The cytosolic and extracellular proteomes of Actinoplanes sp. SE50/110 led to the identification of gene products involved in acarbose metabolism

Wendler, Sergej; Hürtgen, Daniel; Kalinowski, Jörn; Klein, Andreas; Niehaus, Karsten; Schulte, Fabian; Schwientek, Patrick; Wehlmann, Hermann; Wehmeier, Udo F.; Pühler, Alfred

doi:10.1016/j.jbiotec.2012.08.011

Cited by 28 publications

(38 citation statements)

References 46 publications

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“…This effect is supposed to be caused by the reduced metabolic burden, since Cgt was identi ed to be one of the highest abundant proteins in Actinoplanes sp. SE50/110 [87][88][89]. The similar expression patterns shown in this study, support the suggestion, that by deletion of genes co-expressed with the acb gene cluster, the acarbose formation might be improved.…”

Section: Identi Cation Of Genes Co-expressed To the Acb Gene Clustersupporting

confidence: 89%

The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase

Droste

Ortseifen

Schaffert

et al. 2020

Preprint

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Background: Actinoplanes sp. SE50/110 is the natural producer of the diabetes mellitus drug acarbose, which is highly produced during the growth phase and ceases during the stationary phase. In previous works, the growth-dependency of acarbose formation was assumed to be caused by a decreasing transcription of the acarbose biosynthesis genes during transition and stationary growth phase.Results: In this study, transcriptomic data using RNA-seq and state-of-the-art proteomic data from seven time points of controlled bioreactor cultivations were used to analyze expression dynamics during growth of Actinoplanes sp. SE50/110. A hierarchical cluster analysis revealed co-regulated genes, which display similar transcription dynamics over the cultivation time. Aside from the expected metabolic switch from primary to secondary metabolism during transition phase, we observed a significantly decreasing transcript abundance of all acarbose biosynthetic genes, with the strongest decrease for the monocistronically transcribed genes acbA, acbB, acbD and acbE. Our data confirm a similar trend for acb gene transcription and acarbose formation rate.Surprisingly, the proteome dynamics does not follow the respective transcription for all acb genes. This suggests different protein stabilities or post-transcriptional regulation of the Acb proteins, which in turn could indicate bottlenecks in the acarbose biosynthesis. Furthermore, several genes are co-expressed with the acb gene cluster over the course of the cultivation, including eleven transcriptional regulators (e.g. ACSP50_0424), two sigma factors (ACSP50_0644, ACSP50_6006) and further genes, which have not previously been in focus of acarbose research in Actinoplanes sp. SE50/110.Conclusion: In conclusion, we have demonstrated, that a genome wide transcriptome and proteome analysis in a high temporal resolution is well suited to study the acarbose biosynthesis and the transcriptional and post-transcriptional regulation thereof.

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Section: Identi Cation Of Genes Co-expressed To the Acb Gene Clustersupporting

confidence: 89%

The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase

Droste

Ortseifen

Schaffert

et al. 2020

Preprint

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“…The ABC transporter MalEFG was suggested as a possible acarbose-metabolite re-importer and AglEFG might be an additional maltose/maltodextrin importer [10, 54]. The proteins MalE, MalF and MalG were detected in high abundancies in both maltose and glucose-grown Actinoplanes sp.…”

Section: Discussionmentioning

confidence: 99%

“…Minimal medium was supplemented with 2.4 C-mole of the respective sugar as carbon source. The composition of the liquid media is described elsewhere [10]. When needed, chloramphenicol (25 μg mL -1 ), kanamycin (50 μg mL -1 ), apramycin (50 μg mL -1 ) or hygromycin (100 μg/mL) was added to the media.…”

Section: Methodsmentioning

confidence: 99%

The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110

et al. 2017

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BackgroundAcarbose is used in the treatment of diabetes mellitus type II and is produced by Actinoplanes sp. SE50/110. Although the biosynthesis of acarbose has been intensively studied, profound knowledge about transcription factors involved in acarbose biosynthesis and their binding sites has been missing until now. In contrast to acarbose biosynthetic gene clusters in Streptomyces spp., the corresponding gene cluster of Actinoplanes sp. SE50/110 lacks genes for transcriptional regulators.ResultsThe acarbose regulator C (AcrC) was identified through an in silico approach by aligning the LacI family regulators of acarbose biosynthetic gene clusters in Streptomyces spp. with the Actinoplanes sp. SE50/110 genome. The gene for acrC, located in a head-to-head arrangement with the maltose/maltodextrin ABC transporter malEFG operon, was deleted by introducing PCR targeting for Actinoplanes sp. SE50/110. Characterization was carried out through cultivation experiments, genome-wide microarray hybridizations, and RT-qPCR as well as electrophoretic mobility shift assays for the elucidation of binding motifs. The results show that AcrC binds to the intergenic region between acbE and acbD in Actinoplanes sp. SE50/110 and acts as a transcriptional repressor on these genes. The transcriptomic profile of the wild type was reconstituted through a complementation of the deleted acrC gene. Additionally, regulatory sequence motifs for the binding of AcrC were identified in the intergenic region of acbE and acbD. It was shown that AcrC expression influences acarbose formation in the early growth phase. Interestingly, AcrC does not regulate the malEFG operon.ConclusionsThis study characterizes the first known transcription factor of the acarbose biosynthetic gene cluster in Actinoplanes sp. SE50/110. It therefore represents an important step for understanding the regulatory network of this organism. Based on this work, rational strain design for improving the biotechnological production of acarbose can now be implemented.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3941-x) contains supplementary material, which is available to authorized users.

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“…[20] Unfortunately, extracellular proteins,i np articular, those that may be involved in secondary metabolism, are seldom characterized. [20,21] Our findings provide new insight into the complex biology of the extracellular environment and enriches our understanding of the extracellular enzymes involved in the biosynthesis of complex natural products.…”

Section: Angewandte Chemiementioning

confidence: 80%

Catalysis of Extracellular Deamination by a FAD‐Linked Oxidoreductase after Prodrug Maturation in the Biosynthesis of Saframycin A

Tang

Song²,

Zhang³

et al. 2017

Angewandte Chemie

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The biosynthesis of antibiotics in bacteria is usually believed to be an intracellular process,atthe end of which the matured compounds are exported outside the cells.T he biosynthesis of saframycin A( SFM-A), an antitumor antibiotic,r equires ac ryptic fatty acyl chain to guide the construction of ap entacyclic tetrahydroisoquinoline scaffold; however,t he follow-up deacylation and deamination steps remain unknown. Herein we demonstrate that SfmE, am embrane-bound peptidase,h ydrolyzes the fatty acyl chain to release the amino group;and SfmCy2, asecreted oxidoreductase covalently associated with FAD, subsequently performs an oxidative deamination extracellularly.T hese results not only fill in the missing steps of SFM-A biosynthesis,but also reveal that aF AD-binding oxidoreductase catalyzesa nu nexpected deamination reaction through an unconventional extracellular pathway in Streptmyces bacteria.

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The cytosolic and extracellular proteomes of Actinoplanes sp. SE50/110 led to the identification of gene products involved in acarbose metabolism

Cited by 28 publications

References 46 publications

The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase

The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase

The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110

Catalysis of Extracellular Deamination by a FAD‐Linked Oxidoreductase after Prodrug Maturation in the Biosynthesis of Saframycin A

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