Complete genome sequence of high-yield strain S. lincolnensis B48 and identification of crucial mutations contributing to lincomycin overproduction

Wang, Ruida; Kong, Fanzhi; Wu, Haizhen; Hou, Bingbing; Kang, Yajing; Cao, Yuan; Duan, Shiwei; Jiang, Ye; Zhang, Huizhan

doi:10.1016/j.synbio.2020.03.001

Cited by 20 publications

(21 citation statements)

References 53 publications

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“…The latter makes rational metabolic engineering aimed at enhancement of antibiotic production or, as of late, activation of silent secondary metabolite biosynthesis gene clusters a formidable challenge. Genome sequencing of high-producing actinomycete mutants recently revealed different mutations that apparently contribute to the desired phenotype but vary significantly between the strains ( 41 , 42 , 45 ). Hence, it appears difficult to design a common strategy for metabolic engineering of various actinomycetes, and spontaneous mutagenesis coupled with smart selection strategy remains a viable alternative.…”

Section: Discussionmentioning

confidence: 99%

Coupling of the engineered DNA “mutator” to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces

Sekurova

Sun

Zehl

et al. 2021

Nucleic Acids Research

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DNA replication fidelity in Streptomyces bacteria, prolific producers of many medically important secondary metabolites, is understudied, while in Escherichia coli it is controlled by DnaQ, the ϵ subunit of DNA polymerase III (DNA PolIII). Manipulation of dnaQ paralogues in Streptomyces lividans TK24, did not lead to increased spontaneous mutagenesis in this bacterium suggesting that S. lividans DNA PolIII uses an alternative exonuclease activity for proofreading. In Mycobacterium tuberculosis, such activity is attributed to the DnaE protein representing α subunit of DNA PolIII. Eight DnaE mutants designed based on the literature data were overexpressed in S. lividans, and recombinant strains overexpressing two of these mutants displayed markedly increased frequency of spontaneous mutagenesis (up to 1000-fold higher compared to the control). One of these ‘mutators’ was combined in S. lividans with a biosensor specific for antibiotic coelimycin, which biosynthetic gene cluster is present but not expressed in this strain. Colonies giving a positive biosensor signal appeared at a frequency of ca 10–5, and all of them were found to produce coelimycin congeners. This result confirmed that our approach can be applied for chemical- and radiation-free mutagenesis in Streptomyces leading to activation of orphan biosynthetic gene clusters and discovery of novel bioactive secondary metabolites.

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Section: Discussionmentioning

confidence: 99%

Coupling of the engineered DNA “mutator” to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces

Sekurova

Sun

Zehl

et al. 2021

Nucleic Acids Research

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“…SFPI medium (30 g/L corn steep liquor, 20 g/L soluble starch, 10 g/L glucose, 1.5 g/L (NH 4 ) 2 SO 4 , 1 g/L soya flour, and 4 g/L CaCO 3 , pH 7.2) and SFPII (105 g/L glucose, 20 g/L soya flour, 8 g/L NaNO 3 , 6 g/L (NH 4 ) 2 SO 4 , 5 g/L NaCl, 1.5 g/L corn steep liquor, 0.025 g/L K 2 HPO 4 , and 8 g/L CaCO 3 , pH 7.8) were used for fermentation. The growth conditions of E. coli, M. luteus, and S lincolnensis strains were described in our previous study [5,32]. The media were supplemented with apramycin of 50 μg/ml and kanamycin of 50 μg/ml as appropriate.…”

Section: Strains Plasmids and Mediamentioning

confidence: 99%

“…To construct a rex disruption strain, a CRISPR/Cas9-based genetic editing method was performed as previously reported [32,33]. A 1.1-kb fragment of rex-upstream region and a 1.1-kb fragment of rex-downstream region were amplified by polymerase chain reaction (PCR) using primer pairs Urex-F/R and Drex-F/R.…”

Section: Construction Of the Rex Disruption Complementation And Overexpression Strainsmentioning

confidence: 99%

A putative redox‐sensing regulator Rex regulates lincomycin biosynthesis in Streptomyces lincolnensis

et al. 2021

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Lincomycin is an important antimicrobial agent which is widely used in clinical and animal husbandry. The biosynthetic pathway of lincomycin comes to light in the past 10 years, however, the regulatory mechanism is still unclear. In this study, a redox-sensing regulator Rex from Streptomyces lincolnensis (Rex lin ) was identified and characterized to affect cell growth and lincomycin biosynthesis. Disruption of rex resulted in an increase in cell growth, but a decrease in lincomycin production. The results of quantitative real-time polymerase chain reaction showed that Rex lin can promote transcription of the regulatory gene lmbU and the structural genes lmbA, lmbC, lmbJ, lmbV, and lmbW. However, electrophoretic mobility shift assay analysis demonstrated that Rex lin can not bind to the promoter regions of these genes above. Findings in this study broadened our horizons in the regulatory mechanism of lincomycin production and laid a foundation for strain improvement of antibiotic producers.

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“…To construct a lmbU disruption strain, the internal region of lmbU (465 bp) was deleted via a CRISPR/Cas9-based genetic editing method (37). The lmbU-specific single-molecule-guide RNA (sgRNA) was amplified by PCR using the primer pair sgUF/R with pKCcas9dO as template.…”

Section: Construction Of Lmbu Disruption Strain δLmbumentioning

confidence: 99%

LmbU directly regulates SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780, which are located outside the lmb cluster and inhibit lincomycin biosynthesis in Streptomyces lincolnensis

Hou

et al. 2021

Preprint

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The productions of antibiotics are usually regulated by cluster-situated regulators (CSRs), which can directly regulate the genes within the corresponding biosynthetic gene cluster (BGC). However, few studies have looked into the regulation of CSRs on the targets outside the BGC. Here, we screened the targets of LmbU in the whole genome of S. lincolnensis, and found 14 candidate targets, among of which, 8 targets can bind to LmbU by EMSAs. Reporter assays in vivo revealed that LmbU repressed transcription of SLINC_RS02575 and SLINC_RS05540, while activated transcription of SLINC_RS42780. In addition, disruptions of SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 promoted the production of lincomycin, and qRT-PCR showed that SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 inhibited transcription of the lmb genes, indicating that all the three regulators can negatively regulate lincomycin biosynthesis. What's more, the homologues of LmbU and its targets SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 are widely found in actinomycetes, while the distributions of DNA-binding sites (DBS) of LmbU are diverse, indicating the regulatory mechanisms of LmbU homologues in various strains are different and complicated.

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Complete genome sequence of high-yield strain S. lincolnensis B48 and identification of crucial mutations contributing to lincomycin overproduction

Cited by 20 publications

References 53 publications

Coupling of the engineered DNA “mutator” to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces

Coupling of the engineered DNA “mutator” to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces

A putative redox‐sensing regulator Rex regulates lincomycin biosynthesis in Streptomyces lincolnensis

LmbU directly regulates SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780, which are located outside the lmb cluster and inhibit lincomycin biosynthesis in Streptomyces lincolnensis

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