Journal articles: 'In vitro biosynthesis'

1

Cheng, Fang, Timo M. Takala, and Per E. J. Saris. "Nisin Biosynthesis in vitro." Journal of Molecular Microbiology and Biotechnology 13, no. 4 (2007): 248–54. http://dx.doi.org/10.1159/000104754.

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Araki, Yasuko, Takayoshi Awakawa, Motomichi Matsuzaki, Rihe Cho, Yudai Matsuda, Shotaro Hoshino, Yasutomo Shinohara, et al. "Complete biosynthetic pathways of ascofuranone and ascochlorin inAcremonium egyptiacum." Proceedings of the National Academy of Sciences 116, no. 17 (April 5, 2019): 8269–74. http://dx.doi.org/10.1073/pnas.1819254116.

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Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, includingAcremonium egyptiacum(synonym:Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses inA. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF inA. egyptiacumby genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

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Preie, Gianfranco Del, Enrico Maggi, Sergio Romagnani, and Mario Ricci. "Human IgE Biosynthesis In Vitro." Clinical Reviews in Allergy 7, no. 2 (June 1989): 193–216. http://dx.doi.org/10.1007/bf02914466.

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Wiltshire, Michael D., and Simon J. Foster. "Identification and Analysis of Staphylococcus aureus Components Expressed by a Model System of Growth in Serum." Infection and Immunity 69, no. 8 (August 1, 2001): 5198–202. http://dx.doi.org/10.1128/iai.69.8.5198-5202.2001.

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ABSTRACT A model system mimicking Staphylococcus aureusbacteremia was developed by growth in serum under microaerobic conditions. Eight genes induced by growth in serum were identified, including an antimicrobial peptide biosynthesis locus, amino acid biosynthetic loci, and genes encoding putative surface proteins. Nine independent insertions were found in the major lysine biosynthesis operon, which encodes eight genes, is repressed by lysine in vitro, and is expressed in vivo.

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Li, Li, Jun Wu, Zixin Deng, T. Mark Zabriskie, and Xinyi He. "Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation." Applied and Environmental Microbiology 79, no. 7 (February 1, 2013): 2349–57. http://dx.doi.org/10.1128/aem.03254-12.

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ABSTRACTBlasticidin S is a peptidyl nucleoside antibiotic produced byStreptomyces griseochromogenesthat exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesisin vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome ofStreptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, forS. lividansblasticidin S deaminase) was identified inS. lividansand shown to govern thisin vivoconversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin Sin vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene inS. lividansLL2 led to successful production of active blasticidin S in the resultant mutant,S. lividansWJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster,blsE,blsF, andblsL, encoding a predicted radicalS-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.

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Mohammed, Yousef, Ding Ye, Mudan He, Houpeng Wang, Zuoyan Zhu, and Yonghua Sun. "Production of Astaxanthin by Animal Cells via Introduction of an Entire Astaxanthin Biosynthetic Pathway." Bioengineering 10, no. 9 (September 11, 2023): 1073. http://dx.doi.org/10.3390/bioengineering10091073.

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Astaxanthin is a fascinating molecule with powerful antioxidant activity, synthesized exclusively by specific microorganisms and higher plants. To expand astaxanthin production, numerous studies have employed metabolic engineering to introduce and optimize astaxanthin biosynthetic pathways in microorganisms and plant hosts. Here, we report the metabolic engineering of animal cells in vitro to biosynthesize astaxanthin. This was accomplished through a two-step study to introduce the entire astaxanthin pathway into human embryonic kidney cells (HEK293T). First, we introduced the astaxanthin biosynthesis sub-pathway (Ast subp) using several genes encoding β-carotene ketolase and β-carotene hydroxylase enzymes to synthesize astaxanthin directly from β-carotene. Next, we introduced a β-carotene biosynthesis sub-pathway (β-Car subp) with selected genes involved in Ast subp to synthesize astaxanthin from geranylgeranyl diphosphate (GGPP). As a result, we unprecedentedly enabled HEK293T cells to biosynthesize free astaxanthin from GGPP with a concentration of 41.86 µg/g dry weight (DW), which represented 66.19% of the total ketocarotenoids (63.24 µg/g DW). Through optimization steps using critical factors in the astaxanthin biosynthetic process, a remarkable 4.14-fold increase in total ketocarotenoids (262.10 µg/g DW) was achieved, with astaxanthin constituting over 88.82%. This pioneering study holds significant implications for transgenic animals, potentially revolutionizing the global demand for astaxanthin, particularly within the aquaculture sector.

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Pan, Guohui, Zhengren Xu, Zhikai Guo, Hindra, Ming Ma, Dong Yang, Hao Zhou, et al. "Discovery of the leinamycin family of natural products by mining actinobacterial genomes." Proceedings of the National Academy of Sciences 114, no. 52 (December 11, 2017): E11131—E11140. http://dx.doi.org/10.1073/pnas.1716245115.

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Nature’s ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF–SH) didomain, specific for sulfur incorporation from the leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF–SH didomains. Further analysis of the representative genomes from each of the clades identified 28 lnm-type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature’s rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature’s biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.

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O'Hanlon, Karen A., Lorna Gallagher, Markus Schrettl, Christoph Jöchl, Kevin Kavanagh, Thomas O. Larsen, and Sean Doyle. "Nonribosomal Peptide Synthetase GenespesLandpes1Are Essential for Fumigaclavine C Production in Aspergillus fumigatus." Applied and Environmental Microbiology 78, no. 9 (February 17, 2012): 3166–76. http://dx.doi.org/10.1128/aem.07249-11.

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ABSTRACTThe identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen,Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway inA. fumigatus. Deletion of eitherpesL(ΔpesL) orpes1(Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion ofpesLresulted in severely reduced virulence in an invertebrate infection model (P< 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster forA. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in bothA. fumigatuswild-type and ΔpesLstrains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesisin vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.

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Fecik, Robert A. "Natural product biosynthesis moves in vitro." Nature Chemical Biology 3, no. 9 (September 2007): 531–32. http://dx.doi.org/10.1038/nchembio0907-531.

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Johnson, M. E., and K. V. Rajagopalan. "In vitro system for molybdopterin biosynthesis." Journal of Bacteriology 169, no. 1 (1987): 110–16. http://dx.doi.org/10.1128/jb.169.1.110-116.1987.

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FITZPATRICK, STEPHEN C., and T. JOSEPH McKENNA. "Dopamine inhibits aldosterone biosynthesis in vitro." Biochemical Society Transactions 16, no. 3 (June 1, 1988): 387. http://dx.doi.org/10.1042/bst0160387.

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Moore, Thomas S., Zhirong Du, and Zhi Chen. "Membrane Lipid Biosynthesis in Chlamydomonas reinhardtii. In Vitro Biosynthesis of Diacylglyceryltrimethylhomoserine." Plant Physiology 125, no. 1 (January 1, 2001): 423–29. http://dx.doi.org/10.1104/pp.125.1.423.

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Kawai, Seiji, Akito Yamada, Yohei Katsuyama, and Yasuo Ohnishi. "Identification of the p-coumaric acid biosynthetic gene cluster in Kutzneria albida: insights into the diazotization-dependent deamination pathway." Beilstein Journal of Organic Chemistry 20 (January 2, 2024): 1–11. http://dx.doi.org/10.3762/bjoc.20.1.

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Recently, we identified the biosynthetic gene cluster of avenalumic acid (ava cluster) and revealed its entire biosynthetic pathway, resulting in the discovery of a diazotization-dependent deamination pathway. Genome database analysis revealed the presence of more than 100 ava cluster-related biosynthetic gene clusters (BGCs) in actinomycetes; however, their functions remained unclear. In this study, we focused on an ava cluster-related BGC in Kutzneria albida (cma cluster), and revealed that it is responsible for p-coumaric acid biosynthesis by heterologous expression of the cma cluster and in vitro enzyme assays using recombinant Cma proteins. The ATP-dependent diazotase CmaA6 catalyzed the diazotization of both 3-aminocoumaric acid and 3-aminoavenalumic acid using nitrous acid in vitro. In addition, the high efficiency of the CmaA6 reaction enabled us to perform a kinetic analysis of AvaA7, which confirmed that AvaA7 catalyzes the denitrification of 3-diazoavenalumic acid in avenalumic acid biosynthesis. This study deepened our understanding of the highly reducing type II polyketide synthase system as well as the diazotization-dependent deamination pathway for the production of avenalumic acid or p-coumaric acid.

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Huang, Ancheng C., Ting Jiang, Yong-Xin Liu, Yue-Chen Bai, James Reed, Baoyuan Qu, Alain Goossens, Hans-Wilhelm Nützmann, Yang Bai, and Anne Osbourn. "A specialized metabolic network selectively modulates Arabidopsis root microbiota." Science 364, no. 6440 (May 9, 2019): eaau6389. http://dx.doi.org/10.1126/science.aau6389.

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Plant specialized metabolites have ecological functions, yet the presence of numerous uncharacterized biosynthetic genes in plant genomes suggests that many molecules remain unknown. We discovered a triterpene biosynthetic network in the roots of the small mustard plant Arabidopsis thaliana. Collectively, we have elucidated and reconstituted three divergent pathways for the biosynthesis of root triterpenes, namely thalianin (seven steps), thalianyl medium-chain fatty acid esters (three steps), and arabidin (five steps). A. thaliana mutants disrupted in the biosynthesis of these compounds have altered root microbiota. In vitro bioassays with purified compounds reveal selective growth modulation activities of pathway metabolites toward root microbiota members and their biochemical transformation and utilization by bacteria, supporting a role for this biosynthetic network in shaping an Arabidopsis-specific root microbial community.

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Li, Zhiyong, Yi Chen, Dauenpen Meesapyodsuk, and Xiao Qiu. "The Biosynthetic Pathway of Major Avenanthramides in Oat." Metabolites 9, no. 8 (August 7, 2019): 163. http://dx.doi.org/10.3390/metabo9080163.

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Avenanthramides are a group of N-cinnamoylanthranilic acids, with health-promoting properties mainly found in oat (Avena sativa L.). However, the biosynthetic mechanism for the main three types of avenanthramides (Avn-A, Avn-B and Avn-C) is not completely understood. In the present study, we report molecular identification and functional characterization of three different types of genes from oat encoding 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) and a caffeoyl-CoA O-methyltransferase (CCoAOMT) enzymes, all involved in the biosynthesis of these avenanthramides. In vitro enzymatic assays using the proteins expressed in Escherichia coli showed that oat 4CL could convert p-coumaric acid, caffeic acid and ferulic acid to their CoA thioesters. Oat HHTs were only responsible for the biosynthesis of Avn-A and Avn-C using hydroxyanthranilic acid as an acyl acceptor and p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor, respectively. Avn-B was synthesized by a CCoAOMT enzyme through the methylation of Avn-C. Collectively, these results have elucidated the molecular mechanisms for the biosynthesis of three major avenanthramides in vitro and paved the way for metabolic engineering of the biosynthetic pathway in heterologous systems to produce nutraceutically important compounds and make possible genetic improvement of this nutritional trait in oat through marker-assisted breeding.

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Bourlon, PM, B. Billaudel, and A. Faure-Dussert. "Influence of vitamin D3 deficiency and 1,25 dihydroxyvitamin D3 on de novo insulin biosynthesis in the islets of the rat endocrine pancreas." Journal of Endocrinology 160, no. 1 (January 1, 1999): 87–95. http://dx.doi.org/10.1677/joe.0.1600087.

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Because 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) is known to activate the biosynthesis of numerous proteins in various tissues, experiments were undertaken to compare the influence of 1,25(OH)2D3 in vitro on both the secretion and biosynthesis of insulin in islets of Langerhans from both 4-week vitamin D3-deficient rats and normal rats. Islets were either incubated or perifused after a 6-h induction period in the presence of various concentrations of 1, 25(OH)2D3 from 10(-12) M, which was inactive in controls, to 10(-6) M. Experiments were performed in the presence of a non-labelled amino acid mixture, to favour protein synthesis. Tritiated tyrosine was added as tracer during glucose stimulation. The newly synthesised proteins, labelled with [3H]tyrosine, were extracted by an acid-alcohol method and separated by gel chromatography adapted for low-molecular-weight proteins. Even in the presence of the amino acid mixture, the insulin response of the islets to 16.7 mM glucose was decreased by vitamin D3 deficiency and improved by 1,25(OH)2D3. This beneficial effect did not occur in basal conditions, but only during glucose stimulation, and was observed in both phases of insulin release. Moreover, these effects disappeared in the presence of 5x10(-4 )M cycloheximide, a protein biosynthesis inhibitor. Islets from vitamin D3-deficient rats exhibited a general decrease in the amount of de novo biosynthesised proteins and of [3H]tyrosine-labelled insulin and proinsulin fractions. A 6-h period of 1,25(OH)2D3 induction significantly improved the amount of de novo biosynthesised proteins, and particularly of newly synthesised insulin in response to a 2-h glucose stimulation. Calculation of the rate of conversion of newly synthesised proinsulin-like material to insulin as the [3H]insulin/[3H]proinsulin-like material ratio provided evidence for a dose-dependent increase, induced by 1, 25(OH)2D3, that could exceed that of normal islets. These data support the hypothesis that 1,25(OH)2D3 in vitro not only facilitated the biosynthetic capacity of the beta cell - which was highly induced during a 16.7-mM glucose stimulation, via a global activation of islets protein biosynthesis - but also produced an acceleration of the conversion of proinsulin to insulin.

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Trepod, Catherine M., and John E. Mott. "Elucidation of Essential and Nonessential Genes in the Haemophilus influenzae Rd Cell Wall Biosynthetic Pathway by Targeted Gene Disruption." Antimicrobial Agents and Chemotherapy 49, no. 2 (February 2005): 824–26. http://dx.doi.org/10.1128/aac.49.2.824-826.2005.

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ABSTRACT Targeted gene disruption by in vitro transposon mutagenesis has been used to identify the genes required for biosynthesis of the Haemophilus influenzae Rd cell wall under standard cultivation conditions. Of the 28 genes known to be associated with the cell wall biosynthetic pathway, 14 were determined to be essential.

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Gorny, James R., and Adel A. Kader. "Elevated CO2 and/or Low O2 Atmospheres Influence ACC Synthase and ACC Oxidase during Long-term Storage of `Golden Delicious' Apple Fruit." HortScience 30, no. 4 (July 1995): 782C—782. http://dx.doi.org/10.21273/hortsci.30.4.782c.

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The objective of this study was to compare and contrast the mode of action by which elevated carbon dioxide and/or reduced oxygen atmospheres inhibit ethylene biosynthesis. `Golden Delicious' apple fruit were placed at 0C in one of the following four atmospheres: 1) air; 2) air + 5% CO2; 3) 2% O2 + 98% N2; or 4) 2% O2 + 5% CO2 + 93% N2 and then sampled monthly for 4 months. Ethylene biosynthesis rates and in vitro ACC synthase activities were closely correlated in all treatments. In vitro ACC synthase activity and ethylene biosynthesis rates were lowest in fruit treated with 5% CO2 + 2% O2, while air-treated fruit had the highest ethylene biosynthesis rate and in vitro ACC synthase activity. Fruit treated with air + 5% CO2, or 2% O2 + 98% N2, had intermediate ethylene and in vitro ACC synthase activities. In vitro ACC oxidase was significantly different among treatments, but not as closely correlated with the ethylene biosynthesis rate as in vitro ACC synthase activity. Western blot analysis of the ACC oxidase protein was performed to determine if activity differences among treatments were correlated with the amount of enzyme present in vivo. ACC synthase and ACC oxidase mRNA transcript of abundance was determined via Northern blot analysis. Results will be discussed regarding how ethylene biosynthesis is inhibited at the molecular level by elevated CO2 and/or reduced O2.

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Mus, Ahmad Asnawi, Lucky Poh Wah Goh, Hartinie Marbawi, and Jualang Azlan Gansau. "The Biosynthesis and Medicinal Properties of Taraxerol." Biomedicines 10, no. 4 (March 30, 2022): 807. http://dx.doi.org/10.3390/biomedicines10040807.

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Taraxerol is a pentacyclic triterpenoid that is actively produced by some higher plants as part of a defense mechanism. The biosynthesis of taraxerol in plants occurs through the mevalonate pathway in the cytosol, in which dimethylallyl diphosphate (DMAPP) and isopentyl pyrophosphate (IPP) are first produced, followed by squalene. Squalene is the primary precursor for the synthesis of triterpenoids, including taraxerol, β-amyrin, and lupeol, which are catalyzed by taraxerol synthase. Taraxerol has been extensively investigated for its medicinal and pharmacological properties, and various biotechnological approaches have been established to produce this compound using in vitro techniques. This review provides an in-depth summary of the hypothesized taraxerol biosynthetic pathway, the medicinal properties of taraxerol, and recent developments on tissue culture for the in vitro production of taraxerol.

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Fang, Jie, Yiping Zhang, Lijuan Huang, Xinying Jia, Qi Zhang, Xu Zhang, Gongli Tang, and Wen Liu. "Cloning and Characterization of the Tetrocarcin A Gene Cluster from Micromonospora chalcea NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics." Journal of Bacteriology 190, no. 17 (June 27, 2008): 6014–25. http://dx.doi.org/10.1128/jb.00533-08.

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ABSTRACT Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of d-tetronitrose, l-amicetose, and l-digitoxose may begin with d-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to ΔchlD3 and ΔchlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.

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Seebeck, Florian P. "In Vitro Reconstitution of Mycobacterial Ergothioneine Biosynthesis." Journal of the American Chemical Society 132, no. 19 (May 19, 2010): 6632–33. http://dx.doi.org/10.1021/ja101721e.

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Flurkey, William H. "In Vitro Biosynthesis of Vicia faba Polyphenoloxidase." Plant Physiology 79, no. 2 (October 1, 1985): 564–67. http://dx.doi.org/10.1104/pp.79.2.564.

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Fontaine, Thierry, Terry K. Smith, Arthur Crossman, John S. Brimacombe, Jean-Paul Latgé, and Michael A. J. Ferguson. "In Vitro Biosynthesis of Glycosylphosphatidylinositol inAspergillus fumigatus†." Biochemistry 43, no. 48 (December 2004): 15267–75. http://dx.doi.org/10.1021/bi0486029.

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Arya, Ranjana, Alka Mehra, Sudha Bhattacharya, Ram A. Vishwakarma, and Alok Bhattacharya. "Biosynthesis of Entamoeba histolytica proteophosphoglycan in vitro." Molecular and Biochemical Parasitology 126, no. 1 (January 2003): 1–8. http://dx.doi.org/10.1016/s0166-6851(02)00252-9.

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Lawen, A., R. Traber, R. Reuille, and M. Ponelle. "In vitro biosynthesis of ring-extended cyclosporins." Biochemical Journal 300, no. 2 (June 1, 1994): 395–99. http://dx.doi.org/10.1042/bj3000395.

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Cyclosporin synthetase, a multifunctional polypeptide, catalyses the biosynthesis of the set of natural cyclosporins. We report that this enzyme is also capable of introducing a beta-alanine into position 7 or 8 of the ring instead of the alpha-alanines present at these positions in cyclosporin A. This leads to 34-membered rings in contrast to the 33-membered ring of the cyclo-undecapeptide cyclosporin A. Both [beta Ala7]CyA and [beta Ala8]CyA show immunosuppressive activity. The cyclosporin synthetase-related enzyme peptolide SDZ 214-103 synthetase, on the other hand, does not incorporate either beta-alanine into position 7 or beta-hydroxy acids into position 8, confirming the previously described higher substrate specificity of this enzyme compared with cyclosporin synthetase [Lawen and Traber (1993) J. Biol. Chem. 268, 20452-20465].

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Carballeira, A., L. M. Fishman, J. Brown, and D. V. A. Trujillo. "In vitro biosynthesis of androgens by pheochromocytomas." Journal of Steroid Biochemistry 25 (January 1986): 61. http://dx.doi.org/10.1016/0022-4731(86)90632-1.

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Edwards, Mary, Paul V. Bulpin, Iain C. M. Dea, and J. S. Grant Reid. "Biosynthesis of legume-seed galactomannans in vitro." Planta 178, no. 1 (1989): 41–51. http://dx.doi.org/10.1007/bf00392525.

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Liao, Cangsong, and Florian P. Seebeck. "In Vitro Reconstitution of Bacterial DMSP Biosynthesis." Angewandte Chemie 131, no. 11 (February 14, 2019): 3591–94. http://dx.doi.org/10.1002/ange.201814662.

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Berkov, Strahil, Ivan Ivanov, Vasil Georgiev, Carles Codina, and Atanas Pavlov. "Galanthamine biosynthesis in plant in vitro systems." Engineering in Life Sciences 14, no. 6 (November 2014): 643–50. http://dx.doi.org/10.1002/elsc.201300159.

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Liao, Cangsong, and Florian P. Seebeck. "In Vitro Reconstitution of Bacterial DMSP Biosynthesis." Angewandte Chemie International Edition 58, no. 11 (February 14, 2019): 3553–56. http://dx.doi.org/10.1002/anie.201814662.

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García-Pérez, Pascual, Leilei Zhang, Begoña Miras-Moreno, Eva Lozano-Milo, Mariana Landin, Luigi Lucini, and Pedro P. Gallego. "The Combination of Untargeted Metabolomics and Machine Learning Predicts the Biosynthesis of Phenolic Compounds in Bryophyllum Medicinal Plants (Genus Kalanchoe)." Plants 10, no. 11 (November 10, 2021): 2430. http://dx.doi.org/10.3390/plants10112430.

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Phenolic compounds constitute an important family of natural bioactive compounds responsible for the medicinal properties attributed to Bryophyllum plants (genus Kalanchoe, Crassulaceae), but their production by these medicinal plants has not been characterized to date. In this work, a combinatorial approach including plant tissue culture, untargeted metabolomics, and machine learning is proposed to unravel the critical factors behind the biosynthesis of phenolic compounds in these species. The untargeted metabolomics revealed 485 annotated compounds that were produced by three Bryophyllum species cultured in vitro in a genotype and organ-dependent manner. Neurofuzzy logic (NFL) predictive models assessed the significant influence of genotypes and organs and identified the key nutrients from culture media formulations involved in phenolic compound biosynthesis. Sulfate played a critical role in tyrosol and lignan biosynthesis, copper in phenolic acid biosynthesis, calcium in stilbene biosynthesis, and magnesium in flavanol biosynthesis. Flavonol and anthocyanin biosynthesis was not significantly affected by mineral components. As a result, a predictive biosynthetic model for all the Bryophyllum genotypes was proposed. The combination of untargeted metabolomics with machine learning provided a robust approach to achieve the phytochemical characterization of the previously unexplored species belonging to the Bryophyllum subgenus, facilitating their biotechnological exploitation as a promising source of bioactive compounds.

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Ahmad, Jianyu, Xu, Noman, Jameel, Na, Yuanyuan, et al. "Overexpression of a Novel Cytochrome P450 Promotes Flavonoid Biosynthesis and Osmotic Stress Tolerance in Transgenic Arabidopsis." Genes 10, no. 10 (September 26, 2019): 756. http://dx.doi.org/10.3390/genes10100756.

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Flavonoids are mainly associated with growth, development, and responses to diverse abiotic stresses in plants. A growing amount of data have demonstrated the biosynthesis of flavonoids through multienzyme complexes of which the membrane‐bounded cytochrome P450 supergene family shares a crucial part. However, the explicit regulation mechanism of Cytochrome P450s related to flavonoid biosynthesis largely remains elusive. In the present study, we reported the identification of a stress-tolerant flavonoid biosynthetic CtCYP82G24 gene from Carthamus tinctorius. The transient transformation of CtCYP82G24 determined the subcellular localization to the cytosol. Heterologously expressed CtCYP82G24 was effective to catalyze the substrate-specific conversion, promoting the de novo biosynthesis of flavonoids in vitro. Furthermore, a qRT-PCR assay and the accumulation of metabolites demonstrated that the expression of CtCYP82G24 was effectively induced by Polyethylene glycol stress in transgenic Arabidopsis. In addition, the overexpression of CtCYP82G24 could also trigger expression levels of several other flavonoid biosynthetic genes in transgenic plants. Taken together, our findings suggest that CtCYP82G24 overexpression plays a decisive regulatory role in PEG-induced osmotic stress tolerance and alleviates flavonoid accumulation in transgenic Arabidopsis.

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El Gamal, Abrahim, Vinayak Agarwal, Stefan Diethelm, Imran Rahman, Michelle A. Schorn, Jennifer M. Sneed, Gordon V. Louie, et al. "Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair." Proceedings of the National Academy of Sciences 113, no. 14 (March 21, 2016): 3797–802. http://dx.doi.org/10.1073/pnas.1519695113.

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Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.

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Beccari, Giovanni, Łukasz Stępień, Andrea Onofri, Veronica M. T. Lattanzio, Biancamaria Ciasca, Sally I. Abd-El Fatah, Francesco Valente, Monika Urbaniak, and Lorenzo Covarelli. "In Vitro Fumonisin Biosynthesis and Genetic Structure of Fusarium verticillioides Strains from Five Mediterranean Countries." Microorganisms 8, no. 2 (February 11, 2020): 241. http://dx.doi.org/10.3390/microorganisms8020241.

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Investigating the in vitro fumonisin biosynthesis and the genetic structure of Fusarium verticillioides populations can provide important insights into the relationships between strains originating from various world regions. In this study, 90 F. verticillioides strains isolated from maize in five Mediterranean countries (Italy, Spain, Tunisia, Egypt and Iran) were analyzed to investigate their ability to in vitro biosynthesize fumonisin B1, fumonisin B2 and fumonisin B3 and to characterize their genetic profile. In general, 80% of the analyzed strains were able to biosynthesize fumonisins (range 0.03–69.84 μg/g). Populations from Italy, Spain, Tunisia and Iran showed a similar percentage of fumonisin producing strains (>90%); conversely, the Egyptian population showed a lower level of producing strains (46%). Significant differences in fumonisin biosynthesis were detected among strains isolated in the same country and among strains isolated from different countries. A portion of the divergent FUM1 gene and of intergenic regions FUM6-FUM7 and FUM7-FUM8 were sequenced to evaluate strain diversity among populations. A high level of genetic uniformity inside the populations analyzed was detected. Apparently, neither geographical origin nor fumonisin production ability were correlated to the genetic diversity of the strain set. However, four strains from Egypt differed from the remaining strains.

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Ramos, Itzel, E. I. Vivas, and D. M. Downs. "Mutations in the Tryptophan Operon Allow PurF-Independent Thiamine Synthesis by Altering Flux In Vivo." Journal of Bacteriology 190, no. 3 (June 8, 2007): 815–22. http://dx.doi.org/10.1128/jb.00582-07.

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ABSTRACT Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.

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Zhao, Wanli, Long Huang, Shu Xu, Junzhi Wu, Fan Wang, Pirui Li, Linwei Li, Mei Tian, Xu Feng, and Yu Chen. "Identification of One O-Methyltransferase Gene Involved in Methylated Flavonoid Biosynthesis Related to the UV-B Irradiation Response in Euphorbia lathyris." International Journal of Molecular Sciences 25, no. 2 (January 8, 2024): 782. http://dx.doi.org/10.3390/ijms25020782.

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Flavonoids are ubiquitous polyphenolic compounds that play a vital role in plants’ defense response and medicinal efficacy. UV-B radiation is a vital environmental regulator governing flavonoid biosynthesis in plants. Many plants rapidly biosynthesize flavonoids as a response to UV-B stress conditions. Here, we investigated the effects of flavonoid biosynthesis via UV-B irradiation in Euphorbia lathyris. We found that exposure of the E. lathyris callus to UV-B radiation sharply increased the level of one O-methyltransferase (ElOMT1) transcript and led to the biosynthesis of several methylated flavonoids. The methyltransferase ElOMT1 was expressed heterologously in E. coli, and we tested the catalytic activity of recombinant ElOMT1 with possible substrates, including caffeic acid, baicalin, and luteolin, in vitro. ElOMT1 could efficiently methylate when the hydroxyl groups were contained in the core nucleus of the flavonoid. This molecular characterization identifies a methyltransferase responsible for the chemical modification of the core flavonoid structure through methylation and helps reveal the mechanism of methylated flavonoid biosynthesis in Euphorbiaceae. This study identifies the O-methyltransferase that responds to UV-B irradiation and helps shed light on the mechanism of flavonoid biosynthesis in Euphorbia lathyris.

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Alcalde, Miguel Angel, Edgar Perez-Matas, Ainoa Escrich, Rosa M. Cusido, Javier Palazon, and Mercedes Bonfill. "Biotic Elicitors in Adventitious and Hairy Root Cultures: A Review from 2010 to 2022." Molecules 27, no. 16 (August 17, 2022): 5253. http://dx.doi.org/10.3390/molecules27165253.

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One of the aims of plant in vitro culture is to produce secondary plant metabolites using plant cells and organ cultures, such as cell suspensions, adventitious, and hairy roots (among others). In cases where the biosynthesis of a compound in the plant is restricted to a specific organ, unorganized systems, such as plant cell cultures, are sometimes unsuitable for biosynthesis. Then, its production is based on the establishment of organ cultures such as roots or aerial shoots. To increase the production in these biotechnological systems, elicitors have been used for years as a useful tool since they activate secondary biosynthetic pathways that control the flow of carbon to obtain different plant compounds. One important biotechnological system for the production of plant secondary metabolites or phytochemicals is root culture. Plant roots have a very active metabolism and can biosynthesize a large number of secondary compounds in an exclusive way. Some of these compounds, such as tropane alkaloids, ajmalicine, ginsenosides, etc., can also be biosynthesized in undifferentiated systems, such as cell cultures. In some cases, cell differentiation and organ formation is necessary to produce the bioactive compounds. This review analyses the biotic elicitors most frequently used in adventitious and hairy root cultures from 2010 to 2022, focusing on the plant species, the target secondary metabolite, the elicitor and its concentration, and the yield/productivity of the target compounds obtained. With this overview, it may be easier to work with elicitors in in vitro root cultures and help understand why some are more effective than others.

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Maniere, G., E. Vanhems, F. Gautron, and JP Delbecque. "Calcium inhibits ovarian steroidogenesis in the blowfly Phormia regina." Journal of Endocrinology 173, no. 3 (June 1, 2002): 533–44. http://dx.doi.org/10.1677/joe.0.1730533.

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Calcium is frequently involved in the stimulation of steroidogenesis in gonads and endocrine glands, generally in association with cAMP. However, our present observations show that it has the opposite effect in the ovary of the blowfly Phormia regina. Our in vitro experiments first showed that extracellular calcium does not play a role during the stimulation of steroidogenesis in fly ovaries; indeed steroidogenesis was activated in vitro as efficiently in a medium with or without calcium, either by pharmacological compounds mimicking cAMP signaling or by active brain extracts. When calcium was experimentally introduced into biosynthetic cells by ionophores or liberated from internal stores by thapsigargin, it did not activate, but clearly inhibited both basal and acute steroidogenesis respectively in previtellogenic and in vitellogenic ovaries. Our experiments also demonstrated that calcium decreases cAMP concentrations in the ovaries of Phormia, by stimulating its degradation, without modifying its biosynthesis. Moreover, inhibitors of calcium-calmodulin phosphodiesterases (PDEs) increased steroid biosynthesis in vitro, whereas inhibitors of calcium-insensitive PDEs did not. These data thus demonstrate that, in blowfly ovaries, calcium ions inhibit cAMP-stimulated steroidogenesis by activating a calmodulin-sensitive (type I) PDE.

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Hasegawa, Morifumi, Ichiro Mitsuhara, Shigemi Seo, Takuya Imai, Jinichiro Koga, Kazunori Okada, Hisakazu Yamane, and Yuko Ohashi. "Phytoalexin Accumulation in the Interaction Between Rice and the Blast Fungus." Molecular Plant-Microbe Interactions® 23, no. 8 (August 2010): 1000–1011. http://dx.doi.org/10.1094/mpmi-23-8-1000.

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Blast fungus–induced accumulations of major rice diterpene phytoalexins (PA), momilactones A and B, and phytocassanes A through E were studied, focusing on their biosynthesis and detoxification. In resistant rice, all PA started to accumulate at 2 days postinoculation (dpi), at which hypersensitive reaction (HR)-specific small lesions became visible and increased 500- to 1,000-fold at 4 dpi, while the accumulation was delayed and several times lower in susceptible rice. Expression of PA biosynthetic genes was transiently induced at 2 dpi only in resistant plants, while it was highly induced in both plants at 4 dpi. Fungal growth was severely suppressed in resistant plants by 2 dpi but considerably increased at 3 to 4 dpi in susceptible plants. Momilactone A treatment suppressed fungal growth in planta and in vitro, and the fungus detoxified the PA in vitro. These results indicate that HR-associated rapid PA biosynthesis induces severe restriction of fungus, allowing higher PA accumulation in resistant rice, while in susceptible rice, failure of PA accumulation at the early infection stage allows fungal growth. Detoxification of PA would be a tactic of fungus to invade the host plant, and prompt induction of PA biosynthesis upon HR would be a trait of resistant rice to restrict blast fungus.

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Marchev, Andrey S., and Milen I. Georgiev. "Plant In Vitro Systems as a Sustainable Source of Active Ingredients for Cosmeceutical Application." Molecules 25, no. 9 (April 25, 2020): 2006. http://dx.doi.org/10.3390/molecules25092006.

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Cosmeceuticals are hybrids between cosmetics and pharmaceuticals which are being designed for a dual purpose: (1) To provide desired esthetical effects and (2) simultaneously treat dermatological conditions. The increased demand for natural remedies and the trends to use natural and safe ingredients resulted in intensive cultivation of medicinal plants. However, in many cases the whole process of plant cultivation, complex extraction procedure, and purification of the targeted molecules are not economically feasible. Therefore, the desired production of natural cosmetic products in sustainable and controllable fashion in the last years led to the intensive utilization of plant cell culture technology. The present review aims to highlight examples of biosynthesis of active ingredients derived through plant in vitro systems with potential cosmeceutical application. The exploitation of different type of extracts used in a possible cosmeceutical formulation, as well as, their activity tested in in vitro/in vivo models is thoroughly discussed. Furthermore, opportunities to manipulate the biosynthetic pathway, hence engineering the biosynthesis of some secondary metabolites, such as anthocyanins, have been highlighted.

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Deng, Changping, Min Shi, Rong Fu, Yi Zhang, Qiang Wang, Yang Zhou, Yao Wang, Xingyuan Ma, and Guoyin Kai. "ABA-responsive transcription factor bZIP1 is involved in modulating biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza." Journal of Experimental Botany 71, no. 19 (June 26, 2020): 5948–62. http://dx.doi.org/10.1093/jxb/eraa295.

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Abstract Phenolic acids and tanshinones are major bioactive ingredients in Salvia miltiorrhiza, which possess pharmacological activities with great market demand. However, transcriptional regulation of phenolic acid and tanshinone biosynthesis remains poorly understood. Here, a basic leucine zipper transcription factor (TF) named SmbZIP1 was screened from the abscisic acid (ABA)-induced transcriptome library. Overexpression of SmbZIP1 positively promoted phenolic acid biosynthesis by enhancing expression of biosynthetic genes such as cinnamate-4-hydroxylase (C4H1). Furthermore, biochemical experiments revealed that SmbZIP1 bound the G-Box-like1 element in the promoter of the C4H1 gene. Meanwhile, SmbZIP1 inhibited accumulation of tanshinones mainly by suppressing the expression of biosynthetic genes including geranylgeranyl diphosphate synthase (GGPPS) which was confirmed as a target gene by in vitro and in vivo experiments. In contrast, the phenolic acid content was reduced and tanshinone was enhanced in CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9]-mediated knockout lines. In addition, the previously reported positive regulator of tanshinone biosynthesis, SmERF1L1, was found to be inhibited in SmbZIP1 overexpression lines indicated by RNA sequencing, and was proven to be the target of SmbZIP1. In summary, this work uncovers a novel regulator and deepens our understanding of the transcriptional and regulatory mechanisms of phenolic acid and tanshinone biosynthesis, and also sheds new light on metabolic engineering in S. miltiorrhiza.

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Fatoni, Ahmad, Reza Agung Sriwijaya, Umy Habiba, and Nurlisa Hidayati. "CuO Nanoparticles : Biosynthesis, Characterization and In Vitro Study." Science and Technology Indonesia 6, no. 1 (January 13, 2021): 25. http://dx.doi.org/10.26554/sti.2021.6.1.25-29.

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The biosynthesis of CuO nanoparticles was studied. The aims of this research were biosynthesis CuO nanoparticles from Cu2+ion solution and leaves aquoeus extract of sweet star fruit and its in vitro study as antibacterial agent of Escherichia coli. CuO nanoparticles was characterized using FTIR spectroscopy and X-Ray diffractometer. CuO nanoparticles was synthesized by reaction between Cu2+ion solution and leaves aquoeus extract of sweet star fruit. Paper disk method was used as the application of the suspension of CuO nanoparticles. The result showed that Cu-O groups detected at wave number of 503, 619, 767 and 821 cm-1. Diffractogram of CuO nanoparticles had sharp and narrow diffraction peaks. The crystallite size of CuO nanoparticles was 4.25 nm. The average inhibition zone of CuO nanoparticles at concentration 10.000, 7.500, 5.000 and 2.500 ppm was 17.43 ± 1.81 ; 15.73 ± 0.40 ; 14.50 ± 2.96 and 9.67 ± 0.28 mm respectively.

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Ishida, Betty K. "ACTIVATED LYCOPENE BIOSYNTHESIS IN TOMATO FRUIT IN VITRO." Acta Horticulturae, no. 487 (March 1999): 445–52. http://dx.doi.org/10.17660/actahortic.1999.487.73.

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Yue, Beatrice Y. J. T., and Judith L. Elvart. "Biosynthesis of glycosaminoglycans by trabecular meshwork cellsin vitro." Current Eye Research 6, no. 8 (January 1987): 959–67. http://dx.doi.org/10.3109/02713688709034867.

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Sun, Yuhui, Frank Hahn, Yuliya Demydchuk, James Chettle, Manuela Tosin, Hiroyuki Osada, and Peter F. Leadlay. "In vitro reconstruction of tetronate RK-682 biosynthesis." Nature Chemical Biology 6, no. 2 (December 20, 2009): 99–101. http://dx.doi.org/10.1038/nchembio.285.

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Cipollo, J. F., A. Awad, C. E. Costello, P. W. Robbins, and C. B. Hirschberg. "Biosynthesis in vitro of Caenorhabditis elegans phosphorylcholine oligosaccharides." Proceedings of the National Academy of Sciences 101, no. 10 (March 1, 2004): 3404–8. http://dx.doi.org/10.1073/pnas.0400384101.

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Zinder, Norton D. "The in vitro biosynthesis of an authentic protein." Trends in Biochemical Sciences 22, no. 8 (August 1997): 318–20. http://dx.doi.org/10.1016/s0968-0004(97)01094-3.

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Nishimura, Yukio, and Keitaro Kato. "In vitro biosynthesis of the lysosomal cathepsin H." Biochemical and Biophysical Research Communications 146, no. 1 (July 1987): 159–64. http://dx.doi.org/10.1016/0006-291x(87)90705-4.

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Strous, Ger J., Peter Van Kerkhof, and Eric G. Berger. "In vitro biosynthesis of two human galactosyltransferase polypeptides." Biochemical and Biophysical Research Communications 151, no. 1 (February 1988): 314–19. http://dx.doi.org/10.1016/0006-291x(88)90595-5.

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Basu, Subhash, Manju Basu, Kamal K. Das, François Daussin, Robert J. Schaeper, Probal Banerjee, Farhat A. Khan, and Isao Suzuki. "Solubilized glycosyltransferases and biosynthesis in vitro of glycolipids." Biochimie 70, no. 11 (November 1988): 1551–63. http://dx.doi.org/10.1016/0300-9084(88)90291-x.

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Journal articles on the topic 'In vitro biosynthesis'

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