Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster

Wendler, Sergej; Otto, Andreas; Ortseifen, Vera; Bonn, Florian; Neshat, Armin; Schneiker-Bekel, Susanne; Walter, Frederik; Wolf, Timo; Zemke, Till; Wehmeier, Udo F.; Hecker, Michael; Kalinowski, Jörn; Becher, Dörte; Pühler, Alfred

doi:10.1016/j.jprot.2015.04.013

Cited by 18 publications

(36 citation statements)

References 138 publications

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“…Furthermore, 36 of the 179 proteins exclusively identi ed in the extracellular fraction were predicted as cytosolic, 64 were predicted as membrane associated or inner membrane proteins and 71 proteins were predicted as extracellular proteins by 50. The identi cation of proteins predicted as cytosolic in the extracellular fraction was reported previously for several bacteria [51,52]. Additionally, it could be shown for Bacillus subtilis that only 21% of over 900 extracellular identi ed proteins show a signal peptide [52].…”

Section: Analysis Of Proteome Data During the Whole Cultivation Procesupporting

confidence: 63%

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: Analysis Of Proteome Data During the Whole Cultivation Procesupporting

confidence: 63%

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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“…SE50/110 was extensively studied in the last decades. Fundamental knowledge about the complete genome (Schwientek et al 2012;Wolf et al 2017b), transcriptome (Schwientek et al 2013;Schwientek et al 2014), proteome (Wendler et al 2013, Wendler et al 2015aOrtseifen 2016), and acarbose-related metabolites (Wendler et al 2014;Ortseifen 2016) was gained, leading to novel interesting targets, like Cgt.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Two transmembrane segments were predicted in Cgt by analyses of Wendler et al (2015a), of which one is located within the N-terminal signal peptide. The second was assumed to be required for a multimerization (Wendler et al 2015a). Hints for such oligomerization were given by the pattern in 2D-gel proteome analysis (Ortseifen 2016;Wendler et al 2013) as well as by the fact that Cgt was completely absent from the proteome of the enriched membrane and membrane shaving fraction (Wendler et al 2015a).…”

Section: Introductionmentioning

confidence: 99%

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Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110

Schaffert

Schneiker-Bekel

Gierhake

et al. 2020

Appl Microbiol Biotechnol

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Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant Δcgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8-16% higher product titers when grown in maltose-containing medium.

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“…SE50/110 was reported, which has a 9.24-Mb circular chromosome hosting 8270 putative protein coding sequences (CDSs) with a G+C content of 71.32% [18] , [19] . Comparative transcriptomics and proteomics were also applied to evaluate the different transcription and expression of acb cluster and other genes of SE50/110 cultivated in different media [20] , [21] , [22] , [23] . The abundant omics information would provide the potential targets for metabolic engineering of SE50/110.…”

Section: Introductionmentioning

confidence: 99%

Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp. SE50/110

Zhao

Xie

Peng

et al. 2017

Synthetic and Systems Biotechnology

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The α-glucosidase inhibitor acarbose is commercially produced by Actinoplanes sp. and used as a potent drug in the treatment of type-2 diabetes. In order to improve the yield of acarbose, an efficient genetic manipulation system for Actinoplanes sp. was established. The conjugation system between E. coli carrying ØC31-derived integrative plasmids and the mycelia of Actinoplanes sp. SE50/110 was optimized by adjusting the parameters of incubation time of mixed culture (mycelia and E. coli), quantity of recipient cells, donor-to-recipient ratio and the concentration of MgCl2, which resulted in a high conjugation efficiency of 29.4%. Using this integrative system, a cloned acarbose biosynthetic gene cluster was introduced into SE50/110, resulting in a 35% increase of acarbose titer from 2.35 to 3.18 g/L. Alternatively, a pIJ101-derived replicating plasmid combined with the counter-selection system CodA(sm) was constructed for gene inactivation, which has a conjugation frequency as high as 0.52%. Meanwhile, almost all 5-flucytosine-resistant colonies were sensitive to apramycin, among which 75% harbored the successful deletion of targeted genes. Using this replicating vector, the maltooligosyltrehalose synthase gene treY responsible for the accumulation of component C was inactivated, and component C was eliminated as detected by LC-MS. Based on an efficient genetic manipulation system, improved acarbose production and the elimination of component C in our work paved a way for future rational engineering of the acarbose-producing strains.

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Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster

Cited by 18 publications

References 138 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

Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110

Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp. SE50/110

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