scholarly journals Enzyme-constrained models and omics analysis of Streptomyces coelicolor reveal metabolic changes that enhance heterologous production

2019 ◽  
Author(s):  
Snorre Sulheim ◽  
Tjaša Kumelj ◽  
Dino van Dissel ◽  
Ali Salehzadeh-Yazdi ◽  
Chao Du ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Metabolic Model ◽  
Heterologous Production ◽  
Comprehensive Picture ◽  
Constrained Models ◽  
Systemic Understanding ◽  
Genome Scale ◽  
Further Development

AbstractMany biosynthetic gene clusters (BGCs) require heterologous expression to realize their genetic potential, including silent and metagenomic BGCs. Although the engineered Streptomyces coelicolor M1152 is a widely used host for heterologous expression of BGCs, a systemic understanding of how its genetic modifications affect the metabolism is lacking and limiting further development. We performed a comparative analysis of M1152 and its ancestor M145, connecting information from proteomics, transcriptomics, and cultivation data into a comprehensive picture of the metabolic differences between these strains. Instrumental to this comparison was the application of an improved consensus genome-scale metabolic model (GEM) of S. coelicolor. Although many metabolic patterns are retained in M1152, we find that this strain suffers from oxidative stress, possibly caused by increased oxidative metabolism. Furthermore, precursor availability is likely not limiting polyketide production, implying that other strategies could be beneficial for further development of S. coelicolor for heterologous production of novel compounds.

scholarly journals Engineering the Erythromycin-Producing Strain Saccharopolyspora erythraea HOE107 for the Heterologous Production of Polyketide Antibiotics

2020 ◽  
Vol 11 ◽  
Author(s):  
Jin Lü ◽  
Qingshan Long ◽  
Zhilong Zhao ◽  
Lu Chen ◽  
Weijun He ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Streptomyces Coelicolor ◽  
Genome Mining ◽  
Bac Library ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Saccharopolyspora Erythraea ◽  
Heterologous Production ◽  
Integration Sites

Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.

scholarly journals Heterologous Expression of a Cryptic Gene Cluster from Streptomyces leeuwenhoekii C34T Yields a Novel Lasso Peptide, Leepeptin

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Juan Pablo Gomez-Escribano ◽  
Jean Franco Castro ◽  
Valeria Razmilic ◽  
Scott A. Jarmusch ◽  
Gerhard Saalbach ◽  
...  
Keyword(s):  
Natural Products ◽  
Heterologous Expression ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Bioinformatic Analysis ◽  
Growth Media ◽  
Content Type ◽  
Lasso Peptide ◽  
Lasso Peptides ◽  
New Family

ABSTRACT Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor. The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides. IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

Heterologous expression of the biosynthetic gene clusters of coumermycin A1, clorobiocin and caprazamycins in genetically modified Streptomyces coelicolor strains

Biopolymers ◽  
2010 ◽  
Vol 93 (9) ◽  
pp. 823-832 ◽  
Author(s):  
Katrin Flinspach ◽  
Lucia Westrich ◽  
Leonard Kaysser ◽  
Stefanie Siebenberg ◽  
Juan Pablo Gomez-Escribano ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Streptomyces Coelicolor ◽  
Genetically Modified ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters

scholarly journals Enzyme-Constrained Models and Omics Analysis of Streptomyces coelicolor Reveal Metabolic Changes that Enhance Heterologous Production

iScience ◽  
2020 ◽  
Vol 23 (9) ◽  
pp. 101525 ◽  
Author(s):  
Snorre Sulheim ◽  
Tjaša Kumelj ◽  
Dino van Dissel ◽  
Ali Salehzadeh-Yazdi ◽  
Chao Du ◽  
...  
Keyword(s):  
Streptomyces Coelicolor ◽  
Metabolic Changes ◽  
Heterologous Production ◽  
Omics Analysis ◽  
Constrained Models

scholarly journals Predicting Strain Engineering Strategies Using iKS1317: A Genome‐Scale Metabolic Model of Streptomyces coelicolor

2019 ◽  
Vol 14 (4) ◽  
pp. 1800180 ◽  
Author(s):  
Tjaša Kumelj ◽  
Snorre Sulheim ◽  
Alexander Wentzel ◽  
Eivind Almaas
Keyword(s):  
Streptomyces Coelicolor ◽  
Metabolic Model ◽  
Strain Engineering ◽  
A Genome ◽  
Genome Scale

scholarly journals Reconstruction of a Genome-Scale Metabolic Model of Streptomyces albus J1074: Improved Engineering Strategies in Natural Product Synthesis

Metabolites ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 304
Author(s):  
Cheewin Kittikunapong ◽  
Suhui Ye ◽  
Patricia Magadán-Corpas ◽  
Álvaro Pérez-Valero ◽  
Claudio J. Villar ◽  
...  
Keyword(s):  
Natural Products ◽  
De Novo ◽  
Streptomyces Coelicolor ◽  
Metabolic Model ◽  
Streptomyces Species ◽  
Model Driven ◽  
A Genome ◽  
Streptomyces Albus ◽  
Genome Scale ◽  
Streptomyces Albus J1074

Streptomyces albus J1074 is recognized as an effective host for heterologous production of natural products. Its fast growth and efficient genetic toolbox due to a naturally minimized genome have contributed towards its advantage in expressing biosynthetic pathways for a diverse repertoire of products such as antibiotics and flavonoids. In order to develop precise model-driven engineering strategies for de novo production of natural products, a genome-scale metabolic model (GEM) was reconstructed for the microorganism based on protein homology to model species Streptomyces coelicolor while drawing annotated data from databases and literature for further curation. To demonstrate its capabilities, the Salb-GEM was used to predict overexpression targets for desirable compounds using flux scanning with enforced objective function (FSEOF). Salb-GEM was also utilized to investigate the effect of a minimized genome on metabolic gene essentialities in comparison to another Streptomyces species, S. coelicolor.

scholarly journals RAVEN 2.0: a versatile toolbox for metabolic network reconstruction and a case study on Streptomyces coelicolor

2018 ◽  
Author(s):  
Hao Wang ◽  
Simonas Marcišauskas ◽  
Benjamín J Sánchez ◽  
Iván Domenzain ◽  
Daniel Hermansson ◽  
...  
Keyword(s):  
Metabolic Networks ◽  
De Novo ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Metabolic Model ◽  
Primary And Secondary Metabolism ◽  
Scale Models ◽  
Improved Performance ◽  
Metabolites Production ◽  
Genome Scale

AbstractRAVEN is a commonly used MATLAB toolbox for genome-scale metabolic model (GEM) reconstruction, curation and constraint-based modelling and simulation. Here we present RAVEN Toolbox 2.0 with major enhancements, including: (i) de novo reconstruction of GEMs based on the MetaCyc pathway database; (ii) a redesigned KEGG-based reconstruction pipeline; (iii) convergence of reconstructions from various sources; (iv) improved performance, usability, and compatibility with the COBRA Toolbox. Capabilities of RAVEN 2.0 are here illustrated through de novo reconstruction of GEMs for the antibiotic-producing bacterium Streptomyces coelicolor. Comparison of the automated de novo reconstructions with the iMK1208 model, a previously published high-quality S. coelicolor GEM, exemplifies that RAVEN 2.0 can capture most of the manually curated model. The generated de novo reconstruction is subsequently used to curate iMK1208 resulting in Sco4, the most comprehensive GEM of S. coelicolor, with increased coverage of both primary and secondary metabolism. This increased coverage allows the use of Sco4 to predict novel genome editing targets for optimized secondary metabolites production. As such, we demonstrate that RAVEN 2.0 can be used not only for de novo GEM reconstruction, but also for curating existing models based on up-to-date databases. Both RAVEN 2.0 and Sco4 are distributed through GitHub to facilitate usage and further development by the community.Author summaryCellular metabolism is a large and complex network. Hence, investigations of metabolic networks are aided by in silico modelling and simulations. Metabolic networks can be derived from whole-genome sequences, through identifying what enzymes are present and connecting these to formalized chemical reactions. To facilitate the reconstruction of genome-scale models of metabolism (GEMs), we have developed RAVEN 2.0. This versatile toolbox can reconstruct GEMs fast, through either metabolic pathway databases KEGG and MetaCyc, or from homology with an existing GEM. We demonstrate RAVEN’s functionality through generation of a metabolic model of Streptomyces coelicolor, an antibiotic-producing bacterium. Comparison of this de novo generated GEM with a previously manually curated model demonstrates that RAVEN captures most of the previous model, and we subsequently reconstructed an updated model of S. coelicolor: Sco4. Following, we used Sco4 to predict promising targets for genetic engineering, which can be used to increase antibiotic production.

scholarly journals Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

2005 ◽  
Vol 71 (5) ◽  
pp. 2452-2459 ◽  
Author(s):  
Alessandra S. Eustáquio ◽  
Bertolt Gust ◽  
Ute Galm ◽  
Shu-Ming Li ◽  
Keith F. Chater ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Attachment Site ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Gene Deletions ◽  
Or Gene ◽  
Genetically Manipulated

ABSTRACT A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage φC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.

Cloning, sequencing and heterologous expression of the medermycin biosynthetic gene cluster of Streptomyces sp. AM-7161: towards comparative analysis of the benzoisochromanequinone gene clusters

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1633-1645 ◽  
Author(s):  
Koji Ichinose ◽  
Makoto Ozawa ◽  
Keiko Itou ◽  
Kanako Kunieda ◽  
Yutaka Ebizuka
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Carrier Protein ◽  
Biosynthetic Gene ◽  
Six Genes

Medermycin is a Streptomyces aromatic C-glycoside antibiotic classified in the benzoisochromanequinones (BIQs), which presents several interesting biosynthetic problems concerning polyketide synthase (PKS), post-PKS tailoring and deoxysugar pathways. The biosynthetic gene cluster for medermycin (the med cluster) was cloned from Streptomyces sp. AM-7161. Completeness of the clone was proved by the heterologous expression of a cosmid carrying the entire med cluster in Streptomyces coelicolor CH999 to produce medermycin. The DNA sequence of the cosmid (36 202 bp) revealed 34 complete ORFs, with an incomplete ORF at either end. Functional assignment of the deduced products was made for PKS and biosynthetically related enzymes, tailoring steps including strereochemical control, oxidation, angolosamine pathway, C-glycosylation, and regulation. The med cluster was estimated to be about 30 kb long, covering 29 ORFs. An unusual characteristic of the cluster is the disconnected organization of the minimal PKS genes: med-ORF23 encoding the acyl carrier protein is 20 kb apart from med-ORF1 and med-ORF2 for the two ketosynthase components. Secondly, the six genes (med-ORF14, 15, 16, 17, 18 and 20) for the biosynthesis of the deoxysugar, angolosamine, are all contiguous. Finally, the finding of a glycosyltransferase gene, med-ORF8, suggests a possible involvement of conventional C-glycosylation in medermycin biosynthesis. Comparison among the three complete BIQ gene clusters – med and those for actinorhodin (act) and granaticin (gra) – revealed some common genes whose deduced functions are unavailable from database searches (the ‘unknowns’). An example is med-ORF5, a homologue of actVI-ORF3 and gra-ORF18, which was highlighted by a recent proteomic analysis of S. coelicolor A3(2).

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