scholarly journals Heterologous erythromycin production across strain and plasmid construction

2017 ◽  
Vol 34 (1) ◽  
pp. 271-276 ◽  
Author(s):  
Lei Fang ◽  
Marc Guell ◽  
George M. Church ◽  
Blaine A. Pfeifer
Keyword(s):  
Plasmid Construction ◽  
Erythromycin Production

scholarly journals Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Shoji Suzuki ◽  
Norio Kurosawa
Keyword(s):  
Gene Knockout ◽  
Arginine Decarboxylase ◽  
Sulfolobus Acidocaldarius ◽  
Dna Photolyase ◽  
Genetic Studies ◽  
Multiple Gene ◽  
Plasmid Construction ◽  
Pcr Product ◽  
Optimized Protocol ◽  
One Step

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

scholarly journals Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea

2021 ◽  
Vol 9 ◽  
Author(s):  
Ying Liu ◽  
Sabir Khan ◽  
Panpan Wu ◽  
Bowen Li ◽  
Lanlan Liu ◽  
...  
Keyword(s):  
Regulatory Network ◽  
Target Genes ◽  
Antibacterial Activities ◽  
Saccharopolyspora Erythraea ◽  
High Yield ◽  
Yield Strain ◽  
Yield Improvement ◽  
Erythromycin Production

Erythromycins produced by Saccharopolyspora erythraea have broad-spectrum antibacterial activities. Recently, several TetR-family transcriptional regulators (TFRs) were identified to control erythromycin production by multiplex control modes; however, their regulatory network remains poorly understood. In this study, we report a novel TFR, SACE_0303, positively correlated with erythromycin production in Sac. erythraea. It directly represses its adjacent gene SACE_0304 encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene eryAI and resistance gene ermE. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting SACE_0303 as well as eryAI and indirectly repressing ermE. Then, the SACE_0303 binding site within the SACE_0303-SACE_0304 intergenic region was defined. Through genome scanning combined with in vivo and in vitro experiments, three additional SACE_0303 target genes (SACE_2467 encoding cation-transporting ATPase, SACE_3156 encoding a large transcriptional regulator, SACE_5222 encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with SACE_0304 deletion and SACE_0303 overexpression, we performed stepwise engineering of the SACE_0303-mediated mini-regulatory network in a high-yield strain, resulting in enhanced erythromycin production by 67%. In conclusion, the present study uncovered the regulatory network of a novel TFR for control of erythromycin production and provides a multiplex tactic to facilitate the engineering of industrial actinomycetes for yield improvement of antibiotics.

scholarly journals Genetic analysis of erythromycin production in Streptomyces erythreus.

1985 ◽  
Vol 164 (1) ◽  
pp. 425-433 ◽  
Author(s):  
J M Weber ◽  
C K Wierman ◽  
C R Hutchinson
Keyword(s):  
Genetic Analysis ◽  
Erythromycin Production

scholarly journals Protocol for examining and eliminating base editor-induced genome-wide and transcriptome-wide off-target mutations

2021 ◽  
Author(s):  
Lijie Wang ◽  
Wei Xue ◽  
Hongxia Zhang ◽  
Runze Gao ◽  
Houyuan Qiu ◽  
...  
Keyword(s):  
Clinical Applications ◽  
Cytidine Deaminases ◽  
Base Editing ◽  
Plasmid Construction ◽  
Background Levels ◽  
Genome Wide ◽  
Deoxycytidine Deaminase

Abstract Fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications but suffers from off-target (OT) mutations. Here, we applied a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. This step-by-step protocol describes the plasmid construction of tBE system, determination of genome/transcriptome-wide OT mutations and tBE-mediated base editing in vivo.

scholarly journals Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

2007 ◽  
Vol 9 (3) ◽  
pp. 293-303 ◽  
Author(s):  
A REEVES ◽  
I BRIKUN ◽  
W CERNOTA ◽  
B LEACH ◽  
M GONZALEZ ◽  
...  
Keyword(s):  
Saccharopolyspora Erythraea ◽  
Erythromycin Production

scholarly journals Intra-Molecular Homologous Recombination of Scarless Plasmid

2020 ◽  
Vol 21 (5) ◽  
pp. 1697
Author(s):  
Yaping Liang ◽  
Yu Zhang ◽  
Liangwei Liu
Keyword(s):  
Homologous Recombination ◽  
Efficient Method ◽  
Dna Amplification ◽  
Accurate Method ◽  
Accuracy Rate ◽  
Plasmid Construction ◽  
Plasmid Dnas ◽  
Circular Plasmid

Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5′- and 3′-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-AdD. The generality was checked by constructing plasmid pET21a-AdD and pET22b-AdD in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids.

scholarly journals Author Correction: A standard for near-scarless plasmid construction using reusable DNA parts

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Xiaoqiang Ma ◽  
Hong Liang ◽  
Xiaoyi Cui ◽  
Yurou Liu ◽  
Hongyuan Lu ◽  
...  
Keyword(s):  
Plasmid Construction

scholarly journals Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

2009 ◽  
Vol 8 (1) ◽  
pp. 18 ◽  
Author(s):  
Elisabetta Carata ◽  
Clelia Peano ◽  
Salvatore M Tredici ◽  
Francesco Ferrari ◽  
Adelfia Talà ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Saccharopolyspora Erythraea ◽  
Erythromycin Production
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