Application of multiple strategies to enhance oleanolic acid biosynthesis by engineered Saccharomyces cerevisiae.

Oleanolic acid and its derivatives are widely used in the pharmaceutical, agricultural, cosmetic and food industries. Previous studies have shown that oleanolic acid production levels in engineered cell factories are low, which is why oleanolic acid is still widely extracted from traditional medicinal plants. To construct a highly efficient oleanolic acid production strain, rate-limiting steps were regulated by inducible promoters and the expression of key genes in the oleanolic acid synthetic pathway was enhanced. Subsequently, precursor pool expansion, pathway refactoring and diploid construction were considered to harmonize cell growth and oleanolic acid production. The multi-strategy combination promoted oleanolic acid production of up to 4.07 g/L in a 100 L bioreactor, which was the highest level reported.

Peptide nucleic acid as a template for Taq DNA polymerase.

Peptide nucleic acid (PNA), an artificial DNA analog, comprises a purine or pyrimidine base and a pseudo-peptide backbone instead of deoxyribose-phosphate. PNA has been found to have stronger adhesion and higher stability in binding to its complementary DNA than deoxyribose-phosphate. Thus, it could serve as an agent for gene modulation, demonstrating potential in antisense therapy, molecular diagnostics, and nanotechnology. However, the applications of PNA remain limited because its biological activities are not fully known. Here, I demonstrate that a thermostable DNA polymerase, Thermus aquaticus (Taq) polymerase, exhibits transcriptase activity when a PNA oligomer is used as a template and that genetic information of the oligomer can be amplified by PCR using DNA primers. Furthermore, the insertion of a glutamine peptide stretch in the middle part of the PNA template did not interfere with transcription; it was transcribed into a guanosine or adenosine stretch. Intriguingly, this amino acid-to-DNA transcription did not occur when glycine residues were inserted. A synthetic PNA oligomer can, therefore, function as a template for a DNA polymerase, and polyglutamine peptides can be transcribed into guanosine or adenosine. These findings provide a cornerstone to reveal all amino acid genetic codes and transcription activity in the future.

Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges.

Oncolytic viruses have emerged as a promising strategy for cancer therapy due to their dual ability to selectively infect and lyse tumor cells and to induce systemic anti-tumor immunity. Among various candidate viruses, coxsackievirus group B (CVBs) have attracted increasing attention in recent years. CVBs are a group of small, non-enveloped, single-stranded, positive-sense RNA viruses, belonging to species human Enterovirus B in the genus Enterovirus of the family Picornaviridae. Preclinical studies have demonstrated potent anti-tumor activities for CVBs, particularly type 3, against multiple cancer types, including lung, breast, and colorectal cancer. Various approaches have been proposed or applied to enhance the safety and specificity of CVBs towards tumor cells and to further increase their anti-tumor efficacy. This review summarizes current knowledge and strategies for developing CVBs as oncolytic viruses for cancer virotherapy. The challenges arising from these studies and future prospects are also discussed in this review.

Engineering the Vasculature of Stem-Cell-Derived Liver Organoids.

The vasculature of stem-cell-derived liver organoids can be engineered using methods that recapitulate embryonic liver development. Hepatic organoids with a vascular network offer great application prospects for drug screening, disease modeling, and therapeutics. However, the application of stem cell-derived organoids is hindered by insufficient vascularization and maturation. Here, we review different theories about the origin of hepatic cells and the morphogenesis of hepatic vessels to provide potential approaches for organoid generation. We also review the main protocols for generating vascularized liver organoids from stem cells and consider their potential and limitations in the generation of vascularized liver organoids.

Plastid Transformation in Potato: An Important Source of Nutrition and Industrial Materials.

For a long time, plastid transformation has been a routine technology only in tobacco due to lack of effective selection and regeneration protocols, and, for some species, due to inefficient recombination using heterologous flanking regions in transformation vectors. Nevertheless, the availability of this technology to economically important crops offers new possibilities in plant breeding to manage pathogen resistance or improve nutritional value. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum), achieved by the optimization of the tissue culture procedures and using transformation vectors carrying homologous potato flanking sequences. This protocol allowed to obtain up to one shoot per shot, an efficiency comparable to that usually accomplished in tobacco. Further, the method described in this chapter has been successfully used to regenerate potato transplastomic plants expressing recombinant GFP protein in chloroplasts and amyloplasts or long double-stranded RNAs for insect pest control.

Plastid Transformation in Sugar Beet: An Important Industrial Crop.

Chloroplast biotechnology has assumed great importance in the past 20 years and, thanks to the numerous advantages as compared to conventional transgenic technologies, has been applied in an increasing number of plant species but still very much limited. Hence, it is of outmost importance to extend the range of species in which plastid transformation can be applied. Sugar beet (Beta vulgaris L.) is an important industrial crop of the temperate zone in which chloroplast DNA is not transmitted trough pollen. Transformation of the sugar beet genome is performed in several research laboratories, conversely sugar beet plastome genetic transformation is far away from being considered a routine technique. We describe here a method to obtain transplastomic sugar beet plants trough biolistic transformation. The availability of sugar beet transplastomic plants should avoid the risk of gene flow between these cultivated genetic modified sugar beet plants and the wild-type plants or relative wild species.

Efficient production of a novel alkaline cold-active phospholipase C from Aspergillus oryzae by molecular chaperon co-expression for crude oil degumming.

A novel alkaline cold-active phospholipase C (PLC) gene (AoPC) from Aspergillus oryzae was cloned. AoPC exhibited the highest sequence similarity of 32.5% with that of a PLC from Arabidopsis thaliana. The gene was co-expressed in Pichia pastoris with molecular chaperone PDI (protein disulfide isomerases), and the highest PLC activity of 82, 782 U mL-1 was achieved in a 5-L fermentor. The recombinant enzyme (AoPC) was most active at pH 8.0 and 25 °C, respectively, and it was stable over a broad pH range of 4.5-9.0 and up to 40 °C. It is the first fungal alkaline PLC. The application of AoPC (with 25% citric acid, w/w) in oil degumming process significantly reduced the phosphorus of crude soybean oil by 93.3% to a commercially acceptable level (<10 mg kg-1). Therefore, the relatively high yield and excellent properties of AoPC may possess it great potential in crude oil refining industry.

Plant Virus Nanoparticles for Vaccine Applications.

In the rapidly evolving field of nanotechnology, plant virus nanoparticles (pVNPs) are emerging as powerful tools in diverse applications ranging from biomedicine to materials science. The proteinaceous structure of plant viruses allows the capsid structure to be modified by genetic engineering and/or chemical conjugation with nanoscale precision. This means that pVNPs can be engineered to display peptides and proteins on their external surface, including immunodominant peptides derived from pathogens allowing pVNPs to be used for active immunization. In this context, pVNPs are safer than VNPs derived from mammalian viruses because there is no risk of infection or reversion to pathogenicity. Furthermore, pVNPs can be produced rapidly and inexpensively in natural host plants or heterologous production platforms. In this review, we discuss the use of pVNPs for the delivery of peptide antigens to the host immune in pre-clinical studies with the final aim of promoting systemic immunity against the corresponding pathogens. Furthermore, we described the versatility of plant viruses, with innate immunostimulatory properties, in providing a huge natural resource of carriers that can be used to develop the next generation of sustainable vaccines.

Characterization and heterologous expression of three DGATs from oil palm (Elaeis guineensis) mesocarp in Saccharomyces cerevisiae.

Oil palm (Elaeis guineensis) can accumulate up to 88% oil in fruit mesocarp. A previous transcriptome study of oil palm fruits indicated that genes coding for three diacylglycerol acyltransferases (DGATs), designated as EgDGAT1_3, EgDGAT2_2 and EgWS/DGAT_1 (according to Rosli et al., 2018) were highly expressed in mesocarp during oil accumulation. In the present study, the corresponding open reading frames were isolated, and characterized by heterologous expression in the mutant yeast H1246, which is devoid of neutral lipid synthesis. Expression of EgDGAT1_3 or EgDGAT2_2 could restore TAG synthesis, confirming that both proteins are true DGAT. In contrast, expression of EgWS/DGAT_1 resulted in the synthesis of fatty acid isoamyl esters (FAIEs) with saturated long-chain and very-long-chain fatty acids. In the presence of exogenously supplied fatty alcohols, EgWS/DGAT_1 was able to produce wax esters, indicating that EgWS/DGAT_1 codes for an acyltransferase with wax ester synthase but no DGAT activity. Finally, the complete wax ester biosynthetic pathway was reconstituted in yeast by coexpressing EgWS/DGAT_1 with a fatty acyl reductase from Tetrahymena thermophila. Altogether, our results characterized two novel DGATs from oil palm as well as a putative wax ester synthase that preferentially using medium chain fatty alcohols and saturated very-long chain fatty acids as substrates.

Genetically Engineered Plasma Membrane Nanovesicles for Cancer-Targeted Nanotheranostics.

A series of ligand-targeted nanosystems have been rapidly exploited to selectively deliver drug molecules to desired cell populations. The conjugation of protein ligands to the nanoparticle (NP) surface endows nanovehicles with active targeting properties. However, the nonspecific covalent coupling of protein ligands to nanocarriers may compromise the protein targeting due to the uncontrolled ligand orientation as well as the decline in ligand activity during linkage process. With this regard, biomimetic synthetic strategies are employed for the preparation of genetically engineered nanovesicles (GNV) from cellular plasma membrane with targeting moieties on the surface in a ligand-oriented manner. Herein, we introduce the biomimetic synthetic strategy and procedures for GNV preparation. This chapter may guide readers to design analogous NPs for cell-specific targeting by displaying particular protein probes (e.g., antibody, nanobody, and single-chain antibody) on the surface of GNVs.

Harnessing Biotechnology for the Development of New Seed Lipid Traits in Brassica.

The seed oil quality of Brassica oilseed species has been improved in the last few decades, using conventional breeding approaches. Modern biotechnology has enabled the significant development of new seed lipid traits in many oil crops. Alternation of seed lipid component with gene knockout by RNAi gene silencing, artificial microRNA or gene editing within the crop is relative straightforward. Introducing a new pathway from an exogenous source via biotechnology enables the creation of a new trait, where the biosynthetic pathway for such a new trait is not available in the host crop. This review updates the recent development of new seed lipid traits in six major Brassica species and highlights the capability of biotechnology to improve the composition of important fatty acids for both industrial and nutritional purposes.

Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean.

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60-80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T4) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T4 seeds compared to NT seed controls. A significant increase in the contents of Fe2+ (15.4%, 21.7%), Zn2+ (7.45%, 11.15%) and Ca2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.

Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii.

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.

Poxviruses as Gene Therapy Vectors: Generating Poxviral Vectors Expressing Therapeutic Transgenes.

Treatments with poxvirus vectors can have long-lasting immunological impact in the host, and thus they have been extensively studied to treat diseases and for vaccine development. More importantly, the oncolytic properties of poxviruses have led to their development as cancer therapeutics. Two poxviruses, vaccinia virus (VACV) and myxoma virus (MYXV), have been extensively studied as virotherapeutics with promising results. Vaccinia virus vectors have advanced to the clinic and have been tested as oncolytic therapeutics for several cancer types with successes in phase I/II clinical trials. In addition to oncolytic applications, MYXV has been explored for additional applications including immunotherapeutics, purging of cancer progenitor cells, and treatments for graft-versus-host diseases. These novel therapeutic applications have encouraged its advancement into clinical trials. To meet the demands of different treatment needs, VACV and MYXV can be genetically engineered to express therapeutic transgenes. The engineering process used in poxvirus vectors can be very different from that of other DNA virus vectors (e.g., the herpesviruses). This chapter is intended to serve as a guide to those wishing to engineer poxvirus vectors for therapeutic transgene expression and to produce viral preparations for preclinical studies.

Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii.

Microalgae constitute a highly diverse group of eukaryotic and photosynthetic microorganisms that have developed extremely efficient systems for harvesting and transforming solar energy into energy-rich molecules such as lipids. Although microalgae are considered to be one of the most promising platforms for the sustainable production of liquid oil, the oil content of these organisms is naturally low, and algal oil production is currently not economically viable. Chlamydomonas reinhardtii (Chlamydomonas) is an established algal model due to its fast growth, high transformation efficiency, and well-understood physiology and to the availability of detailed genome information and versatile molecular tools for this organism. In this review, we summarize recent advances in the development of genetic manipulation tools for Chlamydomonas, from gene delivery methods to state-of-the-art genome-editing technologies and fluorescent dye-based high-throughput mutant screening approaches. Furthermore, we discuss practical strategies and toolkits that enhance transgene expression, such as choice of expression vector and background strain. We then provide examples of how advanced genetic tools have been used to increase oil content in Chlamydomonas. Collectively, the current literature indicates that microalgal oil content can be increased by overexpressing key enzymes that catalyze lipid biosynthesis, blocking lipid degradation, silencing metabolic pathways that compete with lipid biosynthesis and modulating redox state. The tools and knowledge generated through metabolic engineering studies should pave the way for developing a synthetic biological approach to enhance lipid productivity in microalgae.

Application of genetics and biotechnology for improving medicinal plants.

MAIN CONCLUSION: Plant tissue culture has been used for conservation, micropropagation, and in planta overproduction of some pharma molecules of medicinal plants. New biotechnology-based breeding methods such as targeted genome editing methods are able to create custom-designed medicinal plants with different secondary metabolite profiles. For a long time, humans have used medicinal plants for therapeutic purposes and in food and other industries. Classical biotechnology techniques have been exploited in breeding medicinal plants. Now, it is time to apply faster biotechnology-based breeding methods (BBBMs) to these valuable plants. Assessment of the genetic diversity, conservation, proliferation, and overproduction are the main ways by which genetics and biotechnology can help to improve medicinal plants faster. Plant tissue culture (PTC) plays an important role as a platform to apply other BBBMs in medicinal plants. Agrobacterium-mediated gene transformation and artificial polyploidy induction are the main BBBMs that are directly dependent on PTC. Manageable regulation of endogens and/or transferred genes via engineered zinc-finger proteins or transcription activator-like effectors can help targeted manipulation of secondary metabolite pathways in medicinal plants. The next-generation sequencing techniques have great potential to study the genetic diversity of medicinal plants through restriction-site-associated DNA sequencing (RAD-seq) technique and also to identify the genes and enzymes that are involved in the biosynthetic pathway of secondary metabolites through precise transcriptome profiling (RNA-seq). The sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) are the genome editing methods that can produce user-designed medicinal plants. These current targeted genome editing methods are able to manage plant synthetic biology and open new gates to medicinal plants to be introduced into appropriate industries.

Rationally designing antisense therapy to keep up with evolving bacterial resistance.

Antisense molecules used as antibiotics offer the potential to keep up with acquired resistance, by redesigning the sequence of an antisense. Once bacteria acquire resistance by mutating the targeted sequence, new antisense can readily be designed by using sequence information of a target gene. However, antisense molecules require additional delivery vehicles to get into bacteria and be protected from degradation. Based on progress in the last few years it appears that, while redesigning or finding new delivery vehicle will be more difficult than redesigning the antisense cargo, it will perhaps be less difficult than finding new conventional small molecule antibiotics. In this study we propose a protocol that maximizes the combined advantages of engineered delivery vehicle and antisense cargo by decreasing the immediate growth advantage to the pathogen of mutating the entry mechanisms and increasing the advantage to the pathogen of antisense target mutations. Using this protocol, we show by computer simulation an appropriately designed antisense therapy can potentially be effective many times longer than conventional antibiotics before succumbing to resistance. While the simulations describe an in-vitro situation, based on comparison with other in-vitro studies on acquired resistance we believe the advantages of the combination antisense strategy have the potential to provide much more sustainability in vivo than conventional antibiotic therapy.

Impact of fabrics from transgenic flax on cultures of skin cells.

BACKGROUND: The development of a new type of wound dressing material that can support skin regeneration is an important challenge to improve treatment of chronic, non-healing wounds. OBJECTIVES: The objective of this study was to compare the impact of flax fabrics from transgenic plants overexpressing phenolic acids and flavonoids (W92) and polyhydroxybutyrate (M48), as well as fabric from non-transgenic plant (Nike) on cultures of human skin cells. MATERIAL AND METHODS: Flax fabric pieces as well as water extracts from the fabrics were co-cultured with human skin cells: keratinocytes, fibroblasts, dermal microvascular endothelial cells, and with monocytoid cell line (THP1) for 48 h. Cell viability and proliferation were assessed with the sulforhodamine B colorimetric assay. Intracellular reactive oxygen species (ROS) was estimated with the 2'7 dichlorodihydrofluorescein diacetate (DCFH-DA) oxidation method. Endothelial cell migration was measured with the scratch assay. The results were compared with the multi-criteria analysis (MCA) procedure. RESULTS: Tested flax fabrics released flavonoids and polyhydroxybutyrate to cell culture media, as it was determined by means of the high performance liquid chromatography (HPLC) method. Fabrics from transgenic plants W92 and M48 promoted proliferation of keratinocytes and fibroblasts. Water extracts from flax fabric diminished the proliferation of monocytoid cells, decreased oxidative burst in activated THP1 cells and accelerated the velocity of dermal microvascular cell migration. The MCA proved that the sum of beneficial effects estimated in human skin cell cultures was higher (by 47% and by 34% with W92 and M48, respectively) than that of non-transgenic flax fabric (Nike). CONCLUSIONS: The W92 and M48 fabrics should be further studied as candidates for elaboration of new types of bandages, able to improve skin wound healing.

Diabetic cardiomyopathy: molecular mechanisms, detrimental effects of conventional treatment, and beneficial effects of natural therapy.

ABSTARCT: Diabetic complications are among the largely exigent health problems currently. Cardiovascular complications, including diabetic cardiomyopathy (DCM), account for more than 80% of diabetic deaths. Investigators are exploring new therapeutic targets to slow or abate diabetes because of the growing occurrence and augmented risk of deaths due to its complications. Research on rodent models of type 1 and type 2 diabetes mellitus, and the use of genetic engineering techniques in mice and rats have significantly sophisticated for our understanding of the molecular mechanisms in human DCM. DCM is featured by pathophysiological mechanisms that are hyperglycemia, insulin resistance, oxidative stress, left ventricular hypertrophy, damaged left ventricular systolic and diastolic functions, myocardial fibrosis, endothelial dysfunction, myocyte cell death, autophagy, and endoplasmic reticulum stress. A number of molecular and cellular pathways, such as cardiac ubiquitin proteasome system, FoxO transcription factors, hexosamine biosynthetic pathway, polyol pathway, protein kinase C signaling, NF-κB signaling, peroxisome proliferator-activated receptor signaling, Nrf2 pathway, mitogen-activated protein kinase pathway, and micro RNAs, play a major role in DCM. Currently, there are a few drugs for the management of DCM and some of them have considerable adverse effects. So, researchers are focusing on the natural products to ameliorate it. Hence, in this review, we discuss the pathogical, molecular, and cellular mechanisms of DCM; the current diagnostic methods and treatments; adverse effects of conventional treatment; and beneficial effects of natural product-based therapeutics, which may pave the way to new treatment strategies. Graphical Abstract.

Development of wheat genotypes expressing a glutamine-specific endoprotease from barley and a prolyl endopeptidase from Flavobacterium meningosepticum or Pyrococcus furiosus as a potential remedy to celiac disease.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.