Biotechnological Applications of Plastid Foreign Gene Expression

Chloroplast is responsible for the major metabolic process photosynthesis. These organelles have their own genome and in the last three decades, the chloroplast genome has been broadly studied and manipulated through genetic engineering tools. The transfer of genes into chloroplast provides advantage over the insertion of transgene into nuclear genome, including overexpression of foreign protein, no positional effects, absence of epigenetic effects and uniparental inheritance of the transgenes, the ability to express multiple transgenes in operons and the possibility of eliminate the marker gene after the transformation and integration of the foreign gene. Now more than 100 transgenes have been reported stably integrated into the chloroplast genome including genes encoding enzymes with industrial value, biomaterials, biopharmaceuticals, vaccines and genes with agronomic trails. The chloroplast genetic tools have been implemented in several important crops. So, the chloroplast engineering technology has been positioned as the most important for the production of proteins and metabolites with biotechnological applications

High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly

Efecto del aceite de orégano en las propiedades fisicoquímicas, texturales y sensoriales del queso panela

Plant essential oils are increasingly used in the food industry for their antimicrobial, antioxidant and sensory properties. The effects of added oregano essential oil (OEO) in panela cheese (QP) production was evaluated on cheese physicochemical, textural and sensory properties during 15 day’s storage. Two addition levels were used, resulting in three treatments: control with no OEO (QP1); 0.05 g OEO/ L milk (QP2); and 0.10 g OEO/L milk (QP3). In all treatments, cheese pH was highest (P<0.05) on d 1 and lowest on d 15, although it remained higher overall in QP1. Weight and weight loss did not differ between treatments. Color parameters differed minimally between treatments and over time, although increased OEO content pushed b* values towards yellow. Addition of OEO lowered cheese hardness and shear force values. Based on sensory parameters, consumer acceptance was highest (P<0.05) for the control treatment. Addition of OEO generally did not affect cheese physicochemical, textural and sensory characteristics during storage. It did slightly lower hardness and shear force values, and decreased consumer acceptance. If used at adequate levels, oregano essential oil can improve cheese performance during storage without substantially affecting quality parameters.Los aceites esenciales obtenidos de plantas están siendo usados en la industria alimentaria debido a su efectividad antimicrobiana, antioxidante y sensorial. En esta investigación se evaluó el efecto del aceite esencial de orégano (AEO) en la elaboración del queso panela (QP) sobre sus propiedades fisicoquímicas, texturales y sensoriales en 15 días. Tres tratamientos se establecieron: testigo (QP1), AEO en 0.05 g/L (QP2) y 0.10 g/L de leche (QP3). El pH de los quesos fue mayor (P<0.05) al día 1 y menor al día 15, y la acidez menor en QP2 al día 1 y en QP3 al día 4. La luminosidad al día 1 fue mayor (P<0.05) en QP2 y menor en QP1. La dureza aumentó (P<0.05) en QP1 y disminuyó en QP2 al día 1 y 8. El pH, acidez y textura del queso se mantienen con 0.05 g AEO/L, y con 0.1 g AEO/L leche mejora la aceptación sensorial

Chloroplasts: The Future of Large-Scale Protein Production

Chloroplast engineering has matured considerably in recent years. It is emerging as a promising tool to address the challenges related to food security, drug production, and sustainable energy posed by an ever-growing world population. Chloroplasts have proven their potential by efficiently expressing transgenes, encapsulating recombinant proteins, and protecting them from cellular machinery, making it possible to obtain highly functional proteins. This quality has also been exploited by interfering RNA technology. In addition to the practical attributes offered by chloroplast transformation, such as the elimination of position effects, polycistronic expression, and massive protein production, the technique represents an advance in biosafety terms; however, even if its great biotechnological potential, crops that have efficiently transformed are still a proof of concept. Despite efforts, other essential crops have remained recalcitrant to chloroplast transformation, which has limited their expansion. In this chapter, we address the most recent advances in this area and the challenges that must be solved to extend the transformation to other crops and become the de facto tool in plant biotechnology

Study of Plastid Genome Stability in Tobacco Reveals That the Loss of Marker Genes Is More Likely by Gene Conversion Than by Recombination between 34-bp loxP Repeats1[C][OA]

In transformed tobacco (Nicotiana tabacum) plastids, we flank the marker genes with recombinase target sites to facilitate their posttransformation excision. The P1 phage loxP sites are identical 34-bp direct repeats, whereas the phiC31 phage attB/attP sites are 54- and 215-bp sequences with partial homology within the 54-bp region. Deletions in the plastid genome are known to occur by recombination between directly repeated sequences. Our objective was to test whether or not the marker genes may be lost by homologous recombination via the directly repeated target sites in the absence of site-specific recombinases. The sequence between the target sites was the barau gene that causes a golden-yellow (aurea) leaf color, so that the loss of the barau gene can be readily detected by the appearance of green sectors. We report here that transplastomes carrying the barau gene marker between recombinase target sites are relatively stable because no green sectors were detected in approximately 36,000 seedlings (Nt-pSS33 lines) carrying attB/attP-flanked barau gene and in approximately 38,000 seedlings (Nt-pSS42 lines) carrying loxP-flanked barau gene. Exceptions were six uniformly green plants in the Nt-pSS42-7A progeny. Sequencing the region of plastid DNA that may derive from the vector indicated that the barau gene in the six green plants was lost by gene conversion using wild-type plastid DNA as template rather than by deletion via directly repeated loxP sites. Thus, the recombinase target sites incorporated in the plastid genome for marker gene excisions are too short to mediate the loss of marker genes by homologous recombination at a measurable frequency

Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

Background: The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results: pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3′UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3′rps′12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion: This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value

Characterization of Trehalose-6-phosphate Synthase and Trehalose-6-phosphate Phosphatase Genes and Analysis of its Differential Expression in Maize (Zea mays) Seedlings under Drought Stress

Maize is the most important crop around the world and it is highly sensitive to abiotic stress caused by drought, excessive salinity, and extreme temperature. In plants, trehalose has been widely studied for its role in plant adaptation to different abiotic stresses such as drought, high and low temperature, and osmotic stress. Thus, the aim of this work was to clone and characterize at molecular level the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) genes from maize and to evaluate its differential expression in maize seedlings under drought stress. To carry out this, resistant and susceptible maize lines were subjected to drought stress during 72 h. Two full-length cDNAs of TPS and one of TPP were cloned and sequenced. Then, TPS and TPP amino acid sequences were aligned with their homologs from different species, showing highly conserved domains and the same catalytic sites. Relative expression of both genes was evaluated by RT-qPCR at different time points. The expression pattern showed significant induction after 0.5 h in resistant lines and after two to four hours in susceptible plants, showing their participation in drought stress response

Identification of Sorghum (<i>Sorghum bicolor</i> (L.) Moench) Genotypes with Potential for Hydric and Heat Stress Tolerance in Northeastern Mexico

Sorghum (Sorghum bicolor (L.) Moench) is cultivated in regions with frequent drought periods and high temperatures, conditions that have intensified in the last decades. One of the most important photosynthetic components, sensible to hydric stress, is maximum quantum yield for photosystem II (PSII, or Fv/Fm). The objective of the present study was to identify sorghum genotypes with tolerance to hydric and heat stress. The treatments were hydric status (hydric stress or non-hydric stress (irrigation)), the plant’s developmental stages (pre or post-anthesis), and six genotypes. The response variables were Fv/Fm; photosynthetic rate (PN); stomatal conductance (gs); transpiration rate (E); relative water content (RWC); damage to cell membrane (DCM) at temperatures of 40 and 45 °C; and agronomic variables. The experiment was conducted in pots in open sky in Marín, N.L., in the dry and hot northeast Mexico. The treatment design was a split–split plot design, with three factors. Hydric stress diminished the functioning of the photosynthetic apparatus by 63%, due to damage caused to PSII. Pre-anthesis was the most vulnerable stage to hydric stress as it decreased the weight of grains per panicle (85%), number of grains per panicle (69%), and weight of 100 grains (46%). Genotypes LER 1 and LER 2 were identified as tolerant to hydric stress, as they had lower damage to PSII; LER 1 and LEB 2 for their superior RWC; and LER 1 as a thermo tolerant genotype, due to its lower DCM at 45 °C. It was concluded that LER 1 could have the potential for both hydric and heat stress tolerance in the arid northeast Mexico