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1.
A transketolase mutant was first isolated from Corynebacterium glutamicum, an organism of industrial importance. The mutant strain exhibited an absolute requirement for shikimic acid or the aromatic
amino acids and vitamins for growth, and also failed to grow on ribose or gluconic acid as sole carbon source, even with the
aromatic supplement. All of these defective properties were fully restored in spontaneous revertants, indicating the existence
of a single transketolase in C. glutamicum that was indispensable both for aromatic biosynthesis and for utilization of these carbohydrates in vivo. The transketolase
mutant accumulated ribulose extracellularly when cultivated in glucose medium with shikimic acid, but no ribose was detected.
Received: 10 April 1998 / Received revision: 26 May 1998 / Accepted: 14 June 1998 相似文献
2.
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
3.
Cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in Corynebacterium glutamicum 总被引:2,自引:0,他引:2
Transketolase is a key enzyme of the nonoxidative pentose phosphate pathway. The effect of its overexpression on aromatic
amino acid production was investigated in Corynebacterium glutamicum, a typical amino-acid-producing organism. For this purpose, the transketolase gene of the organism was cloned on the basis
of its ability to complement a C. glutamicum transketolase mutant with pleiotropically shikimic-acid-requiring, ribose- and gluconic-acid-negative phenotype. The gene
was shown by deletion mapping and complementation analysis to be located in a 3.2-kb XhoI-SalI fragment of the genome. Amplification of␣the gene by use of low-, middle-, and high-copy-number vectors in a C. glutamicum strain resulted in overexpression of transketolase activities as well as a␣protein of approximately 83kDa in proportion to
the copy numbers. Introduction of the plasmids into a tryptophan and lysine co-producer resulted in copy-dependent increases
in tryptophan production along with concomitant decreases in lysine production. Furthermore, the presence of the gene in high
copy numbers enabled tyrosine, phenylalanine and tryptophan producers to accumulate 5%–20% more aromatic amino acids. These
results indicate that overexpressed transketolase activity operates to redirect the glycolytic intermediates toward the nonoxidative
pentose phosphate pathway in vivo, thereby increasing the intracellular level of erythrose 4-phosphate, a precursor of aromatic
biosynthesis, in the aromatic-amino-acid-producing C. glutamicum strains.
Received: 27 July 1998 / Received last revision: 12 October 1998 / Accepted: 24 October 1998 相似文献
4.
Elucidation of anaplerotic pathways in Corynebacterium glutamicum via 13C-NMR spectroscopy and GC-MS
S. M. Park C. Shaw-Reid A. J. Sinskey G. Stephanopoulos 《Applied microbiology and biotechnology》1997,47(4):430-440
We have obtained direct evidence indicating the presence of pyruvate-carboxylating activity in Corynebacterium glutamicum, a lysine-overproducing bacterium. This evidence was obtained through the use of 13C nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectrometry (GC-MS) of secreted metabolites in
a lysine fermentation. The distribution of 13C label after multiple turns in the tricarboxylic acid cycle was accounted for properly to obtain predictions for [13C] metabolite enrichments that were employed in the interpretation of 13C-NMR and GC-MS data. Of critical importance in arriving at the conclusions was the use of C. glutamicum mutants with deletions of the pyruvate kinase and/or phosphoenolpyruvate carboxylase enzymes. Our results demonstrate the presence of pyruvate-carboxylating pathway(s) in C.␣glutamicum operating simultaneously with phosphoenolpyruvate carboxylase, with the latter enzyme contributing approximately 10 % of the total oxaloacetate synthesis during the
lysine-production phase with pyruvate and gluconate as carbon sources. These findings are important for developing strategies
to increase the total carbon flux for synthesis of amino acids of the aspartate family through metabolic engineering.
Received: 11 June 1996 / Received revision: 30 October 1996 / Accepted: 15 November 1996 相似文献
5.
An IS element, termed ISCg2, was identified in the chromosome of Corynebacterium glutamicum ATCC 13032. After screening a cosmid library of the C. glutamicum ATCC 13032 genome, six copies of ISCg2 including their flanking regions were sequenced and analyzed. ISCg2 is 1636 bp in length and has 26-bp imperfect inverted repeats flanked by 3-bp direct repeats. By comparisons with other IS
elements, ISCg2 was classified as a member of the IS30 family of insertion sequences. The six copies of ISCg2 were identical at the nucleotide level and were located in intergenic, AT-rich regions of the chromosome. The regions in
which the six copies of ISCg2 were inserted displayed significant similarities. This similarity extends over a region of 65 bp, which was assumed to be
the target region for ISCg2. Interestingly, five of the six copies of ISCg2 were located adjacent to genes that may be involved in aspartate and glutamate metabolism or its regulation. Investigation
of the distribution of ISCg2 showed that the IS element is restricted to certain C. glutamicum strains. Analysis of various integration regions indicates active transposition of ISCg2 in C. glutamicum.
Received: 7 April 1999 / Accepted: 17 June 1999 相似文献
6.
The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agar-plate assay. The assay involves the substitution of the main carbon source
in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication
and showed pH and temperature optima of 6.5 and 30 °C respectively.
Received: 2 February 1998 / Received revision: 20 April 1998 / Accepted: 27 April 1998 相似文献
7.
A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA 总被引:10,自引:0,他引:10
An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for
electrotransformation at 18 °C instead of 30 °C, and application of a heat shock immediately following electrotransformation.
Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (108 cfu μg−1) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved
the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation
temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to
2.5 × 106 cfu μg−1 xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum.
Received: 26 March 1999 / Received revision: 9 June 1999 / Accepted: 11 June 1999 相似文献
8.
Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1 总被引:1,自引:0,他引:1
A. Corsetti M. Gobbetti J. Rossi P. Damiani 《Applied microbiology and biotechnology》1998,50(2):253-256
Sourdough lactic acid bacteria, cultivated in wheat flour hydrolysate, produced antimould compounds. The antimould activity
varied greatly among the strains and was mainly detected within obligately heterofermentative Lactobacillus spp. Among these, Lb. sanfrancisco CB1 had the largest spectrum. It inhibited moulds related to bread spoilage such as Fusarium, Penicillium, Aspergillus and Monilia. A mixture of acetic, caproic, formic, propionic, butyric and n-valeric acids, acting in a synergistic way, was responsible for the antimould activity. Caproic acid played a key role in
inhibiting mould growth.
Received: 20 January 1998 / Received revision: 17 April 1998 / Accepted: 27 April 1998 相似文献
9.
The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the
previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50 kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors.
Received: 14 January 1998 / Accepted: 31 March 1998 相似文献
10.
N. T. Mlobeli N. A. Gutierrez I. S. Maddox 《Applied microbiology and biotechnology》1998,50(1):125-128
A strain of Bifidobacterium bifidum was grown on different sugars under pH-controlled conditions to estimate some kinetic parameters for growth and product formation.
Glucose was the preferred sugar in terms of growth rate and yield, sugar utilisation rate and acetate formation rate, while
lactose gave considerably lower values for these parameters. When present in a mixture with glucose, the rate of lactose utilisation
was lower than when present on its own.
Received: 24 February 1998 / Received revision: 15 April 1998 / Accepted: 19 April 1998 相似文献
11.
C. Förster S. Marienfeld F. Wendisch R. Krämer 《Applied microbiology and biotechnology》1998,50(2):219-226
Osmoadaption mechanisms of the biotechnologically important hemiascomycete Ashbya gossypii were investigated, thereby distinguishing between halo- and osmotolerance by exposure to NaCl and mannitol stress. We studied
the growth and ultrastructure of differently treated cells and quantified the intracellular contents of compatible solutes
and inorganic ions. Mannitol affected growth of A. gossypii at concentrations above 0.8 M, whereas NaCl inhibited growth at 0.2 M. NaCl-treated cells differed from control cells in
having smaller vacuoles, which occupied a smaller part of the cell volume. Glycerol was found to be the predominant compatible
solute in A. gossypii; accumulation of inorganic ions could not be detected. Measurement of glycerol uptake under isosmotic conditions as well
as upon hyperosmotic stress revealed the existence of a highly active glycerol-uptake system, which, however, was down-regulated
under hyperosmotic stress. Investigation of glycerol biosynthesis by measuring glycerol-3-phosphate dehydrogenase activity
under hyperosmotic conditions indicated that accumulation of glycerol in A. gossypii is almost solely due to biosynthesis.
Received: 13 January 1998 / Received revision: 7 April 1998 / Accepted: 13 April 1998 相似文献
12.
M.-P. Bousquet R.-M. Willemot P. Monsan E. Boures 《Applied microbiology and biotechnology》1998,50(2):167-173
α-Transglucosidase from Talaromycesduponti was used to synthesize different alkyl-α-d-glucosides from α(1-4) linked carbohydrate donors. The enzymatic preparation, purified by a single step, consisting of hydrophobic
interaction chromatography, was sufficiently pure for very stereospecific synthesis to be achieved. Biphasic and homogeneous
organic media could be compared for such purposes. Yields appeared to be two- to threefold higher in low-water biphasic media.
High concentrations of the glucosyl donor were present in the aqueous phase, while water-immiscible alcohols were used as
glucosyl acceptors. The high efficiency of the method was attributed to the shift of the thermodynamic equilibrium thanks
to the extraction of the product from the aqueous phase, where the reaction occurs, into the organic phase. Operated in a
continuous biphasic reactor, T. dupontiα-transglucosidase showed a good thermostability with a half-life of 23 days at 30 °C.
Received: 26 January 1998 / Received revision: 15 April 1998 / Accepted: 19 April 1998 相似文献
13.
14.
Y. Morita Q. Hasan T. Sakaguchi Y. Murakami K. Yokoyama E. Tamiya 《Applied microbiology and biotechnology》1998,50(6):669-675
Protease activity was detected in the culture medium of Flavobacterium balustinum P104 grown at 10 °C, which was isolated from salmon (Oncorhynchus keta) intestine. The enzyme, designated as CP-70 protease, was purified to homogeneity from the culture broth by ion exchange
and gel filtration chromatographyies. The molecular mass of the protease was 70 kDa, and its isoelectric point was close to
3.5. Maximal activity toward azocasein was observed at 40 °C and from pH 7.0 to 9.0. The activity was strongly inhibited by
phenylmethylsulfonyl fluoride, suggesting that the enzyme is a serine protease. The n-terminal amino acid sequence was Asp-Thr-Arg-Gln-Leu-Leu-Asn-Ala-Asn-Ser-Asp-Leu-Leu-Asn-Thr-Thr-Gly-Asn-Val-Thr-Gly-Leu-Thr-Gly-Ala-Phe-Asn-Gly-Glu-Asn.
A search through the database for sequence homology yielded no significant match. The initial cleavage sites for oxidized
insulin B-chain were found to be the Glu13-Ala14 and Phe24-Phe25 bonds. The result of the cleavage pattern of oxidized insulin
B-chain suggests that CP-70 protease has a broader specificity than the other cold-active proteases against the peptide substrate.
Received: 17 April 1998 / Received last revision: 17 June 1998 / Accepted: 10 July 1998 相似文献
15.
B. L. García A. S. Ball J. Rodríguez M. I. Pérez-Leblic M. E. Arias J. L. Copa-Patiño 《Applied microbiology and biotechnology》1998,50(2):213-218
Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates.
Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical
characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast
(a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme
showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C
while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics
and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage
and for biopulping.
Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998 相似文献
16.
A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able
to grow at 42° C but not at 32° C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to
complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed
that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation
codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32° C but not at 42° C, an indication
that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes
of the divE42 mutant and the TR4 suppressor mutant.
Received: 3 March 1998 / Accepted: 14 July 1998 相似文献
17.
C. L. López-Fernández J. Rodríguez A. S. Ball J. L. Copa-Patiño M. I. Pérez-Leblic M. E. Arias 《Applied microbiology and biotechnology》1998,50(2):284-287
bstract The use of the insoluble polysaccharides Avicel and oat-spelt xylan for the binding and subsequent purification of active
xylanases from Streptomyces chattanoogensis was investigated. Maximum recovery of xylanases was achieved with oat-spelt xylan, using NaCl (2 M) to remove active protein.
The application of this technique to the purification of xylanases resulted in the purification of an endoxylanase (CM-2)
with high specific activity (729.5 U mg−1). The properties of the purified enzyme, exhibiting activity and stability between 40 °C and 60 °C and between pH 5 and 8,
suggest a potential role for both the enzyme and the rapid purification protocol in the removal of hemicelluloses from kraft
pulp prior to bleaching.
Received: 6 April 1998 / Accepted: 8 May 1998 相似文献
18.
D. Segura C. Santana R. Gosh L. Escalante S. Sanchez 《Applied microbiology and biotechnology》1997,48(5):615-620
In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited
the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant
to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic
recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying
resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those
obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium
at 6 days of fermentation.
Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997 相似文献
19.
Very good solvent formation rates were observed when Clostridium beijerinckii NRRL B592 was cultivated on different whole potato media. The increase in whole potato concentration contributed to the increased
final solvent concentrations, while the addition of yeast extract or mineral salts gave negative effects. To obtain good solvent
productivities and high final solvent concentrations during batch fermentation, no enzymatic hydrolysis of the potato starch
was necessary, indicating high activity of the clostridial amylases produced by the strain applied.
Received: 17 April 1998 / Received revision: 22 June 1998 / Accepted: 27 June 1998 相似文献
20.
K. Hofvendahl E. W. J. van Niel B. Hahn-Hägerdal 《Applied microbiology and biotechnology》1999,51(5):669-672
Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures
(up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied
by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation
of lactate dehydrogenase and pyruvate formate-lyase.
Received: 14 December 1998 / Received revision: 12 January 1999 / Accepted: 22 January 1999 相似文献