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1.
An Escherichia coli strain, JM109, was successfully engineered into an efficient hyaluronic acid (HA) producer by co-expressing the only known
class-II HA synthase from a Gram-negative bacterium (Pasteurella multocida) and uridine diphosphate-glucose dehydrogenase from E. coli K5 strain. The engineered strain produced about 0.5 g/L HA in shake flask culture and about 2.0–3.8 g/L in a fed-batch fermentation
process in a 1-L bioreactor. The sharp increase in viscosity associated with HA accumulation necessitated pure oxygen supplement
to maintain fermentation in aerobic regime. Precursor supply during HA synthesis was probed by glucosamine supplement, which
shortens biosynthesis pathway and eliminates one step requiring ATP. HA synthesis was increased with glucosamine supplement
from 2.7 to 3.7 g/L (37%), which was mirrored with a concomitant 42% decrease in pure oxygen input, suggesting a close connection
between energy metabolism and precursor supply. Decoupling HA synthesis from cell growth by using fosfomycin (an inhibitor
for cell wall synthesis) led to a 70% increase in HA synthesis, suggesting detrimental effects on HA synthesis from cell growth
via precursor competition. This study demonstrates a potentially viable process for HA based on a recombinant E. coli strain. In addition, the precursor supply limitation identified in this study suggests new engineering targets in subsequent
metabolic engineering efforts. 相似文献
2.
Withu Choosri Regina Paukner Petra Wührer Dietmar Haltrich Christian Leitner 《World journal of microbiology & biotechnology》2011,27(6):1349-1353
The gene gaoA encoding the copper-dependent enzyme galactose oxidase (GAO) from Fusarium graminearum PH-1 was cloned and successfully overexpressed in E. coli. Culture conditions for cultivations in shaken flasks were optimized, and optimal conditions were found to be double-strength
LB medium, 0.5% lactose as inducer, and induction at the reduced temperature of 25°C. When using these cultivation conditions ~24 mg
of active GAO could be produced in shaken flasks per litre medium. Addition of copper to the fermentation medium decreased
the enzyme production significantly. The His-tagged recombinant enzyme could be purified conveniently with a single affinity
chromatography step. The purified enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 66 kDa and had
kinetic properties similar to those of the fungal wild-type enzyme. 相似文献
3.
Regulation of <Emphasis Type="Italic">lac</Emphasis> Transcription in Antibiotic-treated <Emphasis Type="Italic">E. coli</Emphasis> 总被引:16,自引:0,他引:16
Chloramphenicol stabilizes pre-existing lac mRNA, but inhibits further accumulation by allowing rapid degradation of nascent message. Puromycin accelerates decay of pre-existing and new lac message by discharging protective ribo-somes. Both effects are partially reversed by the suA mutation. 相似文献
4.
Recombinant human interferon-gamma (rhIFN-γ) is a protein of great potential for clinical therapy due to its multiple biological
activities. However, overexpressing rhIFN-γ in Escherichia coli was found to accumulate as cytoplasmic inclusion bodies. In this work, a system for soluble and active expression of rhIFN-γ
was constructed by coexpressing chaperonin GroEL/GroES in E. coli. The rhIFN-γ gene was fused to a pET-28a expression vector, and rhIFN-γ was partially expressed as the soluble form following
coexpression with a second vector producing chaperonin GroEL/GroES. The fermentation of recombinant E. coli harboring rhIFN-γ and GroEL/GroES plasmids was investigated, and the optimized conditions were as follows: culture temperature
of 25°C, incubation time of 8 h, isopropyl-β-d-thio-galactoside concentration of 0.2 mM, and l-arabinose concentration of 0.5 g/L. As a result, the expression level of rhIFN-γ was improved accordingly by 2.2-fold than
the control, while a significantly positive correlation was also found between the ratio of supernatant to precipitate of
rhIFN-γ and the amount of chaperonin. Circular dichroism spectra, fluorescence spectra, size exclusion chromatography, and
chemical cross-linking method were applied to characterize rhIFN-γ, indicating that the three-dimensional structure of rhIFN-γ
was identical to that of the native rhIFN-γ. The enzyme-linked immunosorbent assay for active rhIFN-γ quantification showed
that coexpression yielded 72.91 mg rhIFN-γ per liter fermentation broth. Finally, protein–protein interactions between rhIFN-γ
and chaperonin were analyzed using the yeast two-hybrid system, which provided the direct evidence that chaperonin GroEL/GroES
interacted with rhIFN-γ to increase the soluble expression and presented the potential in producing efficiently recombinant
proteins. 相似文献
5.
Sereikaite J Statkute A Morkunas M Radzevicius K Borromeo V Secchi C Bumelis VA 《Applied microbiology and biotechnology》2007,74(2):316-323
Escherichia coli cells expressing mink (Mustela vison) growth hormone were grown in a batch fermentation process. The expression level was estimated to be 27% of the total cellular
protein after 3 h of induction with 1 mM isopropyl β-d-thiogalactoside (IPTG). If the expression of mink growth hormone (mGH) was induced with 0.2 mM IPTG, the concentration of
target protein was slightly lower and was found to be 23% at the same time after induction. mGH expressed as inclusion bodies
was solubilized in 8 M urea and renatured by dilution protocol at a protein concentration of 1.4–2.1 mg/ml in the presence
of glutathione pair in a final concentration of 11.3 mM. [GSH]/[GSSG] ratio equal to 2/1 was used. Two-step purification process
comprising of ion-exchange chromatography on Q-Sepharose and hydrophobic chromatography on Phenyl-Sepharose was developed.
Some 25–30 mg of highly purified and biologically active mGH was obtained from 4 g of biomass. The method presented in this
study allows producing large quantities of mGH and considering initiation of scientific investigation on mGH effect on mink
in vivo and availability in fur industry. 相似文献
6.
Xylella fastidiosa was the first phytopathogen to be completely sequenced, and its genome revealed several interesting features to be used in functional studies. In the present work, the htpX gene, which encodes a protein involved in the heat shock response in other bacteria, was analyzed by RT-PCR by using cells derived from different cultural conditions. This gene was induced after a temperature upshift to 37°C after growth in minimal medium, XDM, but showed constitutive expression in rich medium or in XDM plus plant extracts. Sequences upstream to the htpX gene, containing a putative regulatory region, were also transferred to E. coli, and the thermoregulation was maintained in the new host, since it was constitutively transcribed at 37°C or 45°C in all culture media tested, but not at 28°C in minimal culture medium. The gene was also cloned into the expression vector pET32Xa/LIC, and the expression of the corresponding protein was confirmed by Western blotting. 相似文献
7.
Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner.
The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate
larvae Galleria
mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host. 相似文献
8.
Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes. 相似文献
9.
Yu YJ Wu SC Chan HH Chen YC Chen ZY Yang MT 《Applied microbiology and biotechnology》2008,81(3):523-532
A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular
mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized
as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed
activity equivalent to the authentic mature TGase.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
10.
In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase. 相似文献
11.
Lee YK Yoon BD Yoon JH Lee SG Song JJ Kim JG Oh HM Kim HS 《Applied microbiology and biotechnology》2007,75(3):567-572
The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed
into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound
extracted from the E. coli transformant exhibited a different R
f value of 0.52 from B. subtilis C9 or authentic surfactin (R
f = 0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing
activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon. 相似文献
12.
The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae. 相似文献
13.
V. N. Verbenko L. V. Kuznetsova E. P. Krupyan V. I. Shalguev 《Russian Journal of Genetics》2009,45(10):1192-1199
Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed
1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA
+ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein. 相似文献
14.
Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of
enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality.
Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers
such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory
strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular
production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies
have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached.
This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that
significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials
and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy. 相似文献
15.
De Mey M De Maeseneire S Soetaert W Vandamme E 《Journal of industrial microbiology & biotechnology》2007,34(11):689-700
Escherichia coli remains the best-established production organism in industrial biotechnology. However, when aerobic fermentation runs at
high growth rates, considerable amounts of acetate are accumulated as by-product. This by-product has negative effects on
growth and protein production. Over the last 20 years, substantial research efforts have been expended on reducing acetate
accumulation during aerobic growth of E. coli on glucose. From the onset it was clear that this quest would not be a simple or uncomplicated one. Simple deletion of the
acetate pathway reduced the acetate accumulation, but other by-products were formed. This mini review gives a clear outline
of these research efforts and their outcome, including bioprocess level approaches and genetic approaches. Recently, the latter
seems to have some promising results. 相似文献
16.
Yoshimune K Ninomiya Y Wakayama M Moriguchi M 《Journal of industrial microbiology & biotechnology》2004,31(9):421-426
The overproduction of d-aminoacylase (d-ANase, 233.8 U/mg), N-acyl-d-glutamate amidohydrolase (d-AGase, 38.1 U/mg) or N-acyl-d-aspartate amidohydrolase (d-AAase, 6.2 U/mg) in Escherichia coli is accompanied by aggregation of the overproduced protein. To facilitate the expression of active enzymes, the molecular chaperones GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), trigger factor (TF), GroELS and DnaKJE or GroELS and TF were coexpressed with the enzymes. d-ANase (313.3 U/mg) and d-AGase (95.8 U/mg) were overproduced in an active form at levels 1.3- and 1.8-fold higher, respectively, upon co-expression of GroELS and TF. An E. coli strain expressing the d-AAase gene simultaneously with the TF gene exhibited a 4.3-fold enhancement in d-AAase activity (32.0 U/mg) compared with control E. coli expressing the d-AAase gene alone. 相似文献
17.
Honglian Wang Xia Zhong Jianchun Li Menglian Zhu Lu Wang Xingli Ji Junming Fan Li Wang 《Molecular biotechnology》2018,60(2):134-140
IgA protease is secreted by various mucosal pathogenic bacteria which can cleave human immunoglobulin A1 (IgA1) in its hinge region. In addition to be considered as a virulence factor, it's reported that IgA protease can also be used for IgA nephropathy (IgAN) treatment. Our previous study identified bacteria H. influenzae 49247 expressed high activity of IgA protease with promised application in IgAN therapy. In this study, we cloned the IgA protease gene of H. influenzae 49247 with degenerate primers. Alignment analysis indicated that H. influenzae 49247 IgA protease showed unique DNA and amino acid sequence but with typical endopeptidase domain and beta transporter domain compared with known IgA proteases from the same species. To facilitate expression and purification, the H. influenzae 49247 IgA protease gene was sub-cloned into the pET28-A(+) vector with insertion of a 6xHis tag downstream of the endopeptidase domain and upstream of the potential autocleavage site. The recombined IgA protease can be constitutively expressed in E. coli and secreted into the culture medium. With a simple nickel affinity binding, the secreted IgA protease can be purified with high purity (95%) and a molecular weight of about 130 kDa. The identity of the IgA protease was validated by the presence of 6xHis tag in the purified protein by western blotting and its ability to cleave human IgA1 molecule. Collectively, the successful cloning, expression and purification of H. influenzae 49247 IgA protease will augment its therapeutic study in IgAN treatment. 相似文献
18.
Background
The pursuit of standardization and reliability in synthetic biology has achieved, in recent years, a number of advances in the design of more predictable genetic parts for biological circuits. However, even with the development of high-throughput screening methods and whole-cell models, it is still not possible to predict reliably how a synthetic genetic construct interacts with all cellular endogenous systems. This study presents a genome-wide analysis of how the expression of synthetic genes is affected by systematic perturbations of cellular functions. We found that most perturbations modulate expression indirectly through an effect on cell size, putting forward the existence of a generic Size-Expression interaction in the model prokaryote Escherichia coli.Results
The Size-Expression interaction was quantified by inserting a dual fluorescent reporter gene construct into each of the 3822 single-gene deletion strains comprised in the KEIO collection. Cellular size was measured for single cells via flow cytometry. Regression analyses were used to discriminate between expression-specific and gene-specific effects. Functions of the deleted genes broadly mapped onto three systems with distinct primary influence on the Size-Expression map. Perturbations in the Division and Biosynthesis (DB) system led to a large-cell and high-expression phenotype. In contrast, disruptions of the Membrane and Motility (MM) system caused small-cell and low-expression phenotypes. The Energy, Protein synthesis and Ribosome (EPR) system was predominantly associated with smaller cells and positive feedback on ribosome function.Conclusions
Feedback between cell growth and gene expression is widespread across cell systems. Even though most gene disruptions proximally affect one component of the Size-Expression interaction, the effect therefore ultimately propagates to both. More specifically, we describe the dual impact of growth on cell size and gene expression through cell division and ribosomal content. Finally, we elucidate aspects of the tight control between swarming, gene expression and cell growth. This work provides foundations for a systematic understanding of feedbacks between genetic and physiological systems.19.
Keratinase from Pseudomonas aeruginosa KS-1 was expressed constitutively as an extracellular protein in Escherichia coli with high specific activity of 3.7 kU/mg. It was purified fourfold as a 33 kDa monomeric protein by Q-Sepharose ion exchange
chromatography with a recovery of 95%. It is a serine protease with optimal activity at pH 9 and 50°C. It was stable from
pH 4 to 12 for 1 h with a t1/2 of 12 min at 70°C. It hydrolyzed haemoglobin > fibrin > feather keratin > azo-casein > casein > meat protein > gelatin. Among
synthetic substrates, it efficiently hydrolyzed N-Suc-ala-ala-pro-phe-pNA, N-Suc-ala-ala-ala-pNA, N-Suc-ala-ala-pro-leu-pNA and also plasmin substrate, d-Val-Leu-Lys-pNA 相似文献
20.
S. Thangminlal Vaiphei Gaurav Pandey K. J. Mukherjee 《Journal of industrial microbiology & biotechnology》2009,36(12):1453-1458
A series of continuous cultures was performed to understand the product formation kinetics of recombinant human interferon
gamma (rhIFN-γ) in Escherichia coli at different dilution rates ranging from 0.1 to 0.3 h−1 in different media. A T7 promoter-based vector was used for expression of IFN-γ in E. coli BL21 (DE3) cells. The recombinant protein was produced as inclusion bodies, thus allowing a rapid buildup of rhIFN-γ inside
the cell, with the specific product yield (Y
p/X
) reaching a maximum value of 182 mg g−1 dry cell weight (DCW). In all the media tested, the specific product formation rate (q
p
) was found to be strongly correlated with the specific growth rate (μ), demonstrating the growth-associated nature of product formation. The q
p
values show no significant decline with time postinduction, even though the recombinant protein has been over produced inside
the cell. The maximum q
p
level of 75.5 mg g−1 h−1 was achieved at the first hour of induction at the dilution rate of 0.3 h−1. Also, this correlation between q
p
and μ was not critically dependent on media composition, which would made it possible to grow cells in defined media in the growth
phase and then push up the specific growth rate just before induction by pulse addition of glucose and yeast extract. This
would ensure the twin objectives of high biomass and high specific productivities, leading to high volumetric product concentration. 相似文献