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1.
A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse α-amylase, is described.
Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis
B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain. To use the NI cells as a host for heterologous expression, we tried to amend the introduced pYAS/12S vector and
obtain a host strain, NI-C, with stable NI phenotype and trp1 marker restored. Southern analysis of genomic DNA of NI-C suggested that the original pYAS/12S was abnormally rearranged
and not completely corrected. Further assay showed that the viability and mitotic ability of the NI-C strain were increased.
While using the NI-C strain as host for plasmid transformation and heterologous expression of mouse α-amylase, we observed
that transformed colonies grew more quickly and secreted more α-amylase than general yeast strains. A further test showed
that the NI-C strain was able to use mouse α-amylase as a positive selection marker to form transformed colonies on nitrogen-starved
plates that contain starch as the sole carbon source. The results imply that the NI-C variant is an improved pep4 strain that can be used for heterologous expression and for the development of new selective markers in the yeast transformation
system.
Received: 7 January 1998 / Received last revision: 7 September 1998 / Accepted: 11 October 1998 相似文献
2.
The complete nucleotide sequence of pER371, a native plasmid in Streptococcus thermophilus ST137, was determined. A putative open reading frame coding for a replication protein, Rep371, was identified. A characteristic
promoter sequence and ribosome-binding site were found upstream of rep371. Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S. thermophilus cryptic plasmids pST1-No.29 and pST1 respectively. The plus-origin sequence and Rep371 are highly homologous to the corresponding
elements of the Staphylococcus aureus plasmids pC194 and pSK89. A novel 140-nucleotide palindromic minus-origin sequence, which is structurally similar but does
not show sequence homology to the palA region of pC194, was identified in pER371. A palindromic sequence capable of forming a putative hairpin structure was identified
and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids. Cloning vectors derived
from pER371 should provide valuable gene-delivery vehicles for the genetic engineering of lactic acid bacteria.
Received: 25 November 1997 / Received last revision: 13 April 1998 / Accepted: 19 April 1998 相似文献
3.
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
4.
A. Phongdara A. Merckelbach P. Keup G. Gellissen C. P. Hollenberg 《Applied microbiology and biotechnology》1998,50(1):77-84
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA
fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with
a calculated M
r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide
sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved
in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998 相似文献
5.
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
6.
M. A. G. Koffas R. Ramamoorthi W. A. Pine A. J. Sinskey G. Stephanopoulos 《Applied microbiology and biotechnology》1998,50(3):346-352
Pyruvate carboxylase is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth,
or lysine and glutamic acid production in industrial fermentations. We used regions of homology from pyruvate carboxylase
sequences of 12 different species (corresponding to the ATP- and pyruvate-binding sites), to design polymerase chain reaction
(PCR) primers for amplifying a fragment of the pyruvate carboxylase (pc) gene from C. glutamicum genomic DNA. This 850-base-pair fragment was used to probe a C. glutamicum cosmid library and four candidate pc cosmids were identified. The fragment was sequenced and the sequence of the complete gene was obtained by several rounds
of primer synthesis, PCR on one of the positive cosmids, and sequencing. The C. glutamicumpc sequence shows 64% homology with the pc gene of Mycobacterium tuberculosis and 44% homology with the human pc gene. Regions of ATP, pyruvate and biotin binding have also been identified.
Received: 16 December 1997 / Received revision: 31 March 1998 / Accepted: 19 April 1998 相似文献
7.
The xylose reductase gene (XYL1) was isolated from Pichia stipitis and Candida shehatae, cloned into YEp-based vectors under the control of ADH2 and PGK1 promoter/terminator cassettes and introduced into Saccharomyces cerevisiae Y294 by electroporation. Shake-flask fermentations were carried out with 5% xylose and 1% galactose, glucose or maltose as
co-substrates. Xylose uptake was similar in both the recombinant strains when different co-substrates were used and slowed
once the co-substrate was depleted. The recombinant strains converted xylose to xylitol with yields approaching the theoretical
maxima. Xylitol production was most rapid when the co-substrate was still present. Approximately 50% of the xylose was not
metabolized due to the depletion of the co-substrate.
Received: 23 December 1999 / Received revision: 30 June 2000 / Accepted: 1 July 2000 相似文献
8.
A. Blanco P. Díaz J. Martínez T. Vidal A. L. Torres F. I. J. Pastor 《Applied microbiology and biotechnology》1998,50(1):48-54
The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced
amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases.
The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the
presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres
showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced
the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished.
Electron-microscope analysis showed that the surface of straw fibres was modified by CelA.
Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998 相似文献
9.
10.
A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino
acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The
expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor
specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two
proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the
organism was grown under aerobic conditions.
Received: 26 August 1997 / Received revision: 6 November 1997 / Accepted: 21 November 1997 相似文献
11.
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
12.
A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the
minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat
GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 × 10−7 and 4.7 × 10−7 transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous
replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly,
both shuttle vectors could also be mobilized efficiently from E. coli into different H.␣pylori recipients, with pHel2 showing an efficiency of 2.0 × 10−5 transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation
of a recA-deficient H. pylori mutant by the cloned H. pylorirecA
+ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.␣pylori demonstrate the general usefulness of␣this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.
Received: 22 April 1997 / Accepted: 4 November 1997 相似文献
13.
M. E. Forster W. Davison M. Axelsson L. Sundin C. E. Franklin S. Gieseg 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1998,168(5):345-352
Two species of Antarctic fish were stressed by moving them from seawater at −1 °C to seawater at 10 °C and holding them for
a period of 10 min. The active cryopelagic species Pagothenia borchgrevinki maintained heart rate while in the benthic species Trematomus bernacchii there was an increase in heart rate. Blood pressure did not change in either species. Both species released catecholamines
into the circulation as a consequence of the stress. P. borchgrevinki released the greater amounts, having mean plasma concentrations of 177 ± 54 nmol · l−1 noradrenaline and 263 ± 131 nmol · l−1 adrenaline at 10 min. Plasma noradrenaline concentrations rose to 47 ± 14 nmol · l−1 and adrenaline to 73 ± 28 nmol · l−1 in T. bernacchii. Blood from P. borchgrevinki was tonometered in the presence of isoprenaline. A fall in extracellular pH suggests the presence of a Na+/H+ antiporter on the red cell membrane, the first demonstration of this in an Antarctic fish. Treatment with the β-adrenergic
antagonist drug sotalol inhibited swelling of red blood cells taken from temperature-stressed P. borchgrevinki, suggesting that the antiporter responds to endogenous catecholamines.
Accepted: 22 January 1998 相似文献
14.
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
15.
P. J. Punt I. A. van Gemeren J. Drint-Kuijvenhoven J. G. M. Hessing G. M. van Muijlwijk-Harteveld A. Beijersbergen C. T. Verrips C. A. M. J. J. van den Hondel 《Applied microbiology and biotechnology》1998,50(4):447-454
The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous
fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bipA, the BiP-encoding gene from Aspergillus niger and Aspergillus awamori. As this result could imply that BiP plays a role in protein overproduction, the effect of modulation of bipA gene expression on protein secretion was studied in several recombinant strains expressing glucoamylase (glaA) fusion genes. For overproduction of BiPA in these strains, extra copies of the bipA gene under the control of an inducible promoter were introduced. To allow analysis of the effect of a decreased bipA expression level on protein secretion, replacement of the wild-type gene for a bipA gene driven by the glaA promoter was attempted. However, this endeavour failed because of the lethality of this replacement. Although the final amount
of secreted recombinant protein did not change significantly in strains with increased BiPA levels, increased levels of unprocessed
fusion protein were detected in the total protein extracts of these strains.
Received: 9 February 1998 / Received last revision: 26 May 1998 / Accepted: 14 June 1998 相似文献
16.
Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates
would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with
Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase
activity was found when a positive transformant was kept at 19 °C instead of 28 °C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation
made for the S. cerevisiae expression system is of general significance.
Received: 8 December 1998 / Received revision: 9 April 1999 / Accepted: 16 April 1999 相似文献
17.
Heterologous protein production in methylotrophic yeasts 总被引:15,自引:0,他引:15
Gellissen G 《Applied microbiology and biotechnology》2000,54(6):741-750
The facultative methylotrophic yeasts Candida boidinii, Pichia methanolica, Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression. They are based on strong and regulatable promoters for expression
control derived from methanol metabolism pathway genes. An increasing number of biotechnological applications attest to their
status as preferred options among the various gene expression hosts. The well-established P. pastoris and H. polymorpha systems have been utilized in especially competitive and consistent industrial-scale production processes. Pharmaceuticals
and technical enzymes produced in these methylotrophs have either already entered the market or are expected to do so in the
near future. The article describes the present status of the methylotrophic yeasts as expression systems, focusing on applied
examples of the recent past.
Received: 9 May 2000 / Received revision: 20 June 2000 / Accepted: 23 June 2000 相似文献
18.
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
19.
M. Va?tilingom M. Stupar J.-M. Grienenberger J. M. Gualberto 《Molecular & general genetics : MGG》1998,258(5):530-537
A gene coding for a protein that shows homologies to prokaryotic ribosomal protein S2 is present in the mitochondrial (mt)
genome of wheat (Triticum aestivum). The wheat gene is transcribed as a single mRNA which is edited by C-to-U conversions at seven positions, all resulting
in alteration of the encoded amino acid. Homologous gene sequences are also present in the mt genomes of rice and maize, but
we failed to identify the corresponding sequences in the mtDNA of all dicotyledonous species tested; in these species the
mitochondrial RPS2 is probably encoded in the nucleus. The protein sequence deduced from the wheat rps2 gene sequence has a long C-terminal extension when compared to other prokaryotic RPS2 sequences. This extension presents
no similarity with any known sequence and is not conserved in the maize or rice mitochondrial rps2 gene. Most probably, after translation, this peptide extension is processed by a specific peptidase to give rise to the mature
wheat mitochondrial RPS2.
Received: 20 November 1997 / Accepted: 29 January 1998 相似文献
20.
P. Lu B. P. Davis J. Hendrick T. W. Jeffries 《Applied microbiology and biotechnology》1998,49(2):141-146
Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5′-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ“pop-out” cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu− Ura+ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5′-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu−Ura− phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids.
Received: 17 June 1997 / Received revision: 10 September 1997 / Accepted: 14 October 1997 相似文献