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1.
Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene ( vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production. 相似文献
2.
Using genetic engineering, the Vitreoscilla (bacterial) hemoglobin gene ( vgb) was integrated stably into the chromosomes of Pseudomonas aeruginosa and Burkholderia sp. strain DNT. This was done for both wild type vgb and two site-directed mutants of vgb that produce Vitreoscilla hemoglobin (VHb) with lowered oxygen affinities; in all cases functional VHb was expressed. Similar to previous results, the wild type VHb improved growth for both species and degradation of 2,4-dinitrotoluene ( Burkholderia sp.) or benzoic acid ( P. aeruginosa) under both normal and low aeration conditions. Both mutant vgbs enhanced these parameters compared to wild type vgb, and the improvement was seen in both species. The enhancements were generally greater at low aeration than at normal aeration. The results demonstrate the possibility that the positive effects provided by VHb may be augmented by protein engineering. 相似文献
3.
To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene ( mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene ( vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM
production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4%
methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM
content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced
with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the
cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme,
the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris. 相似文献
4.
The influence of bacterial hemoglobin, VHb, on dechlorinationand degradation of 2-chlorobenzoate (2-CBA) by recombinant Burkholderia sp. under variable oxygen availability with an initial dissolved oxygenconcentration of 0.27 mM-0.72 mM was investigated in batch and continuous culture. Abilityto express VHb was provided to recombinant Burkholderia by transformationwith the VHb gene, vgb, on plasmid pSC160. 100% of 0.5 mM CBA was degraded incultures with 85% and 70% of total volume as headspace air in closed reactorsby both wild type and recombinant Burkholderia. The recombinant cultures were able todechlorinate and degrade 100% of the 2-CBA in less than 48 hours at 30 °Ccompared to more than 120 hours for wild type cultures. The rate and extent of CBAdegradation by recombinant cultures with 40% of total volume as headspace air was higher than thoseachieved by wild type cells at the end of the 168 hours of incubation period, 98and 73%, respectively. The chloride released: CBA degraded molar ratio for cultures with 40%of total volume headspace air was nearly stoichiometric (molar ratio = 1.0) for recombinantstrains, whereas it was non-stoichiometric (molar ratio = 0.24)for wild type cells. The results suggest a suicidal meta-pathway for wild type cells and a complete dechlorinationand degradation pathway for recombinant cells under hypoxic conditions.The degradation and dechlorination ability of both types of cells was alsoinvestigated in continuous reactor studies by varying the dilution rate under hypoxicconditions. Regarding potential of the recombinant strain for 2-CBA degradation in eitheropen ecosystems or closed bioreactor bioremediation systems, the stability of the plasmidcontaining vgb in the recombinant cells was also studied; the plasmid was100% stable at 0.025 h -1 dilution rate (1.7 d hydraulic retention time),even after one month. 相似文献
5.
The Vitreoscilla hemoglobin (VHb) gene ( vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation
experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was
reduced from 18 to 12%. The maximum viable cell counts of the VHb + strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb − strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb + strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb − strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production
of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition. 相似文献
6.
The gene ( vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb) was electroporated into Gordonia amarae, where it was stably maintained, and expressed at about 4 nmol VHb g −1 of cells. The maximum cell mass (OD 600) of vgb-bearing G. amarae was greater than that of untransformed G. amarae for a variety of media and aeration conditions (2.8-fold under normal aeration and 3.4-fold under limited aeration in rich medium, and 3.5-fold under normal aeration and 3.2-fold under limited aeration in mineral salts medium). The maximum level of trehalose lipid from cultures grown in rich medium plus hexadecane was also increased for the recombinant strain, by 4.0-fold in broth and 1.8-fold in cells under normal aeration and 2.1-fold in broth and 1.4-fold in cells under limited aeration. Maximum overall biosurfactant production was also increased in the engineered strain, by 1.4-fold and 2.4-fold for limited and normal aeration, respectively. The engineered strain may be an improved source for producing purified biosurfactant or an aid to microorganisms bioremediating sparingly soluble contaminants in situ. 相似文献
7.
Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA−, which, together with W3110recA−vgb+, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production. 相似文献
8.
The gene ( vgb) encoding the hemoglobin (VHb) of Vitreoscilla sp. was cloned into Burkholderia sp. and the effect of VHb on the growth characteristics of genetically engineered Burkholderia (YV1) were compared with wild type Burkholderia (R34) using continuous flow reactors (chemostat) at various dilution rates under aerobic conditions. Batch oxygen uptake
rate showed that YV1 has much higher oxygen uptake rate than R34 (i.e. 0.63 mg O 2/g biomass/min vs. 1.43 mg O 2/g biomass/min for R34 and YV1 respectively at a dilution rate of 1.2 day −1). Monod parameters, maximum growth rate (μ max) and half saturation coefficient (K s) were found to be 7.03 day −1 and 691 mg/L for R34 respectively, compared to 5.49 day −1 and 404 mg/L for YV1 respectively. At low dilution rates (<2.5 day −1), when the substrate is present in low concentrations, the growth yield was much higher in YV1 (0.52) than in R34 (0.37).
Although substrate utilization rates were similar between R34 and YV1, the latter showed much higher oxygen uptake rate than
did R34 at all dilution rates. When the stability of VHb was tested on agar plates containing 40 μg/L of kanamycin and 100
μg/L of ampicillin, vgb gene containing VHb plasmid in YV1 was stable over 82 days. When survivability under oxygen limited conditions was tested,
R34 survived only for 11 days whereas YV1 survived over 25 days in liquid media; in agar plate experiments, R34 did not survive
more than 40 days whereas more than 75% of YV1 survived over 110 days. 相似文献
9.
Using the pUT-miniTn 5 vector system developed by the laboratory of K.N. Timmis, the Vitreoscilla hemoglobin gene ( vgb) was integrated into the chromosomes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemoglobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-type P. aeruginosa and degraded benzoic acid faster than the latter strain at both normal and low aeration. In contrast, the vgb-bearing B. cepacia strain had a growth advantage over the wild-type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no
difference in DNT degradation was seen between the two strains at either normal or low aeration. The results demonstrate the
practicality of enhancing bioremediation with vgb stably integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial
effects to be seen. Journal of Industrial Microbiology & Biotechnology (2001) 27, 27–33.
Received 20 October 2000/ Accepted in revised form 04 May 2001 相似文献
10.
Vitreoscilla hemoglobin (VHb) gene vgb equipped with a native promoter Pvgb or a tac promoter Ptac was introduced into Corynebacterium glutamicum ATCC14067, respectively. Ptac was proven to be more suitable for expressing VHb protein in higher concentration in both Escherichia coli and C. glutamicum strains compared with the native vgb promoter Pvgb. VHb-expressing C. glutamicum exhibited higher oxygen uptake rate and enhanced cell growth. Recombinant C. glutamicum harboring vgb gene equipped with Ptac promoter produced 23% more l-glutamate in shake-flask culture and grew to 30% more cell density and formed 22% more l-glutamate in fermentor studies compared with the wild-type strain. When a site-directed mutagenesis in which Tyr405 was replaced
by a phenylalanine residue (Y405F) was performed on glutamine synthesis gene, recombinant C. glutamicum overexpressing the mutated gene glnA′ was able to produce l-glutamine effectively. Co-expression of vgb and glnA′ genes in C. glutamicum produced 17 g/l l-glutamine in shake flask culture, approximately 30% more than that produced by the recombinant harboring only glnA′ gene. In fermentor cultivation, the recombinant yielded 25% more cells and produced 40.5 g/l l-glutamine. In this study, it was clearly demonstrated that VHb significantly enhanced cell growth, l-glutamate, and l-glutamine production by recombinant C. glutamicum. 相似文献
12.
The hemoglobins found in unicellular organisms show a great deal of chemical reactivity, protecting cells against oxidative stress, and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than their VHb counterparts. Here, the expression of VHb, its effect on the growth and antioxidant enzyme status of cells under different culture conditions was studied by cloning the complete regulatory and coding sequences ( vgb) for VHb in Enterobacter aerogenes. Contrary to what has been reported for Escherichia coli, the expression of vgb in E.aerogenes decreased several fold under 10% of atmospheric oxygen (2% oxygen) and its growth was not greatly improved by the presence of VHb. Measured either as viable cells or total cell mass, untransformed E. aerogenes grew better than the recombinant strains. At the late exponential phase, however, the vgb-bearing strain was determined to have a higher cell number and total cell mass than the strain bearing only the plasmid vector with no vgb insert. The VHb expressing strain also had an oxygen uptake rate several fold higher than its counterparts. Given that oxidative stress may occur upon elevated oxygen exposure and be balanced by the action of antioxi-dative compounds, the level of antioxidative response of E. aerogenes expressing VHb was also studied. The VHb expressing strain had substantially (1.5–2.6-fold) higher catalase activity than strains not expressing VHb. Both VHb+ and VHb- strains, however, showed similar levels of superoxide dismutase activity. The activity of both enzymes was also growth phase dependent. Stationary phase cells of all strains showed 2–5-fold higher activity for these enzymes than cells at the exponential phase. 相似文献
13.
Escherichia coli αDH5 harboring Vitreoscilla hemoglobin gene, vgb ( Escherichia:pUC8:16), and control strains (parental and vgb-lacking transformed strains, E. coli:pUC9) were employed for studying the potential ability of copper uptake. The strains were grown on a minimal medium, 5XM9,
that contained different carbon (mannitol, fructose, lactose, and maltose) and nitrogen (casein and yeast extract) sources.
A clear type–response relationship between the level of copper uptake and the type of carbon and nitrogen sources was observed.
Copper uptake ability was better for vgb-bearing cells grown on either mannitol, lactose, fructose, or maltose as carbon source and casein as well as yeast extract
as nitrogen source compared with the control medium. The increase in copper uptake was in the following order: parental cells,
then pUC9-bearing cells, then VHb-expressing cells. 相似文献
14.
Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene ( tnpR) of transposon Tn 801. Chromosomal integration of pME134 was selected in a recombination-deficient ( rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS 21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec
+; from its chromosome the pME134::IS 21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA
+ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa. 相似文献
15.
We report here novel array of gene cassettes found in single variable region of class 1 integron disseminated in Pseudomonas aeruginosa isolated from a teaching hospital in Nanjing, Jiangsu Province, China. 29 of 47 (61%) P. aeruginosa strains were confirmed haboured class 1 integron, and all the positive strains have the same variable region confirmed by
PCR and RFLP methods. The variable region contained an unreported order of four gene cassettes aac(6′)-II- aadA13- cmlA8- oxa-10. Of those, cmlA8 gene was a variant of cmlA5 encoding non-enzymatic protein which putatively confer resistance to chloramphenicol. Susceptibility testing revealed multidrug-resistant
mechanisms were involved in the class 1 integron positive clinical isolates. And the class 1 integron located on an about
15 kb transferable plasmid was certified by conjugation experiment and plasmid DNA analysis. The macro restriction profile
indicated those clinical strains were clonally related.
These authors contributed equally to this work. 相似文献
16.
The PsADH2-promoter of Pichia stipitis alcohol dehydrogenase II (ADH II) gene was employed to control the expression of Vitreoscilla hemoglobin (VHb) gene in Pichia pastoris. As in P. stipitis, the promoter was also induced microaerobically in P. pastoris. The expression level of VHb in P. pastoris at low O 2 tension (<5% air saturation) was 16 nmol/g dry cell wt, i.e. about 24-fold higher than that at 60% air saturation. The expressed
VHb enhanced growth of P. pastoris under microaerobic conditions. The application of O 2-regulated promoter in P. pastoris revealed that induction of high-level expression of heterologous protein is feasible without addition of supplementary compounds. 相似文献
17.
Given the well-established beneficial effects of Vitreoscilla hemoglobin (VHb) on heterologous organisms, the potential of this protein for the production of L -DOPA and dopamine in two bacteria, Citrobacter freundii and Erwinia herbicola, was investigated. The constructed recombinants bearing the VHb gene ( vgb+) had substantially higher levels of cytoplasmic L -DOPA (112 mg/L for C. freundii and 97 mg/L for E. herbicola) than their respective hosts (30.4 and 33.8 mg/L) and the vgb– control strains (35.6 and 35.8 mg/L). Further, the vgb+ recombinants of C. freundii and E. herbicola had 20-fold and about two orders of magnitude higher dopamine levels than their hosts, repectively. The activity of tyrosine phenol-lyase, the enzyme converting L -tyrosine to L -DOPA, was well-correlated to cytoplasmic L -DOPA levels. As cultures aged, higher tyrosine phenol-lyase activity of the vgb+ strains was more apparent. 相似文献
18.
Summary The pathogenesis of Pseudomonas aeruginosa for the respiratory tract has been examined using hamster tracheal organ cultures. Tracheal rings prepared from male Syrian
hamsters, strain LSH/LAK, were infected with P. aeruginosa for 4 h and processed at 4-h intervals for 24 h for examination by light- and electron microscopy. Tissue destruction was
observed within 8 h after infection with 10 8 colony-forming units (cfu)/ml and within 12 h after infection with 10 4 or 10 6 cfu/ml. Ciliated cells that contained abnormal subcellular organelles were expelled from the epithelium. By 20 h the epithelial
borders were composed primarily of nonciliated cells. Transmission- and scanning electron microscopy revealed details of the
cellular destruction and attachment of P. aeruginosa to the ciliated epithelium. Pseudomonas aeruginosa causes a rapid destruction of the epithelium of hamster trachea in cultures. Hamster tracheal organ cultures have been shown
to be useful in studying the pathogenesis of P. aeruginosa for the respiratory tract.
This work was supported by Grants G-430B and G-431B from the Cystic Fibrosis Foundation. 相似文献
19.
Fusarium venenatum A3/5 was transformed using the Aspergillus niger expression plasmid, pIGF, in which the coding sequence for the F. solani f. sp. pisi cutinase gene had been inserted in frame, with a KEX2 cleavage site, with the truncated A. niger glucoamylase gene under control of the A. niger glucoamylase promoter. The transformant produced up to 21 U cutinase l −1 in minimal medium containing glucose or starch as the primary carbon source. Glucoamylase (165 U l −1 or 8 mg l −1) was also produced. Both the transformant and the parent strain produced cutinase in medium containing cutin. 相似文献
20.
Bacteria isolated from spent mushroom substrate (SMS) were evaluated for the suppression of Pyricularia grisea, the causal agent of gray leaf spot of perennial ryegrass ( Lolium perenne) turf. Thirty-two of 849 bacterial isolates (3.8%) from SMS significantly inhibited the mycelial growth of P. grisea in vitro. Six bacterial isolates that afforded maximum inhibition of P. grisea were also refractory to Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, and Fusarium culmorum. Each of the six isolates was identified as Pseudomonas aeruginosa by fatty acid profile analysis. The biocontrol activity of the bacterial isolates was not compromised by a prolonged exposure to UV radiation in vitro. In controlled-environment chamber experiments, all 32 bacterial isolates were tested for suppression of gray leaf spot on Pennfine perennial ryegrass when applied as seed treatment or foliar sprays. Foliar applications of the bacteria (10 8 cfu/ml 0.1% carboxymethylcellulose), but not the seed treatment, significantly reduced disease severity and incidence. The three most efficient isolates from foliar application treatments, which were among the six bacterial isolates identified as P. aeruginosa, were further evaluated for suppression of gray leaf spot as a function of timing of application. The three isolates of P. aeruginosa suppressed gray leaf spot in perennial ryegrass in Cone-tainers when applied at 1, 3, and 7 days prior to inoculation with P. grisea both in controlled-environment chamber experiments, and in potted ryegrass plants maintained in the field. All application intervals, regardless of the bacterial isolate, provided significant reduction of gray leaf spot severity. Suppression of gray leaf spot by isolates of P. aeruginosa under controlled-environment chamber conditions was not different from that observed in potted ryegrass plants maintained in the field. In field experiments, an isolate of P. aeruginosa provided significant suppression of gray leaf spot when applied at 1, 7, and 14 days prior to inoculation with P. grisea. The bacterium proved effective against gray leaf spot of perennial ryegrass maintained as fairway and rough heights. These results indicate that P. aeruginosa may be a potential biocontrol agent for gray leaf spot of perennial ryegrass turf. 相似文献
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