Transient expression and purification of β-caryophyllene synthase in <i>Nicotiana benthamiana</i> to produce β-caryophyllene in vitro

Muthusamy, Sarala Devi; Vetukuri, Ramesh R.; Lundgren, Anneli; Ganji, Suresh; Zhu, Lu; Brodelius, Peter E.; Kanagarajan, Selvaraju

doi:10.7717/peerj.8904

Cited by 12 publications

(12 citation statements)

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“…Genomic and full-length open reading frames of six BnGTR1 and six BnGTR2 paralogs were amplified from genomic DNA and cDNA of cv. Kumily, respectively, using gene specific primers according to published protocols (Kim et al, 2020 ; Muthusamy et al, 2020 ), with minor modifications, and confirmed by sequencing. Genomic DNA sequences of different paralogs from the BnGTR1 and BnGTR2 were aligned to find conserved target sites among the paralogs of each gene.…”

Section: Methodsmentioning

confidence: 99%

Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

Sandgrind

Moss

et al. 2021

Front. Plant Sci.

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Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3–4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l−1) and 2,4-D (0.5 mg l−1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l−1 TDZ in combination with auxin 0.5 mg l−1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.

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Section: Methodsmentioning

confidence: 99%

Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

Sandgrind

Moss

et al. 2021

Front. Plant Sci.

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“…Many studies have reported sesquiterpenoid production in plants. Artemisia annua beta-caryophyllene synthase was transferred into Agrobacterium through a viral vector in a previous study, and then agroinfiltrated into N. benthamiana leaves to produce 26.5 mg of β-caryophyllene ( Muthusamy et al., 2020 ). Transient co-expression of the parthenolide pathway candidate genes in N. benthamiana was used to reconstitute the co-expression, and up to 1.4 µg of the final product was generated ( Liu et al., 2014 ).…”

Section: Discussionmentioning

confidence: 99%

“…These include amorpha-4,11-dienesynthase (ADS), epi-cedrol synthase, β-caryophyllene synthase, (E)-farnesene synthase, germacrene A synthase, and squalene synthase. ADS is the only enzyme involved in artemisinin production ( Liu et al., 2011 ; Kanagarajan et al., 2012 ; Cankar et al., 2015 ; Muthusamy et al., 2020 ). Numerous studies have been conducted to increase mono- and sesquiterpene biosynthesis by inhibiting the expression of mono- and sesquiterpene synthase(s) striving for GPPS and FPP using antisense technology or RNA interference ( Li et al., 2020 ).…”

Section: Discussionmentioning

confidence: 99%

Combinatorial transient gene expression strategies to enhance terpenoid production in plants

et al. 2022

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IntroductionThe monoterpenoid linalool and sesquiterpenoid costunolide are ubiquitous plant components that have been economically exploited for their respective essential oils and pharmaceutical benefits. In general, monoterpenes and sesquiterpenes are produced by the plastid 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytosolic mevalonate (MVA) pathways, respectively. Herein, we investigated the individual and combinatorial potential of MEP and MVA pathway genes in increasing linalool and costunolide production in Nicotiana benthamiana.MethodsFirst, six genes from the MEP (1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, geranyl pyrophosphate synthase, and linalool synthase) and MVA (acetoacetyl-CoA-thiolase, hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, germacrene A synthase, germacrene A oxidase, and costunolide synthase) pathways were separately cloned into the modular cloning (MoClo) golden gateway cassette. Second, the cassettes were transformed individually or in combination into the leaves of N. benthamiana by agroinfiltration.Results and discussionFive days post infiltration (DPI), all selected genes were transiently 5- to 94-fold overexpressed. Quantification using gas chromatography-Q-orbitrap-mass spectrometry (GC-Q-Orbitrap-MS) determined that the individual and combinatorial expression of MEP genes increased linalool production up to 50–90ng.mg-1 fresh leaf weight. Likewise, MVA genes increased costunolide production up to 70–90ng.mg-1 fresh leaf weight. Our findings highlight that the transient expression of MEP and MVA pathway genes (individually or in combination) enhances linalool and costunolide production in plants.

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“…Genomic and full-length open reading frames of six BnGTR1 and six BnGTR2 paralogs were amplified from genomic DNA and cDNA of cv. Kumily, respectively, using gene specific primers according to published protocols (Kim et al, 2020;Muthusamy et al, 2020), with minor modifications, and confirmed by sequencing. Genomic DNA sequences of different paralogs from the BnGTR1 and BnGTR2 were aligned to find conserved target sites among the paralogs of each gene.…”

Section: Identification and Cloning Of Gtr Genes Sgrna Design And Vec...mentioning

confidence: 99%

Genome editing of oilseed species by CRISPR/Cas9 for trait improvement

Sandgrind¹

2022

Acta Universitatis Agriculturae Sueciae

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The demand for vegetable oils for food and other applications is rapidly increasing due to a growing population and an urgent need to replace fossil oils. Furthermore, there are several challenges facing agricultural production, such as a shortage of arable land, climate change, and water pollution due to nutrient leaching from extensive use of fertilizers and tillage. More sustainable agricultural systems, better use of side-streams, domestication of novel oil crops, and improvement of existing oil crops are important for global food security and the supply of raw materials for different industries. In Sweden, rapeseed is the main oil crop, but is mainly cultivated in the southern region of Sweden due to its weak cold tolerance. The seed cake is rich in protein, but is underutilized due to high levels of anti-nutritional compounds such as glucosinolates. Field cress (Lepidium campestre) is a novel cold-hardy and high-yielding oilseed crop that can be undersown with a spring cereal in cool climates as a cover crop for reducing nutrient leaching and soil erosion, while producing a valuable seed oil and improving the cereal yield. Development of the CRISPR/Cas9 genome editing system has revolutionized the field of biology and led to major advances in plant science. This novel breeding tool offers great opportunities for accelerated trait improvement of crops with high precision. In this thesis, we have developed efficient protocols for protoplast transfection and regeneration of field cress and rapeseed, and demonstrated their usefulness in genome editing via CRISPR/Cas9 by mutating multiple glucosinolate transporter genes in rapeseed and field cress for reducing seed glucosinolate contents, and by mutating key genes for improving the oil quality of field cress.

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Transient expression and purification of β-caryophyllene synthase in Nicotiana benthamiana to produce β-caryophyllene in vitro

Cited by 12 publications

References 59 publications

Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

Combinatorial transient gene expression strategies to enhance terpenoid production in plants

Genome editing of oilseed species by CRISPR/Cas9 for trait improvement

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