Genetic Engineering Approaches Research Articles

Genetic and metabolic engineering approaches for enhanced biodesulfurization of petroleum fractions

Sulfur, an abundant component of crude oil, causes severe damage to the environment, poses risks to human health, and poisons the catalysts used in combustion engines. Hydrodesulfurization, the conventionally used method, is not sufficient to remove thiophenes like dibenzothiophene (DBT) and other aromatic heterocyclic compounds. The push for “ultra-clean” fuels, with sulfur content less than 15 ppm, drives the need for deep desulfurization. Thus, in conjunction with hydrodesulfurization, efficient and eco-friendly methods of deep desulfurization, like biodesulfurization, are desirable. In biodesulfurization, naturally desulfurizing microorganisms are used, with genetic engineering and biotechnology, to reduce the sulfur content of crude oil to below 15 ppm. In this review, we describe genetic and metabolic engineering approaches reported to date to develop more efficient methods to carry out biodesulfurization, making it a practically applicable reality.

Proteomic analysis reveals molecular changes following genetic engineering in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome. The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response. These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications.

P3.02F.05 A Genetic Engineering Approach to Interrogate Myosin 10 As a Driver of Non-Small Cell Lung Cancer Metastasis

P3.02F.05 A Genetic Engineering Approach to Interrogate Myosin 10 As a Driver of Non-Small Cell Lung Cancer Metastasis

Transcriptome sequencing and identification of full-length genes involved in the biosynthesis of anticancer compounds Oleanolic acid and Ursolic acid in Achyranthes aspera L.

Achyranthes aspera is renowned for its rich medicinal properties since the Ayurvedic era. This plant is known for the presence of experimentally validated anticancer compounds like oleanolic acid and ursolic acid. Our study involved sequencing the RNA from the root tissue of A. aspera to elucidate the genes responsible for synthesizing these two critical secondary metabolites. Through RNA-Seq analysis, we assembled approximately 167,698 transcripts, averaging 847 base pairs in length, with an N50 value of 1509 bp. From this data, we mapped 604 sequences involved in the metabolism of terpenoids and polyketide pathways. Among them, 241 transcripts were mapped to the triterpenoid biosynthesis pathway, which included 127 transcripts involved in OA and UA biosynthesis. From these transcripts, we identified 22 full-length genes coding for all the 21 enzymes required for OA and UA biosynthesis. Identifying these full-length genes will lead to a better understanding of the pathway and adopting genetic engineering approaches.

Innovative Bacterial Therapies and Genetic Engineering Approaches in Colorectal Cancer: A Review of Emerging Strategies and Clinical Implications.

Colorectal cancer (CRC) is considered a widespread cancer, ranking second in mortality and incidence among cancer patients worldwide. CRC develops from adenoma to carcinoma through the dynamic interplay of genetic and environmental factors. The conventional modes of treatment, including operation, chemotherapy, and irradiation, are associated with significant challenges, such as drug resistance and toxicity, necessitating the exploration of new treatment modalities. These difficulties reveal the necessity of the emergence of new therapeutic approaches. This review mainly emphasizes the bacterial-based therapies that have recently developed like the engineered bacteriophage therapy and bacterial immunotherapy that pale the existing chemotherapy in terms of toxicity but are effective in killing tumor cells. Also, it also investigates various molecular genetic engineering strategies such as CRISPR-Cas9, CRISPR prime editing and gene silencing to achieve better targeting of CRC. Implementing these new approaches into the forefront of CRC treatment may bring better, more effective therapy with fewer side effects on patients' quality of life.

Redundancy in Innate Immune Pathways That Promote CD8+ T-Cell Responses in AAV1 Muscle Gene Transfer.

While adeno-associated viral (AAV) vectors are successfully used in a variety of in vivo gene therapy applications, they continue to be hampered by the immune system. Here, we sought to identify innate and cytokine signaling pathways that promote CD8+ T-cell responses against the transgene product upon AAV1 vector administration to murine skeletal muscle. Eliminating just one of several pathways (including DNA sensing via TLR9, IL-1 receptor signaling, and possibly endosomal sensing of double-stranded RNA) substantially reduced the CD8+ T-cell response at lower vector doses but was surprisingly ineffective at higher doses. Using genetic, antibody-mediated, and vector engineering approaches, we show that blockade of at least two innate pathways is required to achieve an effect at higher vector doses. Concurrent blockade of IL-1R1 > MyD88 and TLR9 > MyD88 > type I IFN > IFNaR pathways was often but not always synergistic and had limited utility in preventing antibody formation against the transgene product. Further, even low-frequency CD8+ T-cell responses could eliminate transgene expression, even in MyD88- or IL-1R1-deficient animals that received a low vector dose. However, we provide evidence that CpG depletion of vector genomes and including TLR9 inhibitory sequences can synergize. When this construct was combined with the use of a muscle-specific promoter, transgene expression in muscle was sustained with minimal local or systemic CD8+ T-cell response. Thus, innate immune avoidance/blockade strategies by themselves, albeit helpful, may not be sufficient to prevent destructive cellular responses in muscle gene transfer because of the redundancy of immune-activating pathways.

Photosynthesis: Genetic Strategies Adopted to Gain Higher Efficiency.

The global challenge of feeding an ever-increasing population to maintain food security requires novel approaches to increase crop yields. Photosynthesis, the fundamental energy and material basis for plant life on Earth, is highly responsive to environmental conditions. Evaluating the operational status of the photosynthetic mechanism provides insights into plants' capacity to adapt to their surroundings. Despite immense effort, photosynthesis still falls short of its theoretical maximum efficiency, indicating significant potential for improvement. In this review, we provide background information on the various genetic aspects of photosynthesis, explain its complexity, and survey relevant genetic engineering approaches employed to improve the efficiency of photosynthesis. We discuss the latest success stories of gene-editing tools like CRISPR-Cas9 and synthetic biology in achieving precise refinements in targeted photosynthesis pathways, such as the Calvin-Benson cycle, electron transport chain, and photorespiration. We also discuss the genetic markers crucial for mitigating the impact of rapidly changing environmental conditions, such as extreme temperatures or drought, on photosynthesis and growth. This review aims to pinpoint optimization opportunities for photosynthesis, discuss recent advancements, and address the challenges in improving this critical process, fostering a globally food-secure future through sustainable food crop production.

Biotechnological approaches for enhancement of heavy metal phytoremediation capacity of plants.

Heavy metals are extremely hazardous for human health due to their toxic effects. They are non-biodegradable in nature, thus remain in the environment and enter and accumulate in the human body through biomagnification; hence, there is a serious need of their remediation. Phytoremediation has emerged as a green, sustainable, and effective solution for heavy metal removal and many plant species could be employed for this purpose. Plants are able to sequester substantial quantity of heavy metals, in some cases thousands of ppm, due to their robust physiology enabling high metal tolerance and anatomy supporting metal ion accumulation. Identification and modification of potential target genes involved in heavy metal accumulation have led to improved phytoremediation capacity of plants at the molecular level. The introduction of foreign genes through genetic engineering approaches has further enhanced phytoremediation capacity manifolds. This review gives an insight towards improving the phytoremediation efficiency through a better understanding of molecular mechanisms involved, expression of different proteins, genetic engineering approaches for transgenic production, and genetic modifications. It also comprehends novel omics tools such as genomics, metabolomics, proteomics, transcriptomics, and genome editing technologies for improvement of phytoremediation ability of plants.

NHEJ and HDR can occur simultaneously during gene integration into the genome of Aspergillus niger

Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are two mechanisms in filamentous fungi to repair DNA damages. NHEJ is the dominant response pathway to rapidly join DNA double-strand breaks, but often leads to insertions or deletions. On the other hand, HDR is more precise and utilizes a homologous DNA template to restore the damaged sequence. Both types are exploited in genetic engineering approaches ranging from knock-out mutations to precise sequence modifications.In this study, we evaluated the efficiency of an HDR based gene integration system designed for the pyrG locus of Aspergillus niger. While gene integration was achieved at a rate of 91.4%, we also discovered a mixed-type repair (MTR) mechanism with simultaneous repair of a Cas9-mediated double-strand break by both NHEJ and HDR. In 20.3% of the analyzed transformants the donor DNA was integrated by NHEJ at the 3’ end and by HDR at the 5’ end of the double-strand break. Furthermore, sequencing of the locus revealed different DNA repair mechanisms at the site of the NHEJ event.Together, the results support the applicability of the genome integration system and a novel DNA repair type with implication on the diversity of genetic modifications in filamentous fungi.

Genetic engineering of Salmonella spp. for novel vaccine strategies and therapeutics.

Salmonella enterica is a diverse species that infects both humans and animals. S. enterica subspecies enterica consists of more than 1,500 serovars. Unlike typhoidal Salmonella serovars which are human host-restricted, non-typhoidal Salmonella (NTS) serovars are associated with foodborne illnesses worldwide and are transmitted via the food chain. Additionally, NTS serovars can cause disease in livestock animals causing significant economic losses. Salmonella is a well-studied model organism that is easy to manipulate and evaluate in animal models of infection. Advances in genetic engineering approaches in recent years have led to the development of Salmonella vaccines for both humans and animals. In this review, we focus on current progress of recombinant live-attenuated Salmonella vaccines, their use as a source of antigens for parenteral vaccines, their use as live-vector vaccines to deliver foreign antigens, and their use as therapeutic cancer vaccines in humans. We also describe development of live-attenuated Salmonella vaccines and live-vector vaccines for use in animals.

Trojan Horse virus delivering CRISPR-AsCas12f1 controls plant bacterial wilt caused by Ralstonia solanacearum.

Plant bacterial wilt caused by Ralstonia solanacearum results in huge losses. Accordingly, developing an effective control method for this disease is urgently required. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential biocontrol solution. A filamentous phage RSCq that infects R. solanacearum was isolated in this study through genome mining. We constructed engineered filamentous phages based on RSCq by employing our proposed approach with wide applicability to non-model phages, enabling the exogenous genes delivery into bacterial cells. CRISPR-AsCas12f1 is a miniature class 2 type V-F CRISPR-Cas system. A CRISPR-AsCas12f1-based gene editing system that targets the key virulence regulator gene hrpB was developed, generating the engineered phage RSCqCRISPR-Cas. Similar to the Greek soldiers in the Trojan Horse, our findings demonstrated that the engineered phage-delivered CRISPR-Cas system could disarm the key "weapon," hrpB, of R. solanacearum, in medium and plants. Remarkably, pretreatment with RSCqCRISPR-Cas significantly controlled tobacco bacterial wilt, highlighting the potential of engineered filamentous phages as promising biocontrol agents against plant bacterial diseases.IMPORTANCEBacterial disease, one of the major plant diseases, causes huge food and economic losses. Phage therapy, an environmentally friendly control strategy, has been frequently reported in plant bacterial disease control. However, host specificity, sensitivity to ultraviolet light and certain conditions, and bacterial resistance to phage impede the widespread application of phage therapy in crop production. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential solution to overcome the limitations of lytic phage biocontrol. This study developed a genetic engineering approach with wide applicability to non-model filamentous phages and proved the application possibility of engineered phage-based gene delivery in plant bacterial disease biocontrol for the first.

Impact of Methyl Jasmonate on Terpenoid Biosynthesis and Functional Analysis of Sesquiterpene Synthesis Genes in Schizonepeta tenuifolia.

This study investigates the impact of methyl jasmonate (MeJA) on the volatile oil composition of Schizonepeta tenuifolia and elucidates the function of the StTPS45 gene, a key player in terpenoid biosynthesis. The effect of different concentrations of MeJA (0, 50, 100, 200, and 300 μmol/L) on the growth of S. tenuifolia adventitious bud clusters was analyzed over a 20 d period. Using gas chromatography-mass spectrometry (GC-MS), 17 compounds were identified from the adventitious bud clusters of S. tenuifolia. Significant changes in the levels of major monoterpenes, including increased contents of (+)-limonene and (+)-menthone, were observed, particularly at higher concentrations of MeJA. Analysis of transcriptome data from three groups treated with 0, 100, and 300 μmol/L MeJA revealed significant changes in the gene expression profiles following MeJA treatment. At 100 μmol/L MeJA, most terpene synthase (TPS) genes were overexpressed. Additionally, gene expression and functional predictions suggested that StTPS45 acts as germacrene D synthase. Therefore, StTPS45 was cloned and expressed in Escherichia coli, and enzyme activity assays confirmed its function as a germacrene D synthase. Molecular docking and structural prediction of StTPS45 further suggested specific interactions with farnesyl diphosphate (FPP), aligning with its role in the terpenoid synthesis pathway. These findings provide valuable insights into the modulation of secondary metabolite pathways by jasmonate signaling and underscore the potential of genetic engineering approaches to enhance the production of specific terpenoids in medicinal plants.

Comparative Transcriptome Analysis of Different Mulberry Varieties to Reveal Candidate Genes and Small Secreted Peptides Involved in the Sclerotiniose Response

Mulberry sclerotiniose is a devastating fungal disease of mulberry fruit and has been a limitation for the utility of mulberry fruits and the diversified development of sericulture. In the present study, we presented a workflow for screening candidate sclerotiniose-resistance genes and small secreted peptides (SSPs) based on a genome-wide annotation of SSPs and comparative transcriptome analysis of different mulberry varieties. A total of 1088 SSPs with expression evidence were identified and annotated in mulberry. A comprehensive analysis of the sclerotiniose-related RNA sequencing datasets showed that photosynthesis, plant hormone signaling, and metabolic pathways were the main pathways involved in the response to sclerotiniose. Fifty-two candidate sclerotiniose-response genes (SRGs), including 15 SSPs, were identified based on comparative transcriptome analysis. These SRGs are mainly involved in the hormone signaling pathway and cell wall biogenesis. Transient overexpression in tobacco and the knock-down of five SRGs affected the resistance against Ciboria shiraiana. MaMYB29, MaMES17, and MaSSP15 were primarily determined as negative regulators of plant resistance to C. shiraiana infection. Our results provide a foundation for controlling sclerotiniose in mulberry using genetic engineering and biological approaches such as spraying antifungal peptides.

Development of approaches for genome editing of pea plants using CRISPR/Cas9 prime-editing technique

Mitogen-activated protein kinases (MAPKs) play an important role as intracellular regulators of signal pathways in plants. Some MAPKs have been shown to be induced in the roots of legume plants during symbiosis with nitrogen-fixing rhizobial bacteria. One of such signal regulators, MAPK6, was shown to be involved in the development of symbiosis between Pisum sativum L. pea plants and rhizobia. Using genetic engineering approaches, we overexpressed the MAPK6 gene in transgenic roots, that resulted in an increase in the number of nodules and the biomass of pea plants. New approaches for genome editing of pea plants have been designed using the CRISPR/Cas9 prime-editing technique, when the MAPK6 gene was used as a target. We have analyzed the transgenic roots of pea transformants and observed the presence of the gene encoding Cas9 the pegRNA sequence in the genome of transformants. Therefore, the possibility of using genetic engineering methods to obtain plants with increased efficiency of symbiosis will be investigated in future experiments.

Transgenesis in microalga <i>Chlamydomonas reinhardtii</i>: current approaches

Microalgae are a rich source of biologically active substances of natural origin, which have potential for use in pharmaceutical, agricultural, food and industrial production. Genetic engineering of microalgae opens up great prospects for creating improved strains that produce various food additives, commercial enzymes, as well as proteins for therapeutic purposes — antibodies, hormones and vaccines. Chlamydomonas reinhardtii P.A. Dang. is a unicellular green alga, a reference organism for studying the genetics of photosynthesis and developing new genetic engineering approaches in microalgae. The advantages of C. reinhardtii include the ability to transform all three of its genomes (nuclear, mitochondrial and chloroplast), low cost and ease of cultivation, safety for humans and the presence of a system for post-translational modification of proteins, which makes this organism a potential platform for use in biotechnology. Over the past few years, significant advances have been made in transgenesis of C. reinhardtii, including the use of new techniques based on the CRISPR/Cas9 genome editing technology. In this review, we summarize the available information on current approaches to transgenesis of the unicellular green alga C. reinhardtii: 1) general principles of transgenic constructs design for transformation of the nuclear and chloroplast genome, 2) popular selection markers used, 3) methods of cell transformation, 4) methods of genome editing using the CRISPR/Cas9 system.

Internalization of transferrin-tagged Myxococcus xanthus encapsulins into mesenchymal stem cells.

Currently, various functionalized nanocarrier systems are extensively studied for targeted delivery of drugs, peptides, and nucleic acids. Joining the approaches of genetic and chemical engineering may produce novel carriers for precise targeting different cellular proteins, which is important for both therapy and diagnosis of various pathologies. Here we present the novel nanocontainers based on vectorized genetically encoded Myxococcus xanthus (Mx) encapsulin, confining a fluorescent photoactivatable mCherry (PAmCherry) protein. The shells of such encapsulins were modified using chemical conjugation of human transferrin (Tf) prelabeled with a fluorescein-6 (FAM) maleimide acting as a vector. We demonstrate that the vectorized encapsulin specifically binds to transferrin receptors (TfRs) on the membranes of mesenchymal stromal/stem cells (MSCs) followed by internalization into cells. Two spectrally separated fluorescent signals from Tf-FAM and PAmCherry are clearly distinguishable and co-localized. It is shown that Tf-tagged Mx encapsulins are internalized by MSCs much more efficiently than by fibroblasts. It has been also found that unlabeled Tf effectively competes with the conjugated Mx-Tf-FAM formulations. That indicates the conjugate internalization into cells by Tf-TfR endocytosis pathway. The developed nanoplatform can be used as an alternative to conventional nanocarriers for targeted delivery of, e.g., genetic material to MSCs.

Expression, characterization and antivascular activity of amino acid sequence repeating collagen hexadecapeptide

Type V collagen is an essential component of the extracellular matrix (ECM), and its remodeling releases specific protein fragments that can specifically inhibit endothelial cell responses such as proliferation, migration, and invasion. In this study, we have successfully constructed two engineered strains of Pichia pastoris capable of producing recombinant collagen through a new genetic engineering approach. Through high-density fermentation, the expression of 1605 protein and 1610 protein could reach 2.72 g/L and 4.36 g/L. With the increase of repetition times, the yield also increased. Bioactivity analysis showed that recombinant collagen could block the angiogenic effect of FGF-2 on endothelial cells by eliminating FGF-2-induced endothelial cell migration and invasion. Collectively, the recombinant proteins we successfully expressed have a wide range of potential for inhibiting angiogenesis in the biomaterials and biomedical fields.

Futuristic opportunities for pretreatment processes in biofuel production from microalgae

AbstractMicroalgal biofuel is a promising solution to replace fossil fuel as a renewable and environmental‐friendly energy source, thereby contributing to the United Nations (UN) Sustainable Development Goals (SDGs), in particular SDG‐7, or Affordable and Clean Energy. Unlike energy crops (like oil palm and sugar cane), microalgae benefit from faster growth rate, higher lipid content, smaller land area required, ability to flourish using waste or brackish water, and posing zero competition with food crops. Microalgae‐derived biofuels (like biodiesel, bioethanol, biomethane, and biohydrogen) are sustainable energy sources that can be produced using well‐developed techniques (e.g., transesterification, fermentation, anaerobic digestion, and Fisher–Tropsch process). To prevent dire climate conditions resulting from the global temperature rise of 1.5°C and resolve worldwide energy security issue, our generation will need to establish and implement renewables on a global scale. To improve the industrial production of microalgal biofuel, the efficiencies of biomass and metabolite production to post‐cultivation biofuel synthesis processes must be enhanced. For the cultivation step, there exist three key techniques that can directly change the traits, structure, and behavior of microalgal cells, and induce them to accumulate targeted metabolites rapidly and in large amounts. These techniques are genetic engineering, chemical modulation, and nanomaterial approach. Genetic engineering commonly alters the chloroplast DNA of microalgae to overexpress or down‐regulate key genes in various metabolic pathways so that the cells accumulate more lipids. Chemicals can also be used to modulate microalgal growth and lipid accumulation by inducing oxidative stress or prevent conversion of lipid molecules. Nanomaterials and nanoparticles can also enhance microalgal lipid production by microenvironmental stress induction, vitamin supplementation, and light backscattering. Therefore, in this review, the recent progress as well as the pros and cons of genetic engineering, chemical modulation, and nanomaterial approach in achieving greater biofuel production from microalgae are comprehensively examined.

Advancements in the Application of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs).

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a significant potential for novel therapeutic applications because of their bioactive properties, stability, and specificity. RiPPs are synthesized on ribosomes, followed by intricate post-translational modifications (PTMs), crucial for their diverse structures and functions. PTMs, such as cyclization, methylation, and proteolysis, play crucial roles in enhancing RiPP stability and bioactivity. Advances in synthetic biology and bioinformatics have significantly advanced the field, introducing new methods for RiPP production and engineering. These methods encompass strategies for heterologous expression, genetic refactoring, and exploiting the substrate tolerance of tailoring enzymes to create novel RiPP analogs with improved or entirely new functions. Furthermore, the introduction and implementation of cutting-edge screening methods, including mRNA display, surface display, and two-hybrid systems, have expedited the identification of RiPPs with significant pharmaceutical potential. This comprehensive review not only discusses the current advancements in RiPP research but also the promising opportunities that leveraging these bioactive peptides for therapeutic applications presents, illustrating the synergy between traditional biochemistry and contemporary synthetic biology and genetic engineering approaches.

Expression of Multicopy Tandem Recombinant Ginseng Hexapeptide in Bacillus subtilis and the Evaluation of Antiaging Activity.

Ginseng oligopeptides are naturally occurring small-molecule peptides extracted from ginseng that exhibit positive effects on health and longevity. However, the current industrial production of ginseng oligopeptides primarily relies on plant extraction and chemical synthesis. In this study, we proposed a novel genetic engineering approach to produce active ginseng peptides through multicopy tandem insertion (5 and 15 times). The recombinant ginseng peptides were successfully produced from engineered Bacillus subtilis with an increasing yield from 356.55 to 2900 mg/L as the repeats multiple. Additionally, an oxidative stress-induced aging model caused by H2O2 was established to evaluate whether the recombinant ginseng peptides, without enzymatic hydrolysis into individual peptides, also have positive effects on antiaging. The results demonstrated that all two kinds of recombinant ginseng peptides could also delay cellular aging through various mechanisms, such as inhibiting cell cycle arrest, suppressing the expression of pro-inflammatory factors, and enhancing cellular antioxidant capacity.