Isolation of precise plastid deletion mutants by homology-based excision: A resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation

We describe a simple and efficient homology-based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aadA-based transformation to duplicate a 649 bp region of plastid DNA corresponding to the atpB promoter region. Efficient recombination between atpB repeats deletes the intervening foreign genes and 1984 bp of plastid DNA (co-ordinates 57 424-59 317) containing the rbcL gene. Only five foreign bases are present in ΔrbcL plants illustrating the precision of homology-based excision. Sequence analysis of non-functional rbcL-related sequences in ΔrbcL plants indicated an extra-plastidic origin. Mutant ΔrbcL plants were heterotrophic, pale-green and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aadA-based transformation with the wild-type rbcL gene ruled out mutations in other genes. Excision and re-use of aadA shows that, despite the multiplicity of plastid genomes, homology-based excision ensures complete removal of functional aadA genes. Rescue of the ΔrbcL mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbcL deletion mutant isolated here is readily transformed with the efficient aadA marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site-directed mutant alleles and is not easily achieved using other methods. © 2006 Blackwell Publishing Ltd

Poboljšanje mikroinkapsulacije C-fikocijanina iz mikroalge Arthrospira pomoću sušenja zamrzavanjem uz dodatak različitih materijala za inkapsulaciju

C-phycocyanin (C-PC), a water-soluble blue pigment, was extracted from microalgae Arthrospira sp. C-PC could be a good substitute for synthetic pigments with high antioxidant activity. However, C-PC is unstable due to sensitivity to temperature, light, pH and oxygen; therefore, applications of C-PC in food and other products are limited. Microencapsulation of C-PC using freeze-drying is a solution to this problem and is considered a suitable method for drying the heat-sensitive pigment. C-phycocyanin was extracted from Arthrospira platensis. C-PC microcapsules were modified by freeze-drying, with maltodextrin and gum Arabic used as microencapsulation wall materials at different fractions from 0 to 100%. The physical properties including moisture content and water activity, solubility, hygroscopicity, bulk density, colour appearance, particle morphology and size distribution of the produced powders were evaluated. Thermal stability and antioxidant activity of freeze-dried microencapsulated C-PC powders were also assessed. Freeze-dried microencapsulated C-PC powders with maltodextrin and gum Arabic as wall materials gave high encapsulation efficiency of around 99%. At higher gum Arabic mass fraction, moisture content decreased and water activity improved. Maltodextrin gave higher solubility of C-PC powders whereas gum Arabic led to a similar colour of C-PC to those without microencapsulation. Freeze-dried microencapsulated C-PC powders were composed of different sized microparticles regardless of the combination of wall materials with amorphous glassy shapes. Thermal stability of encapsulated C-PC increased and also showed high antioxidant properties. This study demonstrates that the freeze-dried microencapsulated C-PC powders have pigment stability with antioxidant properties and are resistant to high temperatures. Therefore, they may have a potential for the development of microencapsulated C-PC as a functional ingredient with improved colour and bioactive properties. Such a product can be applied in food, cosmetic, biotechnology and nutraceutical industries.Pozadina istraživanja. C-fikocijanin, plavi pigment topljiv u vodi, izoliran je iz mikroalge Arthrospira sp. Taj se pigment može upotrijebiti kao dobra zamjena za sintetičke pigmente, a ima i izraženu antioksidacijsku aktivnost. Međutim, njegova je primjena u prehrambenim i ostalim proizvodima limitirana, jer je nestabilan pri povišenoj temperaturi, na svjetlosti, pri promjeni pH-vrijednosti i izloženosti kisiku. Moguće rješenje tog problema je mikroinkapsulacija C-fikocijanina postupkom sušenja zamrzavanjem, metodom prikladnom za sušenje pigmenata osjetljivih na toplinu. Eksperimentalni pristup. C-fikocijanin je izoliran iz mikroalge Arthrospira platensis. Mikrokapsule koje su sadržavale C-fikocijanin modificirane su postupkom sušenja zamrzavanjem, s dodatkom različitih udjela inkapsulacijskog materijala, maltodekstrina i gume arabike, u rasponu od 0 do 100 %. Ispitana su fizikalna svojstva dobivenog praha, uključujući udjel vlage, aktivitet vode, topljivost, higroskopnost, nasipnu gustoću, boju, morfologiju čestica i raspodjelu veličine čestica. Osim toga, procijenjeni su njegova toplinska stabilnost i antioksidacijska aktivnost. Rezultati i zaključci. Učinkovitost mikroinkapsulacije praha C-fikocijanina dobivenog sušenjem zamrzavanjem uz dodatak maltodekstrina i gume arabike bila je oko 99 %. Pri većim masenim udjelima gume arabike smanjio se udjel vlage, a povećala aktivnost vode. Dodatkom maltodekstrina dobiven je prah C-fikocijanina veće topljivosti, dok je dodatkom gume arabike boja praha ostala slična onoj prije mikroinkapsulacije. Bez obzira na sastav materijala za inkapsulaciju, mikroinkapsulirani prahovi dobiveni sušenjem zamrzavanjem sadržavali su čestice različitih veličina i amorfne strukture. Toplinska se stabilnost inkapsuliranog C-fikocijanina povećala, a mikrokapsule su imale i izražen antioksidacijski učinak. Novina i znanstveni doprinos. Ovo istraživanje pokazuje da mikroinkapsulirani prahovi C-fikocijanina dobiveni sušenjem zamrzavanjem zadržavaju stabilnost pigmenta i antioksidacijska svojstva, te da su otporni na visoke temperature. Stoga se mogu koristiti za proizvodnju funkcionalnog mikroinkapsuliranog C-fikocijanina s jačim intenzitetom boje i izraženijim bioaktivnim svojstvima. Takve se mikrokapsule mogu upotrijebiti u proizvodnji hrane, kozmetike, biotehnoloških proizvoda i dodataka hrani

Tobacco plastid transformation using the feedback-insensitive anthranilate synthase [α]-subunit of tobacco (ASA2) as a new selectable marker

Genetic engineering of chloroplasts normally requires the stable introduction of bacterial derived antibiotic or herbicide-resistance genes as selective markers. Ecological and health concerns have been raised due to the presence of such genes within the environment or the food supply. One way to overcome this issue is the use of plant genes able to confer a metabolic or developmental advantage to the transformed cells manipulating the plant's biosynthetic pathways. We explored the feasibility of using, for plastid transformation, the selection system based on the feedback-insensitive anthranilate synthase (AS) α-subunit gene of tobacco (ASA2) as a new selective marker and the indole analogue 4-methylindole (4MI) or the tryptophan analogue 7-methyl-DL-tryptophan (7MT) as the selection agents. An expression cassette containing Prrn-ASA2 was effectively integrated into the region between accD and ycf4 of the tobacco plastome by the biolistic process. Plastid transgenic plants were obtained on medium supplemented with 300 μM 7MT or 4MI. Transplastomic plants showed normal phenotype and fertility and the resistance to the selection agents 7MT and 4MI was transmitted maternally. The plastid transformed lines also exhibited a higher level of AS enzyme activity that was less sensitive to Trp-feedback inhibition and, consequently, increased free Trp levels in leaves about 7-fold

Binding of lac repressor-GFP fusion protein to lac operator sites inserted in the tobacco chloroplast genome examined by chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) has been used to detect binding of DNA-binding proteins to sites in nuclear and mitochondrial genomes. Here, we describe a method for detecting protein-binding sites on chloroplast DNA, using modifications to the nuclear ChIP procedures. The method was developed using the lac operator (lacO)/lac repressor (LacI) system from Escherichia coli. The lacO sequences were integrated into a single site between the rbcL and accD genes in tobacco plastid DNA and homoplasmic transplastomic plants were crossed with transgenic tobacco plants expressing a nuclear-encoded plastid-targeted GFP-LacI fusion protein. In the progeny, the GFP-LacI fusion protein could be visualized in living tissues using confocal microscopy, and was found to co-localize with plastid nucleoids. Isolated chloroplasts from the lacO/GFP-LacI plants were lysed, treated with micrococcal nuclease to digest the DNA to fragments of ∼600 bp and incubated with antibodies to GFP and protein A-Sepharose. PCR analysis on DNA extracted from the immunoprecipitate demonstrated IPTG (isopropylthiogalactoside)-sensitive binding of GFP-LacI to lacO. Binding of GFP-LacI to endogenous sites in plastid DNA showing sequence similarity to lacO was also detected, but required reversible cross-linking with formaldehyde. This may provide a general method for the detection of binding sites on plastid DNA for specific proteins

A synthetic gene increases TGFβ3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule

Human transforming growth factor-β3 (TGFβ3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFβ3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFβ3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFβ3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFβ3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFβ3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFβ3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFβ3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFβ3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFβ3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFβ3. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd

Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts

Thioredoxins (Trxs) are small ubiquitous disulphide proteins widely known to enhance expression and solubility of recombinant proteins in microbial expression systems. Given the common evolutionary heritage of chloroplasts and bacteria, we attempted to analyse whether plastid Trxs could also act as modulators of recombinant protein expression in transgenic chloroplasts. For that purpose, two tobacco Trxs (m and f) with different phylogenetic origins were assessed. Using plastid transformation, we assayed two strategies: the fusion and the co-expression of Trxs with human serum albumin (HSA), which was previously observed to form large protein bodies in tobacco chloroplasts. Our results indicate that both Trxs behave similarly as regards HSA accumulation, although they act differently when fused or co-expressed with HSA. Trxs-HSA fusions markedly increased the final yield of HSA (up to 26% of total protein) when compared with control lines that only expressed HSA; this increase was mainly caused by higher HSA stability of the fused proteins. However, the fusion strategy failed to prevent the formation of protein bodies within chloroplasts. On the other hand, the co-expression constructs gave rise to an absence of large protein bodies although no more soluble HSA was accumulated. In these plants, electron micrographs showed HSA and Trxs co-localization in small protein bodies with fibrillar texture, suggesting a possible influence of Trxs on HSA solubilization. Moreover, the in vitro chaperone activity of Trx m and f was demonstrated, which supports the hypothesis of a direct relationship between Trx presence and HSA aggregates solubilization in plants co-expressing both proteins. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.Authors wish to thank MJ Villafranca for excellent plant care and cultivation. This work was supported by grants Res. 17/2004 and IIM10865.RI1 (Proyecto EUROINNOVA) from Gobierno de Navarra (Spain). RSB and PCM were supported by predoctoral fellowships from CSIC and Generalitat Valenciana, respectively. AFS was supported by a postdoctoral fellowship from Public University of Navarra.Sanz-Barrio, R.; Fernandez-San Millan, A.; Corral Martínez, P.; Seguí-Simarro, JM.; Farran, I. (2011). Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts. Plant Biotechnology Journal. 9(6):639-650. https://doi.org/10.1111/j.1467-7652.2011.00608.xS6396509

The chloroplast transformation toolbox: Selectable markers and marker removal

Plastid transformation is widely used in basic research and for biotechnological applications. Initially developed in Chlamydomonas and tobacco, it is now feasible in a broad range of species. Selection of transgenic lines where all copies of the polyploid plastid genome are transformed requires efficient markers. A number of traits have been used for selection such as photoautotrophy, resistance to antibiotics and tolerance to herbicides or to other metabolic inhibitors. Restoration of photosynthesis is an effective primary selection method in Chlamydomonas but can only serve as a screening tool in flowering plants. The most successful and widely used markers are derived from bacterial genes that inactivate antibiotics, such as aadA that confers resistance to spectinomycin and streptomycin. For many applications, the presence of a selectable marker that confers antibiotic resistance is not desirable. Efficient marker removal methods are a major attraction of the plastid engineering tool kit. They exploit the homologous recombination and segregation pathways acting on chloroplast genomes and are based on direct repeats, transient co-integration or co-transformation and segregation of trait and marker genes. Foreign site-specific recombinases and their target sites provide an alternative and effective method for removing marker genes from plastids

Development and Applications of Transplastomic Plants; A Way Towards Eco-Friendly Agriculture

With distribution of genetic materials and advance molecular characteristics, the chloroplast is prokaryotic compartments within the eukaryotic plants that have turned into a crucial source for the genetic engineering and transplastomic plants are becoming more popular means of agricultural development with elevated crop yield. To address global agricultural problems, genetic modification of crop plants is a rapid and promising solution to adapt the environment-friendly and well-controlled farming system. The transplastomic plant with high accumulation of foreign proteins (up to 45-46% TSP) and stable transgene expression with gene containment can be a unique choice for the agricultural innovation of coming centuries. Although the transplastomic plants still facing encumber to ensure the full potential exploitation and expansion as an economical means, the removal of hardness and obstacles of this technology and commercialization can contribute for the sustainable development of future agriculture. In this book chapter, we intend to recapitulate the up to date development and achievement of transplastomic plant including gene transfer procedures in plastid genomes, regulable expression of plastid transgenes, plant trait improvement by foreign gene expression, biopharmaceuticals production, engineering of metabolic pathways in plant, study of transformation mediated RNA editing technologies, bio-safety issues and public concerns on transplastomic plants and overall beneficial aspects. We believe that the utilization of transplastomic plants will ensure an eco-friendly approach in agriculture with minimized hazards and public concerns. © Springer Nature Switzerland AG 2020