Employing a biochemical protecting group for a sustainable indigo dyeing strategy

Hsu, Tammy M; Welner, Ditte Hededam; Russ, Zachary N.; Cervantes, Bernardo; Prathuri, Ramya L; Adams, Paul D.; Dueber, John E.

doi:10.1038/nchembio.2552

Cited by 170 publications

(161 citation statements)

References 66 publications

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“…Indole alkaloids comprise another active component of I. indigotica [48][49][50] with important anti-influenza, anti-inflammatory, and leukocyte inhibition effects 11,23,[51][52][53] . Based on the KEGG maps and previously suggested pathways 25,54,55 , we identified 32 genes that encoded 11 enzymes involved in the biosynthesis of indole alkaloids ( Fig. 3c and Supplementary Table S16).…”

Section: Identification Of Genes Involved In the Biosynthetic Pathwaymentioning

confidence: 99%

A chromosome-scale genome assembly of Isatis indigotica, an important medicinal plant used in traditional Chinese medicine

Kang

Yang

et al. 2020

Hortic Res

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Isatis indigotica (2n = 14) is an important medicinal plant in China. Its dried leaves and roots (called Isatidis Folium and Isatidis Radix, respectively) are broadly used in traditional Chinese medicine for curing diseases caused by bacteria and viruses such as influenza and viral pneumonia. Various classes of compounds isolated from this species have been identified as effective ingredients. Previous studies based on transcriptomes revealed only a few candidate genes for the biosynthesis of these active compounds in this medicinal plant. Here, we report a high-quality chromosome-scale genome assembly of I. indigotica with a total size of 293.88 Mb and scaffold N50 = 36.16 Mb using single-molecule real-time long reads and high-throughput chromosome conformation capture techniques. We annotated 30,323 highconfidence protein-coding genes. Based on homolog searching and functional annotations, we identified many candidate genes involved in the biosynthesis of main active components such as indoles, terpenoids, and phenylpropanoids. In addition, we found that some key enzyme-coding gene families related to the biosynthesis of these components were expanded due to tandem duplications, which likely drove the production of these major active compounds and explained why I. indigotica has excellent antibacterial and antiviral activities. Our results highlighted the importance of genome sequencing in identifying candidate genes for metabolite synthesis in medicinal plants. Data availabilityThe PacBio long reads and Illumina short reads were uploaded to the NCBI SRA database under BioProject PRJNA549758. The final chromosome-scale genome assembly was submitted to the NCBI with accession number VHIU00000000. The genome fasta and gff3 files were uploaded to Figshare.

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Section: Identification Of Genes Involved In the Biosynthetic Pathwaymentioning

confidence: 99%

A chromosome-scale genome assembly of Isatis indigotica, an important medicinal plant used in traditional Chinese medicine

Kang

Yang

et al. 2020

Hortic Res

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“…The plant GTs are, therefore, promising catalysts for glycoside production (for reviews, see: Lim, ; Nidetzky, Gutmann, & Zhong, ). Glycosylated derivatives of small molecules (e.g., flavonoids, terpenoids, peptides) with important applications in the food, fragrance, cosmetic, and chemical industries are synthesized efficiently using GTs (De Bruyn, Maertens, Beauprez, Soetaert, & De Mey, ; Desmet et al, ; Hofer, ; Hsu et al, ; Kim, Yang, Kim, Cha, & Ahn, ; Nidetzky et al, ; Olsson et al, ; Schmölzer, Lemmerer, & Nidetzky, ; Schwab, Fischer, & Wüst, ; Schwab, Fischer, Giri, & Wüst, ; Xiao, Muzashvili, & Georgiev, ).…”

Section: Introductionmentioning

confidence: 99%

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

Lemmerer

Mairhofer²,

Lepak

et al. 2019

Biotech & Bioengineering

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Sugar nucleotide‐dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology‐based strategy of decoupling the enzyme expression from the Escherichia coli BL21(DE3) cell growth was effective in enhancing their individual (approximately fivefold) or combined (approximately twofold) production as correctly folded, biologically active proteins. The approach entails a synthetic host cell, which is able to shut down the production of host messenger RNA by inhibition of the E. coli RNA polymerase. Overexpression of the enzyme(s) of interest is induced by the orthogonal T7 RNA polymerase. Shutting down of the host RNA polymerase is achieved by l‐arabinose‐inducible expression of the T7 phage‐derived Gp2 protein from a genome‐integrated site. The glycosyltransferase genes are encoded on conventional pET‐based expression plasmids that allow T7 RNA polymerase‐driven inducible expression by isopropyl‐β‐ d‐galactoside. Laboratory batch and scaled‐up (20 L) fed‐batch bioreactor cultivations demonstrated improvements in an overall yield of active enzyme by up to 12‐fold as a result of production under growth‐decoupled conditions. In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 and 2.30 U/g cell dry weight, corresponding to ∼5 and ∼1% of total intracellular protein. Fed‐batch production gave sucrose synthase in a yield of 2,300 U/L of culture (830 mg protein/L). Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield (5.3‐fold) and quality (2.3‐fold) of the soluble sucrose synthase. Enzyme preparation from the decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homotetramer. In summary, therefore, we showed that the expression in growth‐arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases.

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“…Pa GT2 showed the highest sequence similarity of 58% with UGT72B1 (25) and 56% with Pt UGT1 from Polygonum tinctorium (26). The overall structure of Pa GT2 was also highly similar with the structures of UGT72B1 and Pt UGT1 (PDB: 5NLM) with rmsds of 1.16 Å for 371 Cα atoms and 1.03 Å for 373 Cα atoms, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A large number of plant UGT gene sequences have been deposited in database; however, crystal structures of only few of them are available. These crystal structures of UGTs were studied for the glycosylation of iso/flavonoids (3, 4, 24) or small molecules, such as trichlorophenol (25), and indoxyl sulfate (26). Consequently, crystal structures of plant UGT with stilbenoids are not available.…”

Section: Introductionmentioning

confidence: 99%

Phytolacca americana PaGT2 is an ambidextrous polyphenol glucosyltransferase

Maharjan

Fukuda

Shimomura

et al. 2019

Preprint

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The health benefits of polyphenols have attracted their use as potential therapeutic agents, food additives, and cosmetics. However, low water solubility of polyphenols limits their cell absorbability, obscuring further exploration. Glycosylation is known to enhance the solubility of polyphenols preserving their pharmacological properties. Here, we show that a uridine diphosphate (UDP) glucosyltransferase from Phytolacca americana (PaGT2) regioselectively catalyzes the transfer of glucose from UDP-glucose to stilbenoids such as piceatannol and flavonoids such as kaempferol. To understand the structure-function relationship of PaGT2, we determined the crystal structure of PaGT2 as well as PaGT2 complexed with donor analogue UDP-2-fluoro glucose and stilbenoid acceptor analogues. While only one conserved histidine residue is recognized as a catalytic residue in known UGTs, the crystal structures of PaGT2 suggested the presence of two catalytically active residues (His18 and His81) at two sides of the catalytic pocket. Although the single catalytic residue mutant His18Ala or His81Ala did not completely impair the glucosylation activity of the enzyme, the double mutant His18Ala/His81Ala failed to form glucoside products. These results showed that both catalytic residues in PaGT2 actively and independently catalyze glucosylation, hence we called PaGT2 as an ambidextrous UGT. The information from PaGT2 will be advantageous for the engineering of efficient biocatalysts for production of therapeutic polyphenols.

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Employing a biochemical protecting group for a sustainable indigo dyeing strategy

Cited by 170 publications

References 66 publications

A chromosome-scale genome assembly of Isatis indigotica, an important medicinal plant used in traditional Chinese medicine

A chromosome-scale genome assembly of Isatis indigotica, an important medicinal plant used in traditional Chinese medicine

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

Phytolacca americana PaGT2 is an ambidextrous polyphenol glucosyltransferase

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