scholarly journals Efficient biosynthesis of nucleoside cytokinin angustmycin A containing an unusual sugar system

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Le Yu ◽  
Wenting Zhou ◽  
Yixuan She ◽  
Hongmin Ma ◽  
You-Sheng Cai ◽  
...  
Keyword(s):  
Natural Product ◽  
Glycosidic Bond ◽  
Natural Product Biosynthesis ◽  
E Coli ◽  
Cofactor Recycling ◽  
The Way

AbstractAngustmycin A has anti-mycobacterial and cytokinin activities, and contains an intriguing structure in which an unusual sugar with C5′-C6′ dehydration is linked to adenine via an N-glycosidic bond. However, the logic underlying the biosynthesis of this molecule has long remained obscure. Here, we address angustmycin A biosynthesis by the full deciphering of its pathway. We demonstrate that AgmD, C, A, E, and B function as d-allulose 6-phosphate 3-epimerase, d-allulose 6-phosphate pyrophosphokinase, adenine phosphoallulosyltransferase, phosphoribohydrolase, and phosphatase, respectively, and that these collaboratively catalyze the relay reactions to biosynthesize angustmycin C. Additionally, we provide evidence that AgmF is a noncanonical dehydratase for the final step to angustmycin A via a self-sufficient strategy for cofactor recycling. Finally, we have reconstituted the entire six-enzyme pathway in vitro and in E. coli leading to angustmycin A production. These results expand the enzymatic repertoire regarding natural product biosynthesis, and also open the way for rational and rapid discovery of other angustmycin related antibiotics.

scholarly journals Toward Biosynthetic Design and Implementation of Escherichia coli-Derived Paclitaxel and Other Heterologous Polyisoprene Compounds

2012 ◽  
Vol 78 (8) ◽  
pp. 2497-2504 ◽  
Author(s):  
Ming Jiang ◽  
Gregory Stephanopoulos ◽  
Blaine A. Pfeifer
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Natural Product ◽  
Cellular Metabolism ◽  
Compound Formation ◽  
Natural Product Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Design And Implementation ◽  
Final Compound

ABSTRACTEscherichia colioffers unparalleled engineering capacity in the context of heterologous natural product biosynthesis. However, as with other heterologous hosts, cellular metabolism must be designed or redesigned to support final compound formation. This task is at once complicated and aided by the fact that the cell does not natively produce an abundance of natural products. As a result, the metabolic engineer avoids complicated interactions with native pathways closely associated with the outcome of interest, but this convenience is tempered by the need to implement the required metabolism to allow functional biosynthesis. This review focuses on engineeringE. colifor the purpose of polyisoprene formation, as it is related to isoprenoid compounds currently being pursued through a heterologous approach. In particular, the review features the compound paclitaxel and early efforts to design and overproduce intermediates throughE. coli.

scholarly journals Generation of two New Macrolactones through Sequential Biotransformation of Dihydroresorcylide

2011 ◽  
Vol 6 (2) ◽  
pp. 1934578X1100600
Author(s):  
Jia Zeng ◽  
Jonathan Valiente ◽  
Jixun Zhan
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Natural Product ◽  
Beauveria Bassiana ◽  
Natural Product Biosynthesis ◽  
Whole Cell ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Engineered Escherichia Coli

Biotransformation is an effective method to generate new derivatives from natural products. Combination of various enzymes or whole-cell biocatalysts creates new opportunities for natural product biosynthesis. Dihydroresorcylide (1) is a phytotoxic macrolactone from Acremonium aeae. It was first chlorinated at C-11 by an engineered Escherichia coli BL21-CodonPlus (DE3)-RIL/pJZ54 strain that overexpresses a fungal flavin-dependent halogenase, and subsequently glycosylated at 12-OH by Beauveria bassiana ATCC 7159, giving rise to a novel derivative, 11-chloro-4′- O-methyl-12- O-β-D-glucosyl-dihydroresorcylide (3). Although 1 can be converted into a new 4′- O-methyl-glucosylated derivative 4 by B. bassiana, this product cannot be further chlorinated by E. coli BL21-CodonPlus (DE3)-RIL/pJZ54 to afford 3. The sequence of these two biotransformation steps was thus restricted and not interchangeable. This sequential biotransformation approach can be applied to other structurally similar natural products to create novel derivatives.

scholarly journals Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

2018 ◽  
Author(s):  
Hong Gao ◽  
Buvani Murugesan ◽  
Janina Hoßbach ◽  
Stephanie K. Evans ◽  
W. Marshall Stark ◽  
...  
Keyword(s):  
Natural Product ◽  
Genetic Studies ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Molecular Approaches ◽  
Genome Integration ◽  
Rare Actinomycetes ◽  
High Level

AbstractFew natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify integrating vectors, derived using phageint/attPloci, that would efficiently integrate site-specifically in the rare Actinomycete,Amycolatopsis marinaDSM45569. Analysis of the genome of A.marinaDSM45569 indicated the presence ofattB-like sequences for TG1 and R4 integrases. The TG1 and R4attBswere active inin vitrorecombination assays with their cognate purified integrases andattPloci. Integrating vectors containing either the TG1 or R4int/attPloci yielded exconjugants in conjugation assays fromE. colitoA. marinaDSM45569. Site-specific recombination of the plasmids into the host TG1 or R4attBsites was confirmed by sequencing. The presence of homologous TG1 and R4attBsites in other species of this genus indicates that vectors based on TG1 and R4 integrases could be widely applicable.ImportanceRare Actinomycetes have the same potential of natural product discovery as Streptomyces, but the potential has not been fully explored due to the lack of efficient molecular biology tools. In this study, we identified two serine integrases, TG1 and R4, which could be used in the rare Actinomycetes species,Amycolatopsis marina, as tools for genome integration. The high level of conservation between theattBsites for TG1 and R4 in a number of Amycolatopsis species suggested that plasmids with the integration systems from these phages should be widely useful in this genus.

New Imidazole Inhibitors of Mycobacterial FtsZ: the Way from High-Throughput Molecular Screening in Grid up to in vitro Verification

2016 ◽  
Vol 12 (3) ◽  
pp. 43-55 ◽  
Author(s):  
P.A. Karpov ◽  
◽  
O.M. Demchuk ◽  
V.M. Britsun ◽  
D.I. Lytvyn ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Screening ◽  
The Way

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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