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.

Functions of Representative Terpenoids and Their Biosynthesis Mechanisms in Medicinal Plants.

Terpenoids are the broadest and richest group of chemicals obtained from plants. These plant-derived terpenoids have been extensively utilized in various industries, including food and pharmaceuticals. Several specific terpenoids have been identified and isolated from medicinal plants, emphasizing the diversity of biosynthesis and specific functionality of terpenoids. With advances in the technology of sequencing, the genomes of certain important medicinal plants have been assembled. This has improved our knowledge of the biosynthesis and regulatory molecular functions of terpenoids with medicinal functions. In this review, we introduce several notable medicinal plants that produce distinct terpenoids (e.g., Cannabis sativa, Artemisia annua, Salvia miltiorrhiza, Ginkgo biloba, and Taxus media). We summarize the specialized roles of these terpenoids in plant-environment interactions as well as their significance in the pharmaceutical and food industries. Additionally, we highlight recent findings in the fields of molecular regulation mechanisms involved in these distinct terpenoids biosynthesis, and propose future opportunities in terpenoid research, including biology seeding, and genetic engineering in medicinal plants.

Genetic Engineering of Potato (Solanum tuberosum) Chloroplasts Using the Small Synthetic Plastome "Mini-Synplastome".

In the rapidly expanding field of synthetic biology, chloroplasts represent attractive targets for installation of valuable genetic circuits in plant cells. Conventional methods for engineering the chloroplast genome (plastome) have relied on homologous recombination (HR) vectors for site-specific transgene integration for over 30 years. Recently, episomal-replicating vectors have emerged as valuable alternative tools for genetic engineering of chloroplasts. With regard to this technology, in this chapter we describe a method for engineering potato (Solanum tuberosum) chloroplasts to generate transgenic plants using the small synthetic plastome (mini-synplastome). In this method, the mini-synplastome is designed for Golden Gate cloning for easy assembly of chloroplast transgene operons. Mini-synplastomes have the potential to accelerate plant synthetic biology by enabling complex metabolic engineering in plants with similar flexibility of engineered microorganisms.

Genetic and functional characterization of multiple thermophilic organosulfur-removal systems reveals desulfurization potentials for waste residue oil cleaning.

Heavy oil and petroleum refining residues usually contain high concentrations of recalcitrant hazardous organosulfur compounds, causing long-term serious global environmental pollution during leakage and combustion. Research conducted here identified a unique thermophilic bacterium Parageobacillus thermoglucosidasius W-36 with the notable ability of waste residue oil desulfurization, utilization and tolerance of multiplex hazardous organosulfur pollutants. Genome information mining revealed multiple desulfurization systems in three organosulfur-utilizing gene clusters. Enzymatic characterization, phylogenetic relationships, transcriptional performance and structural prediction indicated four novel key monooxygenases for diverse organosulfur removal. Importantly, all monooxygenases shared obvious commonalities in the predicted tertiary structure backbone and catalytic characteristics of C-S bond cleavage, implying the potential of genetic engineering for broad-spectrum hazardous organosulfur removal. Therefore, this work demonstrated the important application potential of thermophilic bacteria as a promising alternative biodesulfurization way for waste residue oil cleaning.

Expression of glycerol-3-phosphate acyltransferase increases non-polar lipid accumulation in Nannochloropsis oceanica.

Microalgae are considered a suitable production platform for high-value lipids and oleochemicals. Several species including Nannochloropsis oceanica produce large amounts of essential [Formula: see text]-3 polyunsaturated fatty acids (PUFAs) which are integral components of food and feed and have been associated with health-promoting effects. N. oceanica can further accumulate high contents of non-polar lipids with chemical properties that render them a potential replacement for plant oils such as palm oil. However, biomass and lipid productivities obtained with microalgae need to be improved to reach commercial feasibility. Genetic engineering can improve biomass and lipid productivities, for instance by increasing carbon flux to lipids. Here, we report the overexpression of glycerol-3-phosphate acyltransferase (GPAT) in N. oceanica during favorable growth conditions as a strategy to increase non-polar lipid content. Transformants overproducing either an endogenous (NoGPAT) or a heterologous (Acutodesmus obliquus GPAT) GPAT enzyme targeted to the endoplasmic reticulum had up to 42% and 51% increased non-polar lipid contents, respectively, compared to the wild type. Biomass productivities of transformant strains were not substantially impaired, resulting in lipid productivities that were increased by up to 37% and 42% for NoGPAT and AoGPAT transformants, respectively. When exposed to nutrient stress, transformants and wild type had similar lipid contents, suggesting that GPAT enzyme exerts strong flux control on lipid synthesis in N. oceanica under favorable growth conditions. NoGPAT transformants further accumulated PUFAs in non-polar lipids, reaching a total of 6.8% PUFAs per biomass, an increase of 24% relative to the wild type. Overall, our results indicate that GPAT is an interesting target for engineering of lipid metabolism in microalgae, in order to improve non-polar lipid and PUFAs accumulation in microalgae.

Harnessing the advances of genetic engineering in microalgae for the production of cannabinoids.

Cannabis is widely recognized as a medicinal plant owing to bioactive cannabinoids. However, it is still considered a narcotic plant, making it hard to be accessed. Since the biosynthetic pathway of cannabinoids is disclosed, biotechnological methods can be employed to produce cannabinoids in heterologous systems. This would pave the way toward biosynthesizing any cannabinoid compound of interest, especially minor substances that are less produced by a plant but have a high medicinal value. In this context, microalgae have attracted increasing scientific interest given their unique potential for biopharmaceutical production. In the present review, the current knowledge on cannabinoid production in different hosts is summarized and the biotechnological potential of microalgae as an emerging platform for synthetic production is put in perspective. A critical survey of genetic requirements and various transformation approaches are also discussed.

Genetic engineering of complex feed enzymes into barley seed for direct utilization in animal feedstuff.

Currently, feed enzymes are primarily obtained through fermentation of fungi, bacteria, and other microorganisms. Although the manufacturing technology for feed enzymes has evolved rapidly, the activities of these enzymes decline during the granulating process and the cost of application has increased over time. An alternative approach is the use of genetically modified plants containing complex feed enzymes for direct utilization in animal feedstuff. We co-expressed three commonly used feed enzymes (phytase, ß-glucanase, and xylanase) in barley seeds using the Agrobacterium-mediated transformation method and generated a new barley germplasm. The results showed that these enzymes were stable and had no effect on the development of the seeds. Supplementation of the basal diet of laying hens with only 8% of enzyme-containing seeds decreased the quantities of indigestible carbohydrates, improved the availability of phosphorus, and reduced the impact of animal production on the environment to an extent similar to directly adding exogenous enzymes to the feed. Feeding enzyme-containing seeds to layers significantly increased the strength of the eggshell and the weight of the eggs by 10.0%-11.3% and 5.6%-7.7% respectively. The intestinal microbiota obtained from layers fed with enzyme-containing seeds was altered compared to controls and was dominated by Alispes and Rikenella. Therefore, the transgenic barley seeds produced in this study can be used as an ideal feedstuff for use in animal feed.

Gossypol and related compounds are produced and accumulate in the aboveground parts of the cotton plant, independent of roots as the source.

MAIN CONCLUSION: Use of Ultra-low gossypol cottonseed event as a scion in a graft combination confirmed that roots are not a source of terpenoids in the aboveground parts of a cotton plant. Gossypol and related terpenoids, derived from the same basic biosynthetic pathway, are present in the numerous lysigenous glands in the aboveground parts of a cotton plant. Roots, with sparse presence of such glands, do produce significant amount of gossypol and a different set of terpenoids. These compounds serve a defensive function against various pests and pathogens. This investigation was undertaken to examine whether gossypol produced in the roots can replenish the gossypol content of the cottonseed-glands that are largely devoid of this terpenoid in a genetically engineered event. Graft unions between a scion derived from the RNAi-based, Ultra-low gossypol cottonseed (ULGCS) event, TAM66274, and a rootstock derived from wild-type parental genotype, Coker 312 (Coker), were compared with various other grafts that served as controls. The results showed that the seeds developing within the scion of test grafts (ULGCS/Coker) continued to maintain the ultra-low gossypol levels found in the TAM66274 seeds. Molecular analyses confirmed that while the key gene involved in gland development showed normal activity in the developing embryos in the scion, two genes encoding the enzymes involved in gossypol biosynthesis were suppressed. Thus, the gene expression data confirmed the results obtained from biochemical measurements and collectively demonstrated that roots are not a source of gossypol for the aboveground parts of the cotton plant. These findings, combined with the results from previous investigations, support the assertion that gossypol and related terpenoids are produced in a highly localized manner in various organs of the cotton plant and are retained therein.

Therapeutic Potential of Mesenchymal Stem Cells in the Treatment of Epilepsy and Their Interaction with Antiseizure Medications.

Epilepsy is a life-threatening neurological disease that affects approximately 70 million people worldwide. Although the vast majority of patients may be successfully managed with currently used antiseizure medication (ASM), the search for alternative therapies is still necessary due to pharmacoresistance in about 30% of patients with epilepsy. Here, we review the effects of ASMs on stem cell treatment when they could be, as expected, co-administered. Indeed, it has been reported that ASMs produce significant effects on the differentiation and determination of stem cell fate. In addition, we discuss more recent findings on mesenchymal stem cells (MSCs) in pre-clinical and clinical investigations. In this regard, their ability to differentiate into various cell types, reach damaged tissues and produce and release biologically active molecules with immunomodulatory/anti-inflammatory and regenerative properties make them a high-potential therapeutic tool to address neuroinflammation in different neurological disorders, including epilepsy. Overall, the characteristics of MSCs to be genetically engineered, in order to replace dysfunctional elements with the aim of restoring normal tissue functioning, suggested that these cells could be good candidates for the treatment of epilepsy refractory to ASMs. Further research is required to understand the potential of stem cell treatment in epileptic patients and its interaction with ASMs.

Chimeric Antigen Receptor T-Cell Therapy in Lung Cancer: Potential and Challenges.

Chimeric antigen receptor T (CAR-T) cell therapy has exhibited a substantial clinical response in hematological malignancies, including B-cell leukemia, lymphoma, and multiple myeloma. Therefore, the feasibility of using CAR-T cells to treat solid tumors is actively evaluated. Currently, multiple basic research projects and clinical trials are being conducted to treat lung cancer with CAR-T cell therapy. Although numerous advances in CAR-T cell therapy have been made in hematological tumors, the technology still entails considerable challenges in treating lung cancer, such as on-target, of-tumor toxicity, paucity of tumor-specific antigen targets, T cell exhaustion in the tumor microenvironment, and low infiltration level of immune cells into solid tumor niches, which are even more complicated than their application in hematological tumors. Thus, progress in the scientific understanding of tumor immunology and improvements in the manufacture of cell products are advancing the clinical translation of these important cellular immunotherapies. This review focused on the latest research progress of CAR-T cell therapy in lung cancer treatment and for the first time, demonstrated the underlying challenges and future engineering strategies for the clinical application of CAR-T cell therapy against lung cancer.

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.

Transcriptome repository of North-Western Himalayan endangered medicinal herbs: a paramount approach illuminating molecular perspective of phytoactive molecules and secondary metabolism.

Medicinal plants of the North-Western Himalayan region are known for their unprecedented biodiversity and valuable secondary metabolites that are unique to this dynamic geo-climatic region. From ancient times these medicinal herbs have been used traditionally for their therapeutic potentials. But from the last 2 decades increasing pharmaceutical demand, illegal and unorganized trade of these medicinal plants have accelerated the rate of over-exploitation in a non-scientific manner. In addition, climate change and anthropogenic activities also affected their natural habitat and driving most of these endemic plant species to critically endangered that foresee peril of mass extinction from this eco-region. Hence there is an urgent need for developing alternative sustainable approaches and policies to utilize this natural bioresource ensuring simultaneous conservation. Hither, arise the advent of sequencing-based transcriptomic studies significantly contributes to better understand the background of important metabolic pathways and related genes/enzymes of high-value medicinal herbs, in the absence of genomic information. The use of comparative transcriptomics in conjunction with biochemical techniques in North-Western Himalayan medicinal plants has resulted in significant advances in the identification of the molecular players involved in the production of secondary metabolic pathways over the last decade. This information could be used to further engineer metabolic pathways and breeding programs, ultimately leading to the development of in vitro systems dedicated to the production of pharmaceutically important secondary metabolites at the industrial level. Collectively, successful adoption of these approaches can certainly ensure the sustainable utilization of Himalayan bioresource by reducing the pressure on the wild population of these critically endangered medicinal herbs. This review provides novel insight as a transcriptome-based bioresource repository for the understanding of important secondary metabolic pathways genes/enzymes and metabolism of endangered high-value North-Western Himalayan medicinal herbs, so that researchers across the globe can effectively utilize this information for devising effective strategies for the production of pharmaceutically important compounds and their scale-up for sustainable usage and take a step forward in omics-based conservation genetics.

Tumor Temporal Proteome Profiling Reveals the Immunological Triple Offensive Induced by Synthetic Anti-Cancer Salmonella.

The engineered "obligate" anaerobic Salmonella typhimurium strain YB1 shows a prominent ability to repress tumor growth and metastasis, which has great potential as a novel cancer immunotherapy. However, the antitumor mechanism of YB1 remains unelucidated. To resolve the proteome dynamics induced by the engineered bacteria, we applied tumor temporal proteome profiling on murine bladder tumors after intravenous injection of either YB1 or PBS as a negative control. Our data suggests that during the two weeks treatment of YB1 injections, the cured tumors experienced three distinct phases of the immune response. Two days after injection, the innate immune response was activated, particularly the complement and blood coagulation pathways. In the meantime, the phagocytosis was initiated. The professional phagocytes such as macrophages and neutrophils were recruited, especially the infiltration of iNOS+ and CD68+ cells was enhanced. Seven days after injection, substantial amount of T cells was observed at the invasion margin of the tumor. As a result, the tumor shrunk significantly. Overall, the temporal proteome profiling can systematically reveal the YB1 induced immune responses in tumor, showing great promise for elucidating the mechanism of bacteria-mediated cancer immunotherapy.

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.

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.

Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doubled Haploids.

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.

Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study.

Phenylketonuria (PKU) is a rare disease caused by biallelic mutations in the PAH gene that result in an inability to convert phenylalanine (Phe) to tyrosine, elevated blood Phe levels and severe neurological complications if untreated. Most patients are unable to adhere to the protein-restricted diet, and thus do not achieve target blood Phe levels. We engineered a strain of E. coli Nissle 1917, designated SYNB1618, through insertion of the genes encoding phenylalanine ammonia lyase and L-amino acid deaminase into the genome, which allow for bacterial consumption of Phe within the gastrointestinal tract. SYNB1618 was studied in a phase 1/2a randomized, placebo-controlled, double-blind, multi-centre, in-patient study ( NCT03516487 ) in adult healthy volunteers (n = 56) and patients with PKU and blood Phe level ≥600 mmol l-1 (n = 14). Participants were randomized to receive a single dose of SYNB1618 or placebo (part 1) or up to three times per day for up to 7 days (part 2). The primary outcome of this study was safety and tolerability, and the secondary outcome was microbial kinetics. A D5-Phe tracer (15 mg kg-1) was used to study exploratory pharmacodynamic effects. SYNB1618 was safe and well tolerated with a maximum tolerated dose of 2 × 1011 colony-forming units. Adverse events were mostly gastrointestinal and of mild to moderate severity. All participants cleared the bacteria within 4 days of the last dose. Dose-responsive increases in strain-specific Phe metabolites in plasma (trans-cinnamic acid) and urine (hippuric acid) were observed, providing a proof of mechanism for the potential to use engineered bacteria in the treatment of rare metabolic disorders.

CAR-NK Cells in the Treatment of Solid Tumors.

CAR-T (chimeric antigen receptor T) cells have emerged as a milestone in the treatment of patients with refractory B-cell neoplasms. However, despite having unprecedented efficacy against hematological malignancies, the treatment is far from flawless. Its greatest drawbacks arise from a challenging and expensive production process, strict patient eligibility criteria and serious toxicity profile. One possible solution, supported by robust research, is the replacement of T lymphocytes with NK cells for CAR expression. NK cells seem to be an attractive vehicle for CAR expression as they can be derived from multiple sources and safely infused regardless of donor-patient matching, which greatly reduces the cost of the treatment. CAR-NK cells are known to be effective against hematological malignancies, and a growing number of preclinical findings indicate that they have activity against non-hematological neoplasms. Here, we present a thorough overview of the current state of knowledge regarding the use of CAR-NK cells in treating various solid tumors.

Antigen-Specific Regulatory T Cell Therapy in Autoimmune Diseases and Transplantation.

Regulatory T (Treg) cells are a heterogenous population of immunosuppressive T cells whose therapeutic potential for the treatment of autoimmune diseases and graft rejection is currently being explored. While clinical trial results thus far support the safety and efficacy of adoptive therapies using polyclonal Treg cells, some studies suggest that antigen-specific Treg cells are more potent in regulating and improving immune tolerance in a disease-specific manner. Hence, several approaches to generate and/or expand antigen-specific Treg cells in vitro or in vivo are currently under investigation. However, antigen-specific Treg cell therapies face additional challenges that require further consideration, including the identification of disease-relevant antigens as well as the in vivo stability and migratory behavior of Treg cells following transfer. In this review, we discuss these approaches and the potential limitations and describe prospective strategies to enhance the efficacy of antigen-specific Treg cell treatments in autoimmunity and transplantation.

Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip.

Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.