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1.
Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping 总被引:7,自引:0,他引:7
Analysis of the primary structure of peptide synthetases involved in the non-ribosomal synthesis of peptide antibiotics has
revealed a highly conserved and ordered modular arrangement. A module contains at least two domains, involved in ATP-dependent
substrate activation and thioester formation. The occurrence and arrangement of these functional building blocks is associated
with the number and order of the amino acids incorporated in the peptide product. In this study, we present data on the targeted
exchange of the leucine-activating module within the three-module surfactin synthetase 1 (SrfA-A) of Bacillus subtilis. This was achieved by engineering several hybrid srfA-A genes, which were introduced into the surfactin biosynthesis operon by in vivo recombination. We examined the hybrid genes
for expression and investigated the enzymatic activities of the resulting recombinant peptide synthetases. For the first time,
we demonstrate directly that an individual minimal module, of bacterial or fungal origin, confers its amino acid-specific
activity on a multi-modular peptide synthetase. Furthermore, it is shown that directed incorporation of ornithine at the second
position of the peptide chain induces a global alteration in the conformation of surfactin and may result in premature cyclization
or a branched cyclic structure.
Received: 10 July 1997 / Accepted: 11 September 1997 相似文献
2.
Candida bombicola produces glycolipids containing sophorose and a glycosidically/esterically bound ω- or (ω−1)-hydroxy C16(18) acid. Here we describe novel glycolipids from this source. Glucose and 2-dodecanol were used for the cultivation of the yeast,
one part of the racemic secondary alcohol being connected directly with a glucose or a sophorose unit. A relatively high content
of yeast extract, up to 4 g l−1, and subsequently higher biomass concentrations favoured the production of novel products. The provision of 150 g l−1 glucose and 15 g l−1 2-dodecanol resulted in maximum production of 22 g l−1 novel alkyl glycosides (more than 90% novel products). The molecular structures were analysed by gas chromatography, fast
atom bombardment/mass spectrometry, 1H- and 13C-nuclear magnetic resonance and optical rotation studies. Sophorose and glucose were detected as carbohydrate moieties, (S)-(+)-2-dodecanol (88%) was found to be the major lipid moiety. The new glycolipids are suitable biosurfactants, reducing
the surface tension of water from 72 mN m−1 to 32–38 mN m−1.
Received: 8 December 1997 / Received revision: 19 March 1998 / Accepted: 20 March 1998 相似文献
3.
Production of sophorolipids from whey 总被引:5,自引:0,他引:5
Otto RT Daniel HJ Pekin G Müller-Decker K Fürstenberger G Reuss M Syldatk C 《Applied microbiology and biotechnology》1999,52(4):495-501
Sophorolipids, obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results
showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude
product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example,
surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified
mono- and diacetylated (17-hydroxyoctadecenoic)-1′,4′′-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate
to good surface active properties (SFTmin 39 mN m−1, CMC 130 mg l−1), water solubilities (2–3 g l−1) and low cytotoxicities (LC50 300–700 mg l−1). In contrast, purified sophorolipids were more surface active (SFTmin 36 mN m−1, CMC 10 mg l−1), less water soluble (max. 70 mg l−1) and showed stronger cytotoxic effects (LC50 15 mg l−1). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available
lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases
had no effect.
Received: 23 February 1999 / Received revision: 27 May 1999 / Accepted: 28 May 1999 相似文献
4.
Koizumi S Yonetani Y Maruyama A Teshiba S 《Applied microbiology and biotechnology》2000,53(6):674-679
Improved strains for the production of riboflavin (vitamin B2) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host
strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities
in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream
region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity
of guanosine 5′-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas
that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing
the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to
higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement,
the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin
was produced at the level of 15.3 g l−1 for 72 h in a 5-l jar fermentor without any end product inhibition.
Received: 23 August 1999 / Received revision: 13 October 1999 / Accepted: 5 November 1999 相似文献
5.
Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under
natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It
also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study,
AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green
plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its
hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed
that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m−1 (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m−1. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 106 ± 0.05 × 106 N m−2 (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive
strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer
strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably
lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition
of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging
of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties
of plant glycoproteins that have potential utility as adhesives.
Received: 22 February 2000 / Accepted: 20 April 2000 相似文献
6.
Biosurfactants containing rhamnose and β-hydroxydecanoic acid and called rhamnolipids are reviewed with respect to microbial
producers, their physiological role, biosynthesis and genetics, and especially their microbial overproduction, physicochemical
properties and potential applications. With Pseudomonas species, more than 100 g l−1 rhamnolipids were produced from 160 g l−1 soybean oil at a volumetric productivity of 0.4 g l−1 h−1. The individual rhamnolipids are able to lower the surface tension of water from 72 mN m−1 to 25–30 mN m−1 at concentrations of 10–200 mg l−1. After initial testing, rhamnolipids seem to have potential applications in combating marine oil pollution, removing oil
from sand and in combating zoosporic phytopathogens. Rhamnolipids are also a source of l-rhamnose, which is already used for the industrial production of high-quality flavor components.
Received: 1 July 1998 / Received revision: 11 September 1998 / Accepted: 13 September 1998 相似文献
7.
Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli 总被引:2,自引:0,他引:2
Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder
as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l−1 and 50 g l−1 respectively in 47 h. When concentrated whey solution containing 210 g l−1 lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l−1 and 69 g l−1 respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry
weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate.
Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998 相似文献
8.
A quantitative study of indole-3-acetic acid (IAA) turnover, and the contribution of tryptophan-dependent and tryptophan-independent
IAA-biosynthesis pathways, was carried out using protoplast preparations and shoot apices obtained from wild-type and transgenic,
IAA-overproducing tobacco (Nicotiana tabacum L.) plants, during a phase of growth when the level of endogenous IAA was stable. Based on the rate of disappearance of [13C6]IAA, the half-life of the IAA pool was calculated to be 1.1 h in wild-type protoplasts and 0.8 h in protoplasts from the
IAA-overproducing line, corresponding to metabolic rates of 59 and 160 pg IAA (μg Chl)−1 h−1, respectively. The rate of conversion of tryptophan to IAA was 15 pg IAA (μg Chl)−1 h−1 in wild-type protoplasts and 101 pg IAA (μg Chl)−1 h−1 in protoplasts from IAA-overproducing plants. In both instances, IAA was metabolised more rapidly than it was synthesised
from tryptophan. As the endogenous IAA pools were in a steady state, these findings indicate that IAA biosynthesis via the
tryptophan-independent pathway was 44 pg IAA (μg Chl)−1 h−1 and 59 pg IAA (μg Chl)−1 h−1, respectively, in the wild-type and transformed protoplast preparations. In a parallel study with apical shoot tissue, the
presumed site of IAA biosynthesis, the rate of tryptophan-dependent IAA biosynthesis exceeded the rate of metabolism of [13C6]IAA despite the steady state of the endogenous IAA pool. The most likely explanation for this anomaly is that, unlike the
protoplast system, injection of substrates into the apical tissues did not result in uniform distribution of label, and that
at least some of the [2H5]tryptophan was metabolised in compartments not normally active in IAA biosynthesis. This demonstrates the importance of using
experimental systems where labelling of the precursor pool can be strictly controlled.
Received: 18 January 2000 / Accepted 24 February 2000 相似文献
9.
Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus 总被引:1,自引:0,他引:1
Camilo Canel M. Inês Lopes-Cardoso Serap Whitmer Leslie van der Fits Giancarlo Pasquali Robert van der Heijden J. Harry C. Hoge Robert Verpoorte 《Planta》1998,205(3):414-419
Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan
decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures
established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures
transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the
pathway. Two such lines accumulated over 200 mg · L−1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and
tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines
showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the
medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity,
but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and
appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive
over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway
will yield positive results only in the presence of a favorable epigenetic environment.
Received: 12 June 1997 / Accepted: 24 October 1997 相似文献
10.
Tudzynski P Hölter K Correia T Arntz C Grammel N Keller U 《Molecular & general genetics : MGG》1999,261(1):133-141
A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot
alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding
gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3′-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similiarity to fungal modular peptide synthetases. The protein
contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal
peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide
assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative
oxido-reductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions
of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway.
Received: 26 August 1998 / Accepted: 19 October 1998 相似文献