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Martina Beranová Slavomír Rakouský Zuzana Vávrová Tomáš Skalický 《Plant Cell, Tissue and Organ Culture》2008,94(3):253-259
A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and
cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO4 solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus
(35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium
to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of
microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used
to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria
elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound
facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends
on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency
in flax. 相似文献
3.
Figen Tokatli Canan Tari S. Mehmet Unluturk Nihan Gogus Baysal 《Journal of industrial microbiology & biotechnology》2009,36(9):1139-1148
Aspergillus sojae, which is used in the making of koji, a characteristic Japanese food, is a potential candidate for the production of polygalacturonase
(PG) enzyme, which of a major industrial significance. In this study, fermentation data of an A. sojae system were modeled by multiple linear regression (MLR) and artificial neural network (ANN) approaches to estimate PG activity
and biomass. Nutrient concentrations, agitation speed, inoculum ratio and final pH of the fermentation medium were used as
the inputs of the system. In addition to nutrient conditions, the final pH of the fermentation medium was also shown to be
an effective parameter in the estimation of biomass concentration. The ANN parameters, such as number of hidden neurons, epochs
and learning rate, were determined using a statistical approach. In the determination of network architecture, a cross-validation
technique was used to test the ANN models. Goodness-of-fit of the regression and ANN models was measured by the R
2 of cross-validated data and squared error of prediction. The PG activity and biomass were modeled with a 5-2-1 and 5-9-1
network topology, respectively. The models predicted enzyme activity with an R
2 of 0.84 and biomass with an R
2 value of 0.83, whereas the regression models predicted enzyme activity with an R
2 of 0.84 and biomass with an R
2 of 0.69. 相似文献
4.
The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore
has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to
the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this
strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation
(Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria.
The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like
serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems. 相似文献
5.
Kaieda M Nagayoshi M Hama S Kondo A Fukuda H 《Applied microbiology and biotechnology》2004,65(3):301-305
In the present study, we used gene manipulation to construct a recombinant Aspergillus oryzae strain overexpressing lipase and investigated its application to the optical resolution of chiral compounds. A. oryzae niaD300, which was derived from the wild-type strain RIB40, was used as the host strain. The tglA gene, which encodes a triacylglycerol lipase, was cloned from the A. oryzae niaD300 chromosomal genome, then reintroduced, with and without a secretion-signal sequence, into the genome and expressed under the control of the improved glaA promoter of plasmid pNGA142. The resulting recombinant strain overexpressing A. oryzae lipase was immobilized within biomass-support particles and used as a whole-cell biocatalyst. The immobilized lipase-overexpressing strain with secretion-signal sequence showed high activity and was used to selectively synthesize (R)-1-phenylethyl acetate from (RS)-1-phenylethanol and vinyl acetate. After 48 h reaction at 30°C with molecular sieve 4A, the yield and enantiomeric excess (%ee) of (R)-1-phenylethyl acetate reached approximately 90 and 95%ee, respectively. The whole-cell biocatalyst for optical resolution of chiral compounds produced in this study maintained its activity over 25 batch-reaction cycles. 相似文献
6.
Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of
the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation
of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection
between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively,
under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional
symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a
peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A.
thaliana covering the entire range of its genetic diversity, as well as Arabidopsis
halleri, were found to be susceptible towards Cuscuta
reflexa. 相似文献
7.
Terentiev Y Pico AH Böer E Wartmann T Klabunde J Breuer U Babel W Suckow M Gellissen G Kunze G 《Journal of industrial microbiology & biotechnology》2004,31(5):223-228
An Arxula adeninivorans
integration vector was applied to a range of alternative yeast species including Saccharomyces cerevisiae, Debaryomyces hansenii, Debaryomyces polymorphus, Hansenula polymorpha and Pichia pastoris. The vector harbours a conserved A. adeninivorans-derived 25S rDNA sequence for targeting, the A. adeninivorans-derived TEF1
promoter for expression control of the reporter sequence, and the Escherichia coli-derived hph gene conferring resistance against hygromycin B for selection of recombinants. Heterologous gene expression was assessed using a green fluorescent protein (GFP) reporter gene. The plasmid was found to be integrated into the genome of the various hosts tested; recombinant strains of all species exhibited heterologous gene expressions of a similar high level. 相似文献
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Jaewoo Yoon Shinya Kimura Jun-ichi Maruyama Katsuhiko Kitamoto 《Applied microbiology and biotechnology》2009,82(4):691-701
Aspergillus oryzae has received attention as a host for heterologous protein production. However, A. oryzae has 134 protease genes, which is recognized to be one of the major reasons for the proteolytic degradation of heterologously
produced proteins. We previously reported that double disruption of the protease genes (tppA and pepE) improved heterologous protein (human lysozyme) production by A. oryzae. In this study, we performed successive round of five protease genes (tppA, pepE, nptB, dppIV, and dppV) disruption in A. oryzae by pyrG marker recycling with highly efficient gene-targeting background (ΔligD). The multiple disruption of protease genes were confirmed by Southern blot analysis. Furthermore, the quintuple protease
gene disruptants showed the maximum production level of bovine chymosin (CHY) that was 34% higher than those of the double
protease gene disruptant (ΔtppA ΔpepE). Consequently, we successfully constructed a multiple protease gene disruptant bearing enhanced levels of CHY productivity.
We presented the first evidence that the quintuple disruption of the protease genes improved the production level of a heterologous
protein by A. oryzae.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
11.
Se Whan Park Moon Gyu Chung Hwa Young Lee Jeong Yoon Kim Young Ha Rhee 《Journal of microbiology (Seoul, Korea)》2008,46(6):662-669
An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly
synthesized mature form of Phaz1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing
Phaz1 and its own N-terminal signal peptide (PrePhaz1) enabled the secretion of active Phaz1 into the extracellular medium.
However, the PrePhaz1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid
during the cultivation. In contrast, INPNC-Phaz1 and INPNC-PrePhaz1 fusion constructs did not affect growth of host cells.
INPNC-Phaz1 was successfully displayed on the cell surface with its fusion form, but did not retain Phaz1 activity. In the
case of INPNC-PrePhaz1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and
active Phaz1 was consequently released into the culture medium. The amount of Phaz1 derived from E. coli (INPNC-PrePhaz1) was almost twice as great as that directly expressed from E. coli (PrePhaz1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently
secreted into the extracellular medium when cultivated at 37°C. 相似文献
12.
Inoculation of bioreactors with shake-flask cultures present the organism with an immediate shift from an environment with
little O2 to one in which O2 is typically at 100% saturation. The inoculation of such shake-flasks cultures into bioreactors sparged with 1 vvm air or
1 vvm air/O2 mix i.e. 50% O2 enrichment is an oxidatively stressful event, as judged by immediate increases in the intracellular concentrations of superoxide
anion radical (O2·−) (from 4,600 to 11,600 RLU mg DCW−1 and 5,500 to 23,000 RLU mg DCW−1 respectively) and changes in the activities of the major antioxidant enzymes superoxide dismutase and catalase in all cultures.
There are further effects on metabolic indices, particularly decreased nutrient consumption in oxygenated cultures (from 0.16
to 0.12 g starch g DCW h−1) and decreased protein production, indicating that inoculation of the bioreactor exerts a global burden on the cellular metabolic
networks. 相似文献
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Christopher D. Skory 《Current microbiology》2003,46(1):0059-0064
A thalium chloride-resistant (TlClr) mutant strain and a sodium chloride-resistant (NaClr) mutant strain of the diazotrophic cyanobacterium Anabaena variabilis have been isolated by spontaneous and chemical mutagenesis by using TlCl, a potassium (K+) analog, and nitrosoguanidine (NTG), respectively. The TlClr mutant strain was found to be defective in K+ transport and showed resistance against 10 μM TlCl. However, it also showed sensitivity against NaCl (LD50, 50 mM). In contrast, neither wild-type A. variabilis nor its NaClr mutant strain could survive in the presence of 10 μM TlCl and died even at 1 μM TlCl. The TlClr mutant strain exhibited almost negligible K+ uptake, indicating the lack of a K+ uptake system. High K+ uptake was, however, observed in the NaClr mutant strain, reflecting the presence of an active K+ uptake system in this strain.
DCMU, an inhibitor of PS II, inhibited the K+ uptake in wild-type A. variabilis and its TlClr and NaClr mutant strains, suggesting that K+ uptake in these strains is an energy-dependent process and that energy is derived from photophosphorylation. This contention
is further supported by the inhibition of K+ uptake under dark conditions. Furthermore, the inhibition of K+ uptake by KCN, DNP, and NaN3 also suggests the involvement of oxidative phosphorylation in the regulation of an active K+ uptake system.
The whole-cell protein profile of wild-type A. variabilis and its TlClr and NaClr mutant strains growing in the presence of 50 mM KCl was made in the presence and absence of NaCl. Lack of transporter proteins in TlClr mutant strain suggests that these proteins are essentially required for the active transport and accumulation of K+ and make this strain NaCl sensitive. In contrast, strong expression of the transporter proteins in NaClr mutant strain and its weak expression in wild-type A. variabilis is responsible for their resistance and sensitivity to NaCl, respectively. Therefore, it appears that the increased salt
tolerance of the NaClr mutant strain was owing to increased K+ uptake and accumulation, whereas the salt sensitivity of the TlClr mutant strain was owing to the lack of K+ uptake and accumulation.
Received: 7 March 2002 / Accepted: 8 April 2002 相似文献
16.
SecA is a central component of the bacterial Sec preprotein translocase. Besides the housekeeping SecA (SecA1), some mostly
pathogenic Gram-positive bacteria possess an accessory SecA (SecA2) that is involved in the export of a few substrates only.
Here we show that neither of the two secA homologous genes present in the genome of the non-pathogenic bacterium Corynebacterium glutamicum can be deleted, unless a copy of the respective gene is provided in trans on a plasmid. This finding is in marked contrast to all other cases examined so far making C. glutamicum the first reported bacterium possessing two essential SecA proteins. 相似文献
17.
Microbial cell surface display of foreign proteins has been widely developed for many potential applications in live vaccine
construction, whole-cell biocatalysts, and bioadsorption. To investigate the feasibility of displaying heterologous proteins
on the surface of attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different display systems were built upon a truncated ice nucleation
protein (INP) from Pseudomonas syringae ICMP3023 whose N- and C-terminal domains were considered to be the putative membrane-anchoring motifs. Green fluorescent
protein (GFP), as a reporter, was fused with the display systems in different forms of N-GFP, NC-GFP, and N-GFP-C. Analysis
of the total expression level and surface localization of GFP demonstrated that the truncated P. syringae INP could be used to display foreign protein in V. anguillarum, while the system of N-GFP showed the higher levels of total expression and surface display based on unit cell density among
the three and might be available for further carrier vaccine development. 相似文献
18.
Melzer G Junne S Wohlgemuth R Hempel DC Götz P 《Journal of industrial microbiology & biotechnology》2008,35(6):485-493
The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH. 相似文献
19.
Vrancken K De Keersmaeker S Geukens N Lammertyn E Anné J Van Mellaert L 《Applied microbiology and biotechnology》2007,73(5):1150-1157
Streptomyces is an interesting host for the secretory production of recombinant proteins because of its innate capacity to secrete proteins
at high level in the culture medium. In this report, we evaluated the importance of the phage-shock protein A (PspA) homologue
on the protein secretion yield in Streptomyces lividans. The PspA protein is supposed to play a role in the maintenance of the proton motive force (PMF). As the PMF is an energy
source for both Sec- and Tat-dependent secretion, we evaluated the influence of the PspA protein on both pathways by modulating
the pspA expression. Results indicated that pspA overexpression can improve the Tat-dependent protein secretion as illustrated for the Tat-dependent xylanase C and enhanced
green fluorescent protein (EGFP). The effect on Sec-dependent secretion was less pronounced and appeared to be protein dependent
as evidenced by the increase in subtilisin inhibitor (Sti-1) secretion but the lack of increase in human tumour necrosis factor
(hTNFα) secretion in a pspA-overexpressing strain. 相似文献
20.
Genome sequence analysis of Xanthomonas
oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this
study, biochemical analyses of xanthan produced by a defined set of X. oryzae
gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization
and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL.
In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work. 相似文献