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Teixeira JA Gonçalves DB de Queiroz MV de Araújo EF 《Journal of applied microbiology》2011,111(4):818-825
Aims: To obtain recombinant strains of Penicillium griseoroseum that produce high levels of pectin lyase (PL) and polygalacturonase (PG) simultaneously. Methods and Results: A strain with high production of PL was transformed with the plasmid pAN52pgg2, containing the gene encoding PG of P. griseoroseum, under control of the gpd promoter gene from Aspergillus nidulans. Southern blot analysis demonstrated that all strain had at least one copy of pAN52pgg2 integrated into the genome. The recombinant strain P. griseoroseum T20 produced levels of PL and PG that were 266‐ and 27‐fold greater, respectively, than the wild‐type strain. Furthermore, the extracellular protein profile of recombinant T20 showed two protein bands of c. 36 and 38 kDa, associated with PL and PG, respectively. Conclusions: This recombinant strain T20 produces PL and PG using carbon sources of low costs, and an enzyme preparation that is free of cellulolytic and proteolytic activities. Significance and Impact of the Study: PL and PG play an important role in the degradation of pectin. Owing to their use in the juice and wines industries, there is a growing interest in the inexpensive production of these enzymes. This work describes an efficient system of protein expression and secretion using the fungus P. griseoroseum. 相似文献
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Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp) gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming. 相似文献
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Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis
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Giovanni B. Morlino Lorenza Tizzani Reinhard Fleer Laura Frontali Michele M. Bianchi 《Applied microbiology》1999,65(11):4808-4813
Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1β, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system. 相似文献
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Jia Zheng Ning Guo Fu-lai Lin Li-shuang Wu Hong-bo Zhou 《World journal of microbiology & biotechnology》2014,30(2):579-584
Pichia pastoris pGAP (glyceraldehyde dehydrogenase promoter) expression system was widely used for the expression and production of heterologous proteins. Screening multi-copy recombinants was an effective strategy to improve the heterologous protein production in P. pastoris. Because multiple gene insertion events occurred with a low frequency, hundreds to thousands of antibiotic-resistance recombinants need to be screened. The common way of improving screening efficiency was to increase antibiotic concentration in screening plates. Here we developed a screening method by selecting small colonies from low-concentration antibiotic screening plates. This strategy greatly improved the probability of obtaining multi-copy mannanase gene (man) recombinants and it could replace the common strategy by increasing antibiotic concentration in screening plates. The further study in liquid shake flask cultures revealed that cell concentrations, growth rates and substrate consumption rates of recombinants gradually decreased with the increase in man copy number. This indicated that such a screening strategy was effective to screen multi-copy recombinants based on colony size. 相似文献
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Cardoso PG Ribeiro JB Teixeira JA de Queiroz MV de Araújo EF 《Journal of industrial microbiology & biotechnology》2008,35(3):159-166
The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process
of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar
cane juice showed PL activities of 4,804 or 5,202 U ml−1 respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by
this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular
proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately
40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to
express pectin lyase at levels comparable to, or exceeding, previously reported data. 相似文献
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Elena Cámara Sergi Monforte Joan Albiol Pau Ferrer 《Biotechnology and bioengineering》2019,116(7):1710-1720
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Li Chen Ali Mohsin Ju Chu Yingping Zhuang Yamei Liu Meijin Guo 《Biotechnology and Bioprocess Engineering》2017,22(6):767-773
Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system. 相似文献
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Carvalho ND Arentshorst M Kooistra R Stam H Sagt CM van den Hondel CA Ram AF 《Applied microbiology and biotechnology》2011,89(2):357-373
Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting
them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences
of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made
in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded
protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target
genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion
of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein
detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels
of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation. 相似文献
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A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon 总被引:15,自引:0,他引:15
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Fellermann K Stange DE Schaeffeler E Schmalzl H Wehkamp J Bevins CL Reinisch W Teml A Schwab M Lichter P Radlwimmer B Stange EF 《American journal of human genetics》2006,79(3):439-448
Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin-gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2-10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with < or = 3 copies have a significantly higher risk of developing colonic CD than did individuals with > or = 4 copies (odds ratio 3.06; 95% confidence interval 1.46-6.45). An HBD-2 gene copy number of < 4 was associated with diminished mucosal HBD-2 mRNA expression (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression. 相似文献
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Louise Morin Marlien van der Merwe Diana Hartley Petra Müller 《Mycological Research》2009,113(6-7):725-736
Several specimens of an aecial rust fungus were collected on Senecio madagascariensis during a field survey carried out in KwaZulu-Natal, South Africa. As telia were not present in the specimens collected, DNA sequence analyses were undertaken to determine the identity of the rust species. ITS and β-tub1 sequencing confirmed that one of the isolates recovered is Puccinia lagenophorae sensu lato. On the other hand, sequencing and RFLP analysis revealed the presence of two divergent copies of ITS and β-tub1 in all the other six isolates investigated. In both phylogenetic trees, one copy of the gene region grouped within a well supported clade with sequences of P. lagenophorae accessions from different geographical origins and hosts, and the Australian rusts Puccinia saccardoi and Puccinia stylidii. The other copy of these gene regions grouped within a separate clade comprising European accessions of Puccinia dioicae (ITS) and Uromyces sommerfeltii (β-tub1) that occur on Asteraceae hosts. Multiple copies of these gene regions were not observed in Australian isolates of P. lagenophorae. Our study provides some evidence that an interspecific hybrid rust fungus, with P. lagenophorae as one of its parents, may occur on S. madagascariensis in South Africa. The identity of the other parent remains unknown. 相似文献
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Haojie Yu Xin Yan Weiliang Shen Qing Hong Ji Zhang Yujia Shen Shunpeng Li 《Current microbiology》2009,59(6):573-578
In the Pichia pastoris expression system, increasing the copy number of the expression cassette often has the effect of increasing the amount of
protein expressed. To improve the expression level of methyl parathion hydrolase (MPH), we constructed two integration vectors
with four and eight direct repeats of the expression cassette using an in vitro multimerization approach. After two successive
integrations, at least 12 copies of the MPH expression cassette were integrated into the P. pastoris chromosome. Under shake-flask conditions, over 55 mg active MPH/l was secreted into the medium by the multicopy clones. The
extracellular enzyme activity was about 10-fold higher for the multicopy clones than for clones containing a single copy of
the gene. Further investigations revealed that the multicopy MPH expression cassette could remain stably integrated and functional
over five generations. Note that the expression vector pRF constructed in our study can be not only used to construct multiple
copies of the expression cassette in vitro, but also integrated into the P. pastoris genome without introducing any antibiotic resistance gene, which is desirable for production of biotherapeutic proteins. 相似文献