scholarly journals A computational workflow for the expansion of heterologous biosynthetic pathways to natural product derivatives

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmin Hafner ◽  
James Payne ◽  
Homa MohammadiPeyhani ◽  
Vassily Hatzimanikatis ◽  
Christina Smolke
Keyword(s):  
Biosynthetic Pathway ◽  
De Novo ◽  
Pharmaceutical Compounds ◽  
De Novo Biosynthesis ◽  
Benzylisoquinoline Alkaloid ◽  
Medicinal Use ◽  
Starting Point ◽  
History Of ◽  
Computational Workflow ◽  
Pharmaceutical Molecules

AbstractPlant natural products (PNPs) and their derivatives are important but underexplored sources of pharmaceutical molecules. To access this untapped potential, the reconstitution of heterologous PNP biosynthesis pathways in engineered microbes provides a valuable starting point to explore and produce novel PNP derivatives. Here, we introduce a computational workflow to systematically screen the biochemical vicinity of a biosynthetic pathway for pharmaceutical compounds that could be produced by derivatizing pathway intermediates. We apply our workflow to the biosynthetic pathway of noscapine, a benzylisoquinoline alkaloid (BIA) with a long history of medicinal use. Our workflow identifies pathways and enzyme candidates for the production of (S)-tetrahydropalmatine, a known analgesic and anxiolytic, and three additional derivatives. We then construct pathways for these compounds in yeast, resulting in platforms for de novo biosynthesis of BIA derivatives and demonstrating the value of cheminformatic tools to predict reactions, pathways, and enzymes in synthetic biology and metabolic engineering.

scholarly journals Identification of 3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase

2021 ◽  
Vol 22 (13) ◽  
pp. 7236
Author(s):  
Endah Dwi Hartuti ◽  
Takaya Sakura ◽  
Mohammed S. O. Tagod ◽  
Eri Yoshida ◽  
Xinying Wang ◽  
...  
Keyword(s):  
Drug Target ◽  
De Novo ◽  
Dihydroorotate Dehydrogenase ◽  
Chemical Library ◽  
New Class ◽  
De Novo Biosynthesis ◽  
Starting Point ◽  
Novel Drugs ◽  
Low Toxicity ◽  
Fourth Step

Plasmodium falciparum’s resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development.

scholarly journals Phylogenetic debugging of a complete human biosynthetic pathway transplanted into yeast

2019 ◽  
Author(s):  
Neta Agmon ◽  
Jasmine Temple ◽  
Zuojian Tang ◽  
Tobias Schraink ◽  
Maayan Baron ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
De Novo ◽  
Enzyme Regulation ◽  
Metabolite Level ◽  
De Novo Biosynthesis ◽  
Human Ortholog ◽  
Pathway Regulation ◽  
Traditional Approaches ◽  
Suppressor Analysis ◽  
Insight Into

Abstract Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the ‘humanized’ yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level ‘setpoints’ in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.

scholarly journals The de novo Biosynthesis of Vitamin B6 Is Required for Disease Resistance Against Botrytis cinerea in Tomato

2014 ◽  
Vol 27 (7) ◽  
pp. 688-699 ◽  
Author(s):  
Yafen Zhang ◽  
Bo Liu ◽  
Xiaohui Li ◽  
Zhigang Ouyang ◽  
Lei Huang ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Botrytis Cinerea ◽  
Plant Disease Resistance ◽  
Defense Response ◽  
Vitamin B6 ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Salvage Pathway ◽  
Biosynthetic Genes ◽  
De Novo Biosynthesis

Vitamin B6 (VB6), an essential cofactor for numerous metabolic enzymes, has recently been shown to act as a potent antioxidant and play important roles in developmental processes and stress responses. However, little is known about the possible function of VB6 in plant disease resistance response against pathogen infection. In the present study, we explored the possible involvement of VB6 in defense response against Botrytis cinerea through functional analysis of tomato VB6 biosynthetic genes. Three de novo VB6 biosynthetic genes (SlPDX1.2, SlPDX1.3, and SlPDX2) and one salvage pathway gene (SlSOS4) were identified and the SlPDX1.2, SlPDX1.3, and SlPDX2 genes were shown to encode functional enzymes involved in de novo biosynthesis of VB6, as revealed by complementation of the VB6 prototrophy in yeast snz1 and sno1 mutants. Expression of SlPDX1.2, SlPDX1.3, and SlSOS4 genes was induced by infection with B. cinerea. Virus-induced gene silencing-mediated knockdown of SlPDX1.2 or SlPDX1.3 but not SlPDX2 and SlSOS4 led to increased severity of disease caused by B. cinerea, indicating that the VB6 de novo biosynthetic pathway but not the salvage pathway is involved in tomato defense response against B. cinerea. Furthermore, the SlPDX1.2- and SlPDX1.3-silenced tomato plants exhibited reduced levels of VB6 contents and reactive oxygen species scavenging capability, increased levels of superoxide anion and H2O2 generation, and increased activity of superoxide dismutase after infection by B. cinerea. Our results suggest that VB6 and its de novo biosynthetic pathway play important roles in regulation of defense response against B. cinerea through modulating cellular antioxidant capacity.

scholarly journals Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

2018 ◽  
Author(s):  
Huan Fang ◽  
Dong Li ◽  
Jie Kang ◽  
Pingtao Jiang ◽  
Jibin Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
De Novo ◽  
Vitamin B ◽  
E Coli ◽  
De Novo Biosynthesis ◽  
Optimization Of Fermentation ◽  
Aerobic And Anaerobic

ABSTRACTThe only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea, and the only unknown step in its biosynthesis is the production of the intermediate adenosylcobinamide phosphate. Here, using genetic and metabolic engineering, we generated an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. Excitingly, the BluE and CobC enzymes from Rhodobacter capsulatus transform L-threonine into (R)-1-Amino-2-propanol O-2-Phosphate, which is then condensed with adenosylcobyric acid to yield adenosylcobinamide phosphate by either CobD from the aeroic R. capsulatus or CbiB from the anerobic Salmonella typhimurium. These findings suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increased the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg/g DCW via metabolic engineering and optimization of fermentation conditions. Beyond our scientific insights about the aerobic and anaerobic pathways and our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

Guanosine metabolism in Neurospora crassa

1980 ◽  
Vol 58 (5) ◽  
pp. 369-376 ◽  
Author(s):  
W. L. Greer ◽  
L. Pendyala ◽  
A. M. Wellman
Keyword(s):  
Neurospora Crassa ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Nutritional Supplement ◽  
Inhibitory Effect ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Wild Type ◽  
Adenine Phosphoribosyltransferase ◽  
Purine Synthesis ◽  
De Novo Biosynthesis

Two aspects of guanosine metabolism in Neurospora have been investigated, (a) The inability of adenine mutants (blocked prior to IMP synthesis) to use guanosine as a nutritional supplement; and (b) the inhibitory effect of guanosine on the utilization of hypoxanthine as a purine source for growth by these mutants. Studies on the utilization of guanosine indicated that the proportion of adenine derived from guanosine may be limiting for the growth of adenine mutants. In wild type, adenine is produced through the biosynthetic pathway when grown in the presence of guanosine The amount of adenine produced through the de novo biosynthesis in wild type increases with increasing concentrations of guanosine in the medium. However, the total purine synthesis does not increase. Guanosine inhibits the uptake of hypoxanthine severely. In addition, guanosine and its nucleotide derivatives also inhibit the hypoxanthine phosphoribosyltransferase activity, at the same time stimulating the adenine phosphoribosyltransferase activity. Guanosine's effects on the uptake of hypoxanthine and its conversion to the nucleotide form may be the reasons why guanosine inhibits the utilization of hypoxanthine but not adenine by these mutants.

scholarly journals Acute Intermittent Porphyria in a Man with Dual Enzyme Deficiencies

2020 ◽  
Vol 2020 ◽  
pp. 1-6
Author(s):  
G. N. Cerbino ◽  
L. Abou Assali ◽  
L. S. Varela ◽  
L. Tomassi ◽  
A. Batlle ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Metabolic Disorders ◽  
De Novo ◽  
Porphyria Cutanea Tarda ◽  
Acute Intermittent Porphyria ◽  
Uroporphyrinogen Decarboxylase ◽  
History Of ◽  
Biochemical Testing ◽  
De Novo Variant ◽  
Deficient Activity

Porphyrias are a heterogeneous group of metabolic disorders that result from the altered activity of specific enzymes of the heme biosynthetic pathway and are characterized by accumulation of pathway intermediates. Porphyria cutanea tarda (PCT) is the most common porphyria and is due to deficient activity of uroporphyrinogen decarboxylase (UROD). Acute intermittent porphyria (AIP) is the most common of the acute hepatic porphyrias, caused by decreased activity of hydroxymethylbilane synthase (HMBS). An Argentinean man with a family history of PCT who carried the UROD variant c.10_11insA suffered severe abdominal pain. Biochemical testing was consistent with AIP, and molecular analysis of HMBS revealed a de novo variant: c.344 + 2_ + 5delTAAG. This is one of the few cases of porphyria identified with both UROD and HMBS mutations and the first confirmed case of porphyria with dual enzyme deficiencies in Argentina.

scholarly journals De Novo Biosynthesis and Radiolabeling of Mammalian Digitalis-Like Factors

2004 ◽  
Vol 50 (3) ◽  
pp. 612-620 ◽  
Author(s):  
Hassan M A M Qazzaz ◽  
(Tim) Zhimin Cao ◽  
Duane D Bolanowski ◽  
Barbara J Clark ◽  
Roland Valdes
Keyword(s):  
Biosynthetic Pathway ◽  
De Novo ◽  
Cholesterol Biosynthesis ◽  
Mammalian Species ◽  
Tumor Cell Line ◽  
Structural Features ◽  
Reversed Phase ◽  
De Novo Biosynthesis ◽  
Cellular Machinery ◽  
Coa Reductase

Abstract Background: Digoxin-like immunoreactive factors (DLIFs) are endogenous mammalian cardenolides with structural features similar to those of the plant-derived digitalis compounds. DLIFs and their structurally related forms (Dh-DLIFs) may serve as effectors of ion-transport activity mediated by their interaction with Na,K-ATPase and thus play a role as a new hormonal axis. Although some evidence implicates the adrenal gland as a tissue source for the DLIFs, little is known about the biosynthetic pathway producing these compounds. We now demonstrate de novo biosynthesis of DLIF by incorporation of radioactive carbon (14C) into the structures of both DLIF and Dh-DLIF. Methods: We used a combination of reversed-phase HPLC techniques to separate the radioactive DLIF components after incorporation of 14C into their structure by use of either [1,2-14C]acetic acid or [4-14C]cholesterol as precursors and a Y-1 mouse adrenocortical tumor cell line. We also stimulated and suppressed production of steroidogenesis by use of cAMP analogs and Mevastatin, respectively, to demonstrate the dependence of DLIF production on the cholesterol-dependent biosynthetic pathway. A combination of chromatographic mobility, immunoassays specific for digoxin and dihydrodigoxin, and deglycosylation using 5-sulfosalicylic acid were used to identify the DLIF and Dh-DLIF components. Results: With cholesterol as precursor, the cells produced DLIF (7.5 mCi/mmol) with a labeling efficiency of 10%, whereas with acetate the cells produced DLIF (72.2 mCi/mmol) with a labeling efficiency of 0.08% of the total DLIF produced. The radiolabeled DLIF and Dh-DLIF molecules had identical chromatographic mobilities and stoichiometric removal of sugars as the previously characterized DLIFs isolated from different mammalian species and tissues. With radioactive cholesterol as precursor, the 14C was incorporated into the DLIF-genin portion of the compounds and not the sugars. Interestingly, treatment of Y-1 cells with 8-bromoadenosine 3′:5′-cAMP to stimulate steroidogenesis did not increase production of DLIF or Dh-DLIF but did increase production of progesterone. Mevastatin (5 μmol), an inhibitor of the enzyme hydroxymethylglutaryl-CoA reductase and thus of cholesterol biosynthesis, gave an 85% decrease in the production of 14C-DLIF and progesterone, but only a modest 15% decrease in 14C-Dh-DLIF production. Conclusions: These data demonstrate that the adrenal cell has the cellular machinery necessary for de novo biosynthesis of DLIF and Dh-DLIF starting from a simple carbon pool and also support the concept that cholesterol is a major precursor of the DLIF compounds. This cell culture model provides a source of radiolabeled DLIF compounds for future experimental work.

The Modernities of H. Lawrence Freeman

2019 ◽  
Vol 72 (3) ◽  
pp. 719-779
Author(s):  
David Gutkin
Keyword(s):  
Nineteenth Century ◽  
New York ◽  
Reception History ◽  
Black Press ◽  
Late Nineteenth Century ◽  
Starting Point ◽  
History Of ◽  
Late Nineteenth ◽  
White Press ◽  
Grand Opera

H. Lawrence Freeman's “Negro Jazz Grand Opera,” Voodoo, was premiered in 1928 in Manhattan's Broadway district. Its reception bespoke competing, racially charged values that underpinned the idea of the “modern” in the 1920s. The white press critiqued the opera for its allegedly anxiety-ridden indebtedness to nineteenth-century European conventions, while the black press hailed it as the pathbreaking work of a “pioneer composer.” Taking the reception history of Voodoo as a starting point, this article shows how Freeman's lifelong project, the creation of what he would call “Negro Grand Opera,” mediated between disparate and sometimes apparently irreconcilable figurations of the modern that spanned the late nineteenth century through the interwar years: Wagnerism, uplift ideology, primitivism, and popular music (including, but not limited to, jazz). I focus on Freeman's inheritance of a worldview that could be called progressivist, evolutionist, or, to borrow a term from Wilson Moses, civilizationist. I then trace the complex relationship between this mode of imagining modernity and subsequent versions of modernism that Freeman engaged with during the first decades of the twentieth century. Through readings of Freeman's aesthetic manifestos and his stylistically syncretic musical corpus I show how ideas about race inflected the process by which the qualitatively modern slips out of joint with temporal modernity. The most substantial musical analysis examines leitmotivic transformations that play out across Freeman's jazz opera American Romance (1924–29): lions become subways; Mississippi becomes New York; and jazz, like modernity itself, keeps metamorphosing. A concluding section considers a broader set of questions concerning the historiography of modernism and modernity.

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