CRISPR/Cas9-induced β-carotene Hydroxylase Mutation in Dunaliella Salina CCAP19/18

2021 ◽  
Author(s):  
Lina Hu ◽  
Shu ying FENG ◽  
Gaofeng Liang ◽  
Jingxia Du ◽  
Aifang Li ◽  
...  
Keyword(s):  
High Performance ◽  
Dunaliella Salina ◽  
Gene Editing ◽  
Expression System ◽  
Transformation Method ◽  
Practical Significance ◽  
Exon 1 ◽  
Targeted Gene Editing ◽  
Foreign Genes ◽  
Β Carotene

Abstract Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of CRISPR/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.

scholarly journals CRISPR/Cas9-induced β-carotene hydroxylase mutation in Dunaliella salina CCAP19/18

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lina Hu ◽  
Shuying Feng ◽  
Gaofeng Liang ◽  
Jingxia Du ◽  
Aifang Li ◽  
...  
Keyword(s):  
High Performance ◽  
Dunaliella Salina ◽  
Gene Editing ◽  
Expression System ◽  
Transformation Method ◽  
Practical Significance ◽  
Exon 1 ◽  
Short Palindromic Repeat ◽  
Foreign Genes ◽  
Β Carotene

AbstractDunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.

scholarly journals An exploration of the rapid transformation method for Dunaliella salina system

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Guannan Song ◽  
Wan Wang ◽  
Lina Hu ◽  
Yu Liu ◽  
Aifang Li ◽  
...  
Keyword(s):  
Dunaliella Salina ◽  
Genetic Manipulation ◽  
Expression System ◽  
Transformation Method ◽  
Pcr Analysis ◽  
Foreign Gene ◽  
High Efficient ◽  
Set Up ◽  
Transformation Methods ◽  
Rapid Transformation

Abstract As a new expression system, Dunaliella salina (D. salina) has bright prospects and applications in various fields. However, its application is currently restricted because of the low expression and instability of foreign gene in D. salina cells. During genetic operation, transformation is a crucial step for genes expression in D. salina system. Although several transformation methods are existing currently, many inherent deficiencies and limitations still can be found in actual practice. Thus, we attempted to set up a rapid transformation method using the change of salt concentrations for D. salina. Based on osmotic pressure difference, exogenous genes can be spontaneously transferred into D. salina cells. After that, transformed D. salina cells were subjected to histochemical and molecular analysis. The results showed that the reporter gene, beta-glucuronidase genes were successfully expressed in the positive transformants, and detected in all of transformed cells by PCR analysis. Moreover, different transformation parameters, containing the salt gradient, time, dye dosage and Triton X-100 concentration, were optimized to obtain an optimal transformation result. Taken together, we preliminarily established a rapid transformation method with the features of fast, simple, economic, and high-efficient. This method will provide a strong genetic manipulation tool for the future transformation of D. salina system.

Cloning and Characterization of SpcrtR and Its Expression in Escherichia Coli for Zeaxanthin Production

2021 ◽  
Author(s):  
Jun Ma ◽  
Yuxi Yin ◽  
Chenqiang Qian ◽  
Jiaxin Chen ◽  
Di-Feng Ren
Keyword(s):  
Western Blotting ◽  
High Performance ◽  
Expression System ◽  
Specific Primers ◽  
E Coli ◽  
Sds Page ◽  
Clone And Express ◽  
Rate Limiting ◽  
Β Carotene

Abstract Zeaxanthin is produced by a series of enzyme catalytic reactions. β-carotene hydroxylase is a key rate-limiting enzyme that catalyzes the conversion of β-carotene to zeaxanthin. The purpose of this study was to clone and express the Spirulina platensis β-carotene hydroxylase gene (SpcrtR) in E. coli. SpcrtR was amplified using specific primers and cloned in vector pGEX-6p-1, the SpcrtR protein (35kDa) was expressed from pGEX-6p-1-SpcrtR in E. coli. Gene expression was analyzed by SDS-PAGE and western blotting. The accumulation of carotenoids was detected by high-performance liquid chromatography (HPLC). SDS-PAGE and western blotting analysis confirmed that SpcrtR protein (35kDa) was expressed in E. coli expression system. Further, the HPLC results demonstrated that SpcrtR can partially catalyze β-carotene to produce zeaxanthin in E. coli. Zeaxanthin and β-carotene yields in recombinant E.coli were 85.71% (±0.4%) and 14.7% (±0.6%), respectively. The results in our study verified that SpCRTR enzyme partially catalyzes the substrate β-carotene to form zeaxanthin. Overall, SpCRTR provides a new choice for enzymatic zeaxanthin production.

scholarly journals Concentration of Bioactive Phenolic Compounds in Olive Mill Wastewater by Direct Contact Membrane Distillation

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1808
Author(s):  
Rosa Tundis ◽  
Carmela Conidi ◽  
Monica R. Loizzo ◽  
Vincenzo Sicari ◽  
Rosa Romeo ◽  
...  
Keyword(s):  
Direct Contact ◽  
High Performance ◽  
Biological Properties ◽  
Membrane Distillation ◽  
Olive Mill Wastewater ◽  
High Concentration ◽  
Direct Contact Membrane Distillation ◽  
Reducing Activity ◽  
Β Carotene ◽  
Olive Mill

Olive mill wastewater (OMW), generated as a by-product of olive oil production, is considered one of the most polluting effluents produced by the agro-food industry, due to its high concentration of organic matter and nutrients. However, OMW is rich in several polyphenols, representing compounds with remarkable biological properties. This study aimed to analyze the chemical profile as well as the antioxidant and anti-obesity properties of concentrated fractions obtained from microfiltered OMW treated by direct contact membrane distillation (DCMD). Ultra-high performance liquid chromatography (UHPLC) analyses were applied to quantify some phenols selected as phytochemical markers. Moreover, α-Amylase, α-glucosidase, and lipase inhibitory activity were investigated together with the antioxidant activity by means of assays, namely β-carotene bleaching, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) diammonium salts, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and Ferric Reducing Activity Power (FRAP) tests. MD retentate—which has content of about five times greater of hydroxytyrosol and verbascoside and about 7 times greater of oleuropein than the feed—was more active as an antioxidant in all applied assays. Of interest is the result obtained in the DPPH test (an inhibitory concentration 50% (IC50) of 9.8 μg/mL in comparison to the feed (IC50 of 97.2 μg/mL)) and in the ABTS assay (an IC50 of 0.4 μg/mL in comparison to the feed (IC50 of 1.2 μg/mL)).

scholarly journals Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jonathan O’Keeffe Ahern ◽  
Irene Lara-Sáez ◽  
Dezhong Zhou ◽  
Rodolfo Murillas ◽  
Jose Bonafont ◽  
...  
Keyword(s):  
Delivery System ◽  
Gene Editing ◽  
Transfection Efficiency ◽  
Attractive Alternative ◽  
Safe Delivery ◽  
Guide Rna ◽  
Recessive Dystrophic Epidermolysis Bullosa ◽  
Targeted Gene Editing ◽  
Genomic Editing ◽  
Polymeric Vectors

AbstractRecent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

scholarly journals Molecular and phylogenetic analysis reveals new diversity of Dunaliella salina from hypersaline environments

2021 ◽  
pp. 1-11
Author(s):  
Andrea Highfield ◽  
Angela Ward ◽  
Richard Pipe ◽  
Declan C. Schroeder
Keyword(s):  
Genetic Diversity ◽  
Phylogenetic Analysis ◽  
Dunaliella Salina ◽  
Phylogenetic Analyses ◽  
Ssu Rdna ◽  
Lsu Rdna ◽  
Hypersaline Environments ◽  
Rdna Its ◽  
Β Carotene ◽  
Selection Of

Abstract Twelve hyper-β carotene-producing strains of algae assigned to the genus Dunaliella salina have been isolated from various hypersaline environments in Israel, South Africa, Namibia and Spain. Intron-sizing of the SSU rDNA and phylogenetic analysis of these isolates were undertaken using four commonly employed markers for genotyping, LSU rDNA, ITS, rbcL and tufA and their application to the study of Dunaliella evaluated. Novel isolates have been identified and phylogenetic analyses have shown the need for clarification on the taxonomy of Dunaliella salina. We propose the division of D. salina into four sub-clades as defined by a robust phylogeny based on the concatenation of four genes. This study further demonstrates the considerable genetic diversity within D. salina and the potential of genetic analyses for aiding in the selection of prospective economically important strains.

Effects of various environmental factors on β-carotene production by Dunaliella salina. —note—

1992 ◽  
Vol 73 (2) ◽  
pp. 178
Author(s):  
Yukiho Yamaoka ◽  
Osamu Takimura ◽  
Hiroyuki Fuse ◽  
Kazuo Kamimura ◽  
Eichi Manabe ◽  
...  
Keyword(s):  
Environmental Factors ◽  
Dunaliella Salina ◽  
Β Carotene

scholarly journals A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Cécile Beck ◽  
Philippe Desprès ◽  
Sylvie Paulous ◽  
Jessica Vanhomwegen ◽  
Steeve Lowenski ◽  
...  
Keyword(s):  
High Performance ◽  
Neurological Diseases ◽  
Expression System ◽  
Cross Reactivity ◽  
Encephalitis Virus ◽  
Serological Tests ◽  
Multiplex Immunoassay ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Negative Controls

West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using theDrosophilaS2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.

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