scholarly journals Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli

PLoS ONE ◽  
2016 ◽  
Vol 11 (3) ◽  
pp. e0151087 ◽  
Author(s):  
Pouya Javidpour ◽  
Samuel Deutsch ◽  
Vivek K. Mutalik ◽  
Nathan J. Hillson ◽  
Christopher J. Petzold ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Anammox Bacteria ◽  
Biosynthetic Genes ◽  
E Coli

scholarly journals Characterization of Leader Processing Shows That Partially Processed Mersacidin Is Activated by AprE After Export

2021 ◽  
Vol 12 ◽  
Author(s):  
Jakob H. Viel ◽  
Amanda Y. van Tilburg ◽  
Oscar P. Kuipers
Keyword(s):  
Antimicrobial Activity ◽  
Synthetic Biology ◽  
Heterologous Expression ◽  
Expression System ◽  
Heterologous Expression System ◽  
Proteolytic Activation ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Precursor Peptide ◽  
Potential Applications

The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli, improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing.

scholarly journals TDP-l-Megosamine Biosynthesis Pathway Elucidation and Megalomicin A Production in Escherichia coli

2010 ◽  
Vol 76 (12) ◽  
pp. 3869-3877 ◽  
Author(s):  
Mariana Useglio ◽  
Salvador Peirú ◽  
Eduardo Rodríguez ◽  
Guillermo R. Labadie ◽  
John R. Carney ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Mass Spectrometric ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
Biosynthesis Pathway ◽  
Biosynthetic Genes

ABSTRACT In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.

scholarly journals Heterologous Expression and Purification of ? Galactosidase Protein Using Affinity Chromatography

2013 ◽  
Vol 5 (3) ◽  
pp. 499-513
Author(s):  
M. Z. Alam ◽  
L. Ragionieri ◽  
M. A. S. Santos ◽  
A. Iqbal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Affinity Chromatography ◽  
Recombinant Dna ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Lacz Gene ◽  
Recombinant Dna Technology ◽  
E Coli ◽  
Elution Buffer

Enzymes and other protein purification using recombinant DNA technology have become popular due to scarcity of natural protein. Saccharomyces cerevisiae is a demanding host, since it facilitates protein expression by its relative simplicity, safe organisms, inexpensive and has many properties of eukaryotic expression system. As an alternative host we express E. coli lacZ gene with GST tag in Saccharomyces cerevisiae and successfully purified from soluble extracts. The concentration of soluble GST-? galactosidase protein was approximately 0.57 mg/ml of elution buffer yielded from 50 ml yeast cell culture. The ?-galactosidase protein from insoluble extract was low due to the increasing solubility of GST tag. Keywords: ?-galactosidase; Heterologous expression; GST tag; Affinity chromatography. © 2013 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi: http://dx.doi.org/10.3329/jsr.v5i3.13820 J. Sci. Res. 5 (3), 499-513 (2013)  

scholarly journals Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

2009 ◽  
Vol 75 (22) ◽  
pp. 7291-7293 ◽  
Author(s):  
Gopal Prasad Ghimire ◽  
Hei Chan Lee ◽  
Jae Kyung Sohng
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Isopentenyl Diphosphate ◽  
Isopentenyl Diphosphate Isomerase ◽  
Streptomyces Peucetius ◽  
E Coli ◽  
Expression Of Genes ◽  
Genes Encoding

ABSTRACT Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.

scholarly journals Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis

2016 ◽  
Vol 82 (21) ◽  
pp. 6414-6422 ◽  
Author(s):  
Ryuki Miyauchi ◽  
Chiho Ono ◽  
Takashi Ohnuki ◽  
Yoichiro Shiba
Keyword(s):  
Genetic Engineering ◽  
Heterologous Expression ◽  
Production System ◽  
Therapeutic Potential ◽  
Biosynthetic Gene Cluster ◽  
Efficient Production ◽  
Biosynthetic Gene ◽  
Content Type ◽  
E Coli ◽  
Glycosidase Inhibitor

ABSTRACTThe fungusThelonectria discophoraSANK 18292 produces the iminosugar nectrisine, which has a nitrogen-containing heterocyclic 5-membered ring and acts as a glycosidase inhibitor. In our previous study, an oxidase (designated NecC) that converts 4-amino-4-deoxyarabinitol to nectrisine was purified fromT. discophoracultures. However, the genes required for nectrisine biosynthesis remained unclear. In this study, the nectrisine biosynthetic gene cluster inT. discophorawas identified from the contiguous genome sequence around thenecCgene. Gene disruption and complementation studies and heterologous expression of the gene showed thatnecA,necB, andnecCcould be involved in nectrisine biosynthesis, during which amination, dephosphorylation, and oxidation occur. It was also demonstrated that nectrisine could be produced by recombinantEscherichia colicoexpressing thenecA,necB, andnecCgenes. These findings provide the foundation to develop a bacterial production system for nectrisine or its intermediates through genetic engineering.IMPORTANCEIminosugars might have great therapeutic potential for treatment of many diseases. However, information on the genes for their biosynthesis is limited. In this study, we report the identification of genes required for biosynthesis of the iminosugar nectrisine inThelonectria discophoraSANK 18292, which was verified by disruption, complementation, and heterologous expression of the genes involved. We also demonstrate heterologous production of nectrisine by recombinantE. coli, toward developing an efficient production system for nectrisine or its intermediates through genetic engineering.

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