Coordinate Expression and Independent Subcellular Targeting of Multiple Proteins from a Single Transgene

Amrani, Abdelhak El; Barakate, Abdellah; Askari, Barak M.; Li, Xuejun; Roberts, Alison G.; Ryan, Martin D.; Halpin, Claire

doi:10.1104/pp.103.032649

Cited by 69 publications

(58 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The risk of TGS due to the presence of several copies of the same promoter could be significantly reduced by expression of a multigene expression construct consisting of the genes encoding the three biosynthetic enzymes and the selective marker under control of one single promoter. By separation of the four proteins with the viral 2A peptide (El Amrani et al 2004), the individual enzymes are predicted to be co-translationally cleaved to yield CYP79A1, CYP71E1, UGT85B1 and the selective marker protein. Alternatively, or in combination with the 2A polyprotein strategy, the use of an inducible promoter would allow selection of transgenic explants without the risk of counter selection for reduced expression of the transgenes.…”

Section: Discussionmentioning

confidence: 99%

Lessons learned from metabolic engineering of cyanogenic glucosides

Morant¹,

Jørgensen²,

Jørgensen³

et al. 2007

Metabolomics

View full text Add to dashboard Cite

Plants produce a plethora of secondary metabolites which constitute a wealth of potential pharmaceuticals, pro-vitamins, flavours, fragrances, colorants and toxins as well as a source of natural pesticides. Many of these valuable compounds are only synthesized in exotic plant species or in concentrations too low to facilitate commercialization. In some cases their presence constitutes a health hazard and renders the crops unsuitable for consumption. Metabolic engineering is a powerful tool to alter and ameliorate the secondary metabolite composition of crop plants and gain new desired traits. The interplay of a multitude of biosynthetic pathways and the possibility of metabolic cross-talk combined with an incomplete understanding of the regulation of these pathways, explain why metabolic engineering of plant secondary metabolism is still in its infancy and subject to much trial and error. Cyanogenic glucosides are ancient defense compounds that release toxic HCN upon tissue disruption caused e.g. by chewing insects. The committed steps of the cyanogenic glucoside biosynthetic pathway are encoded by three genes. This unique genetic simplicity and the availability of the corresponding cDNAs have given cyanogenic glucosides pioneering status in metabolic engineering of plant secondary metabolism. In this review, lessons learned from metabolic engineering of cyanogenic glucosides in Arabidopsis thaliana (thale cress), Nicotiana tabacum cv Xanthi (tobacco), Manihot esculenta Crantz (cassava) and Lotus japonicus (bird's foot trefoil) are presented. The importance of metabolic channelling of toxic intermediates as mediated by metabolon formation in avoiding unintended metabolic cross-talk and unwanted pleiotropic effects is emphasized. Likewise, the potential of metabolic engineering of plant secondary metabolism as a tool to elucidate, for example, the impact of secondary metabolites on plant-insect interactions is demonstrated.

show abstract

Section: Discussionmentioning

confidence: 99%

Lessons learned from metabolic engineering of cyanogenic glucosides

Morant¹,

Jørgensen²,

Jørgensen³

et al. 2007

Metabolomics

View full text Add to dashboard Cite

show abstract

“…The two PCR fragments were then ligated and interspaced with a sequence encoding the 2A sequence in pC130035Su in accordance to the USER cloning method delineated by Nour-Eldin et al (34) and Geu-Flores et al (39), yielding pC1302-35SPro-T2-2A-CytoEpi-35STerm (T2-2A-CytoEpi). The 2A linker region of T2-2A-CytoEpi encodes QGSGQTLNFDLLKLAGD-VESNPG2PMD, where underline designates the 2A sequence, and arrow indicates the auto-cleavage site (36,37). 2) The reverse order, encoding (from N to C terminus) cytosolic epimerase followed by the 2A sequence and Golgi-targeted GalNAc-T2, was made by PCR-amplifying 4 with primers PwbppFor and …”

Section: Methodsmentioning

confidence: 99%

Engineering Mammalian Mucin-type O-Glycosylation in Plants

Yang

Drew

Jørgensen

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.

show abstract

“…deletion of chromosomal fragments, mutation of several closely related alleles or NHEJ-mediated native gene replacements) may call for expression of several monomers. This can be achieved by producing transgenic plants in multiple rounds of transformation (DafnyYelin and Tzfira, 2007;Naqvi et al, 2010), by using polyprotein vectors (Halpin et al, 1999;El Amrani et al, 2004) or multigene vectors . A dedicated plant ZFN-expression system, which enables the expression of up to four independent ZFN monomers, was developed by Tovkach et al, (2009).…”

Section: Induction Of Genomic Dsbs -Enzyme Expression Systemsmentioning

confidence: 99%

From Agrobacterium to viral vectors: genome modification of plant cells by rare cutting restriction enzymes

Marton¹,

Honig²,

Omid³

et al. 2013

Int. J. Dev. Biol.

View full text Add to dashboard Cite

Researchers and biotechnologists require methods to accurately modify the genome of higher eukaryotic cells. Such modifications include, but are not limited to, site-specific mutagenesis, site-specific insertion of foreign DNA, and replacement and deletion of native sequences. Accurate genome modifications in plant species have been rather limited, with only a handful of plant species and genes being modified through the use of early genome-editing techniques. The development of rare-cutting restriction enzymes as a tool for the induction of site-specific genomic double-strand breaks and their introduction as a reliable tool for genome modification in animals, animal cells and human cell lines have paved the way for the adaptation of rare-cutting restriction enzymes to genome editing in plant cells. Indeed, the number of plant species and genes which have been successfully edited using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered homing endonucleases is on the rise. In our review, we discuss the basics of rare-cutting restriction enzyme-mediated genome-editing technology with an emphasis on its application in plant species.

show abstract

Coordinate Expression and Independent Subcellular Targeting of Multiple Proteins from a Single Transgene

Cited by 69 publications

References 34 publications

Lessons learned from metabolic engineering of cyanogenic glucosides

Lessons learned from metabolic engineering of cyanogenic glucosides

Engineering Mammalian Mucin-type O-Glycosylation in Plants

From Agrobacterium to viral vectors: genome modification of plant cells by rare cutting restriction enzymes

Contact Info

Product

Resources

About