uptake was assayed in these strains and in other mutant strains
in which the regulation of nitrogen metabolism is impaired. Our data suggest that the AmtB protein is an ammonium transporter,
which is mainly regulated by NtrC in response to nitrogen availability.
Received: 2 February 1998 / Accepted: 20 March 1998 相似文献
20.
The effect of biomass concentration on the formation of Aspergillus oryzaeα-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of α-amylase formation in chemostats decreased significantly
with increasing biomass concentration in the range of approx. 2–12 g dry weight kg−1. When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzaeα-amylase gene (creA) was deleted, no significant decrease in the specific rate of α-amylase formation was observed. On the basis of the experimental
results, it is suggested that the low value of the specific α-amylase productivity observed at high biomass concentration
is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium.
Received: 13 January 2000 / Received revision: 30 June 2000 / Accepted: 1 July 2000 相似文献