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Author: search.filters.author.Pandey, AshutoshSubject: search.filters.subject.Metabolic engineering

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    The R2R3‑MYB transcription factor MtMYB134 orchestrates favonol biosynthesis in Medicago truncatula
    (Springer Nature Publishing AG, 2021) Naik, Jogindra; Rajput, Ruchika; Pucker, Boas; Stracke, Ralf; Pandey, Ashutosh
    Flavonols are plant specialized metabolites with vital roles in plant development and defense and are known as diet compound beneficial to human health. In leguminous plants, the regulatory proteins involved in flavonol biosynthesis are not well characterized. Using a homology-based approach, three R2R3-MYB transcription factor encoding genes have been identified in the Medicago truncatula reference genome sequence. The gene encoding a protein with highest similarity to known flavonol regulators, MtMYB134, was chosen for further experiments and was characterized as a functional flavonol regulator from M. truncatula. MtMYB134 expression levels are correlated with the expression of MtFLS2, encoding a key enzyme of flavonol biosynthesis, and with flavonol metabolite content. MtMYB134 was shown to activate the promoters of the A. thaliana flavonol biosynthesis genes AtCHS and AtFLS1 in Arabidopsis protoplasts in a transactivation assay and to interact with the Medicago promoters of MtCHS2 and MtFLS2 in yeast 1-hybrid assays. To ascertain the functional aspect of the identified transcription factor, we developed a sextuple mutant, which is defective in anthocyanin and flavonol biosynthesis. Ectopic expression of MtMYB134 in a multiple myb A. thaliana mutant restored flavonol biosynthesis. Furthermore, overexpression of MtMYB134 in hairy roots of M. truncatula enhanced the biosynthesis of various flavonol derivatives. Taken together, our results provide insight into the understanding of flavonol biosynthesis regulation in M. truncatula and provides MtMYB134 as tool for genetic manipulation to improve flavonol synthesis.
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    CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit
    (Elsevier B.V., 2020) Kaur, Navneet; Alok, Anshu; Shivani; Kumar, Pankaj; Kaur, Navjot; Awasthi, Praveen; Chaturvedi, Siddhant; Pandey, Pankaj; Pandey, Ashutosh; Pandey, Ajay K.; Tiwari, Siddharth
    Banana is one of the most economically important fruit crops worldwide. Genetic improvement in banana is a challenging task due to its parthenocarpic nature and triploid genome. Genetic modification of crops via the CRISPR/Cas9 module has emerged as a promising tool to develop important traits. In the present work, a CRISPR/Cas9-based approach was used to develop the β-carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome). The fifth exon of the lycopene epsilon-cyclase (LCYε) gene was targeted. The targeting specificity of the designed guide-RNA was also tested by its ability to create indels in the LCYε gene at the A genome of cv. Rasthali (AAB genome). Sequence analysis revealed multiple types of indels in the genomic region of Grand Naine LCYε (GN-LCYε). Metabolic profiling of the fruit pulp of selected edited lines showed enhanced accumulation of β-carotene content up to 6-fold (~24 μg/g) compared with the unedited plants. These lines also showed either an absence or a drastic reduction in the levels of lutein and α-carotene, suggesting metabolic reprogramming, without any significant effect on the agro-morphological parameters. In addition, differential expression of carotenoid pathway genes was observed in the edited lines in comparison to unedited plants. Overall, this is the first report in banana to improve nutritional trait by using a precise genome editing approach.