Comparative analysis of transcriptional activities of heterologous promoters in the rare actinomycete Actinoplanes friuliensis

Wagner, Nina; Oßwald, Corina; Biener, Richard; Schwartz, Dirk

doi:10.1016/j.jbiotec.2009.05.002

Cited by 15 publications

(20 citation statements)

References 22 publications

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“…To ensure that these promoters can be used to drive the transcription of heterologous genes and also compare their activities with the ones reported in literature, the intergenic region between the gene located upstream and the gapdh operon (or the rpsL operon) were subsequently fused with the Streptomycete reporter gene, catechol 2, 3-dioxygenase ( xylE ), which catalyzes the conversion of colorless catechol to yellow-color 2-hydroxymuconic semialdehyde. The xylE activity assay (20) confirmed their much stronger activities than the control promoter, ermE* p , (Figure 1b), which is the mutated variant of the promoter of the erythromycin resistance gene from Saccharopolyspora erythraea , and is believed to be one of the strongest constitutive promoters in Streptomycetes (21, 22). The strong activities of gapdh p and the promoters of various translation elongation factors have also been observed in many other microorganisms, such as fungi, bacteria, micro-algae, and protozoa (23–29).…”

Section: Resultsmentioning

confidence: 84%

Refactoring the Silent Spectinabilin Gene Cluster Using a Plug-and-Play Scaffold

et al. 2013

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Natural products (secondary metabolites) are a rich source of compounds with important biological activities. Eliciting pathway expression is always challenging but extremely important in natural product discovery because individual pathway is tightly controlled through unique regulation mechanism and hence often remains silent in the routine culturing conditions. To overcome the drawback of the traditional approaches that lack general applicability, we developed a simple synthetic biology approach that decouples pathway expression from complex native regulations. Briefly, the entire silent biosynthetic pathway is refactored using a plug-and-play scaffold and a set of heterologous promoters that are functional in a heterologous host under the target culturing condition. Using this strategy, we successfully awakened the silent spectinabilin pathway from Streptomyces orinoci. This strategy bypasses the traditional laborious processes to elicit pathway expression and represents a new platform for discovering novel natural products.

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Section: Resultsmentioning

confidence: 84%

Refactoring the Silent Spectinabilin Gene Cluster Using a Plug-and-Play Scaffold

et al. 2013

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“…The widely used ermEp*, which is a heterogenous promoter from Saccharopolyspora erythraea, has multiple Ϫ10 and Ϫ35 sites and bidirectional promoter activities (5). Such structural complexity hinders its characterization and evaluation (9,19). Therefore, developing simple and well-characterized promoters is necessary for metabolic engineering in streptomycetes.…”

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confidence: 99%

An Engineered Strong Promoter for Streptomycetes

Wang

et al. 2013

Appl Environ Microbiol

227

223

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Well-characterized promoters are essential tools for metabolic engineering and synthetic biology. In Streptomyces coelicolor, the native kasOp is a temporally expressed promoter strictly controlled by two regulators, ScbR and ScbR2. In this work, first, kasOp was engineered to remove a common binding site of ScbR and ScbR2 upstream of its core region, thus generating a stronger promoter, kasOp 3 . Second, another ScbR binding site internal to the kasOp 3 core promoter region was abolished by random mutation and screening of the mutant library to obtain the strongest promoter, kasOp* (where the asterisk is used to distinguish the engineered promoter from the native promoter). The activities of kasOp* were compared with those of two known strong promoters, ermEp* and SF14p, in three Streptomyces species. kasOp* showed the highest activity at the transcription and protein levels in all three hosts. Furthermore, relative to ermEp* and SF14p, kasOp* was shown to confer the highest actinorhodin production level when used to drive the expression of actII-ORF4 in S. coelicolor. Therefore, kasOp* is a simple and well-defined strong promoter useful for gene overexpression in streptomycetes.

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“…The construct pALM201 containing salR2 under transcriptional control of ermE *p was, however, not able to restore salinosporamide A production in the salR2 − mutant. Although this vegetative promoter is frequently used for genetic complementation studies in Streptomyces , ermE *p has been shown to exhibit low activity in non-streptomycete actinobacteria such as Actinoplanes and Amycolatopsis (Wagner et al, 2009). We next tested the promoter of the apramycin resistance gene aac(3)IV, since previous studies indicated high activity in the related Actinoplanes friuliensis (Wagner et al, 2009).…”

Section: Resultsmentioning

confidence: 99%

Selective Overproduction of the Proteasome Inhibitor Salinosporamide A via Precursor Pathway Regulation

Lechner

Eustáquio

Gulder

et al. 2011

Chemistry & Biology

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The chlorinated natural product salinosporamide A is a potent 20S proteasome inhibitor currently in clinical trials as an anticancer agent. To deepen our understanding of salinosporamide biosynthesis, we investigated the function of a LuxR-type pathway-specific regulatory gene, salR2, and observed a selective effect on the production of salinosporamide A over its less active aliphatic analogs. SalR2 was shown to specifically activate genes involved in the biosynthesis of the halogenated precursor chloroethylmalonyl-CoA, which is a dedicated precursor of salinosporamide A. Specifically, SalR2 activates transcription of two divergent operons – one of which contains the unique S-adenosyl-L-methionine-dependent chlorinase encoding gene salL. By applying this knowledge towards rational engineering, we were able to selectively double salinosporamide A production. This study exemplifies the specialized regulation of a polyketide precursor pathway and its application to the selective overproduction of a specific natural product congener.

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Comparative analysis of transcriptional activities of heterologous promoters in the rare actinomycete Actinoplanes friuliensis

Cited by 15 publications

References 22 publications

Refactoring the Silent Spectinabilin Gene Cluster Using a Plug-and-Play Scaffold

Refactoring the Silent Spectinabilin Gene Cluster Using a Plug-and-Play Scaffold

An Engineered Strong Promoter for Streptomycetes

Selective Overproduction of the Proteasome Inhibitor Salinosporamide A via Precursor Pathway Regulation

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