The Experts below are selected from a list of 843 Experts worldwide ranked by ideXlab platform
Jörn Kalinowski - One of the best experts on this subject based on the ideXlab platform.
-
Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110
Applied Microbiology and Biotechnology, 2020Co-Authors: Lena Schaffert, Julian Droste, Marcus Persicke, Alfred Puhler, Susanne Schneiker-bekel, Jessica Gierhake, Winfried Rosen, Jörn KalinowskiAbstract: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 ∆ cgt , 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.
-
essentiality of the maltase amle in maltose utilization and its transcriptional regulation by the repressor amlr in the acarbose producing bacterium Actinoplanes sp se50 110
Frontiers in Microbiology, 2019Co-Authors: Lena Schaffert, Susanne Schneikerbekel, Julian Droste, David Brandt, Marcus Persicke, Alfred Puhler, Tobias Busche, Saskiamichelle Dymek, Jörn KalinowskiAbstract:Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. Bioinformatic analysis located a putative maltase gene amlE (ACSP50_2474, previously named malL (Wendler et al., 2015a)), in an operon with an upstream PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. Targeted gene deletion mutants of amlE and amlR were constructed by use of CRISPR/Cas9 technology. By growth experiments and functional assays of ∆amlE, we could show that AmlE is essential for maltose utilization in Actinoplanes sp. SE50/110. Neither a gene encoding a maltose phosphorylase (MalP) nor MalP enzyme activity were detected in the wild type. By this, the maltose/maltodextrin system appears to be fundamentally different from other described prokaryotic systems. By sequence similarity analysis and functional assays from the species Streptomyces lividans TK23, S. coelicolor A3(2) and S. glaucescens GLA.O, first hints for a widespread lack of MalP and presence of AmlE in the class Actinobacteria were given. Transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ∆amlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ∆amlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes. We ascribe these effects to ACSP50_2473, which is localized downstream of amlE and presumably involved in the metabolism of the second messenger c-di-GMP. It can be assumed, that maltose does not only represent the most important carbon source of Actinoplanes sp. SE50/110, but that its metabolism is coupled to the nucleotide messenger system of c-di-GMP.
-
The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110
'Springer Science and Business Media LLC', 2017Co-Authors: Timo Wolf, Julian Droste, Alfred Puhler, Susanne Schneiker-bekel, Tetiana Gren, Vera Ortseifen, Till Zemke, Jörn KalinowskiAbstract:Abstract Background Acarbose 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. Results The 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. Conclusions This 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
-
RESEARCH ARTICLE Open Access The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110
2016Co-Authors: Patrick Schwientek, Jörn Kalinowski, Udo F Wehmeier, Rafael Szczepanowski, Christian Rückert, Andreas Klein, Klaus Selber, Jens Stoye, Alfred PuhlerAbstract:Background: Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. Results: Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32 % and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence
-
complete genome sequence of the actinobacterium Actinoplanes friuliensis hag 010964 producer of the lipopeptide antibiotic friulimycin
Journal of Biotechnology, 2014Co-Authors: Christian Rückert, Alfred Puhler, D Schwartz, Rafael Szczepanowski, Andreas Albersmeier, Alexander Goesmann, Nicole Fischer, Anne Steinkamper, R Biener, Jörn KalinowskiAbstract:Actinoplanes friuliensis HAG 010964 (DSM 7358) was isolated from a soil sample from the Friuli region in Italy and characterized as a producer of the antibiotic friulimycin. The complete genome sequence includes genomic information of secondary metabolite biosynthesis and of its lifestyle. Genbank/EMBL/DDBJ Accession Nr: CP006272 (chromosome).
Kalinowski Jörn - One of the best experts on this subject based on the ideXlab platform.
-
pSETT4, an Improved φC31-Based Integrative Vector System for Actinoplanes sp. SE50/110
'American Society for Microbiology', 2020Co-Authors: Schaffert Lena, Jacob Lucas, Schneiker-bekel Susanne, Persicke Marcus, März Camilla, Rückert Christian, Pühler Alfred, Kalinowski JörnAbstract:Schaffert L, Jacob L, Schneiker-Bekel S, et al. pSETT4, an Improved φC31-Based Integrative Vector System for Actinoplanes sp. SE50/110. Microbiology Resource Announcements. 2020;9(39): e00596-20.ABSTRACT The pSETT4 vector integrates into the Actinoplanes sp. SE50/110 chromosome via the bacteriophage φC31 integrase and allows cloning of a gene of interest by Golden Gate assembly (BsaI). T4 terminators surround the expression cassette to isolate the transcriptional unit and to prevent antisense transcription. The system can be used in other Actinomycetales by exchanging the promoter.
-
Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110
'Springer Science and Business Media LLC', 2020Co-Authors: Schaffert Lena, Schneiker-bekel Susanne, Persicke Marcus, Pühler Alfred, Gierhake Jessica, Droste Julian, Rosen Winfried, Kalinowski JörnAbstract:Schaffert L, Schneiker-Bekel S, Gierhake J, et al. Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110. Applied Microbiology and Biotechnology. 2020;104:5395–5408.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 ∆cgt, 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
-
A maltose-regulated large genomic region is activated by the transcriptional regulator MalT in Actinoplanes sp. SE50/110
'Springer Science and Business Media LLC', 2020Co-Authors: Droste Julian, Schaffert Lena, Schneiker-bekel Susanne, Pühler Alfred, Wolf Timo, Kulisch Martin, Kalinowski JörnAbstract:Droste J, Kulisch M, Wolf T, et al. A maltose-regulated large genomic region is activated by the transcriptional regulator MalT in Actinoplanes sp. SE50/110. Applied microbiology and biotechnology. 2020;104:9283–9294.Actinoplanes sp. SE50/110 is the industrially relevant producer of acarbose, which is used in the treatment of diabetes mellitus. Recent studies elucidated the expression dynamics in Actinoplanes sp. SE50/110 during growth. From these data, we obtained a large genomic region (ACSP50_3900 to ACSP50_3950) containing 51 genes, of which 39 are transcribed in the same manner. These co-regulated genes were found to be stronger transcribed on maltose compared with glucose as a carbon source. The transcriptional regulator MalT was identified as an activator of this maltose-regulated large genomic region (MRLGR). Since most of the genes are poorly annotated, the function of this region is farther unclear. However, comprehensive BLAST analyses indicate similarities to enzymes involved in amino acid metabolism. We determined a conserved binding motif of MalT overlapping the -35 promoter region of 17 transcription start sites inside the MRLGR. The corresponding sequence motif 5'-TCATCC-5nt-GGATGA-3' displays high similarities to reported MalT binding sites in Escherichia coli and Klebsiella pneumoniae, in which MalT is the activator of mal genes. A malT deletion and an overexpression mutant were constructed. Differential transcriptome analyses revealed an activating effect of MalT on 40 of the 51 genes. Surprisingly, no gene of the maltose metabolism is affected. In contrast to many other bacteria, MalT is not the activator of mal genes in Actinoplanes sp. SE50/110. Finally, the MRLGR was found partly in other closely related bacteria of the family Micromonosporaceae. Even the conserved MalT binding site was found upstream of several genes inside of the corresponding regions. KEY POINTS : MalT is the maltose-dependent activator of a large genomic region in ACSP50_WT. The consensus binding motif is similar to MalT binding sites in other bacteria. MalT is not the regulator of genes involved in maltose metabolism in ACSP50_WT
-
The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase.
'Springer Science and Business Media LLC', 2020Co-Authors: Droste Julian, Schaffert Lena, Schneiker-bekel Susanne, Persicke Marcus, Pühler Alfred, Ortseifen Vera, Kalinowski JörnAbstract:Droste J, Ortseifen V, Schaffert L, et al. The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase. BMC genomics. 2020;21: 818.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 an expected metabolic switch from primary to secondary metabolism during transition phase, we observed a continuously decreasing transcript abundance of all acarbose biosynthetic genes from the early growth phase until stationary phase, 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
-
Essentiality of the Maltase AmlE in Maltose Utilization and Its Transcriptional Regulation by the Repressor AmlR in the Acarbose-Producing Bacterium Actinoplanes sp. SE50/110
'Frontiers Media SA', 2019Co-Authors: Schaffert Lena, Schneiker-bekel Susanne, Persicke Marcus, Pühler Alfred, Droste Julian, Dymek Saskia, Busche Tobias, Brandt David, Kalinowski JörnAbstract:Schaffert L, Schneiker-Bekel S, Dymek S, et al. Essentiality of the Maltase AmlE in Maltose Utilization and Its Transcriptional Regulation by the Repressor AmlR in the Acarbose-Producing Bacterium Actinoplanes sp. SE50/110. Frontiers in Microbiology. 2019;10: 2448.Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. Bioinformatic analysis located a putative maltase gene amlE (ACSP50_2474, previously named malL; Wendler et al., 2015a), in an operon with an upstream PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. Targeted gene deletion mutants of amlE and amlR were constructed by use of the CRISPR/Cas9 technology. By growth experiments and functional assays of ΔamlE, we could show that AmlE is essential for the maltose utilization in Actinoplanes sp. SE50/110. Neither a gene encoding a maltose phosphorylase (MalP) nor MalP enzyme activity were detected in the wild type. By this, the maltose/maltodextrin system appears to be fundamentally different from other described prokaryotic systems. By sequence similarity analysis and functional assays from the species Streptomyces lividans TK23, S. coelicolor A3(2) and S. glaucescens GLA.O, first hints for a widespread lack of MalP and presence of AmlE in the class Actinobacteria were given. Transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ΔamlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ΔamlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes. We ascribe these effects to ACSP50_2473, which is localized downstream of amlE and presumably involved in the metabolism of the second messenger c-di-GMP. It can be assumed, that maltose does not only represent the most important carbon source of Actinoplanes sp. SE50/110, but that its metabolism is coupled to the nucleotide messenger system of c-di-GMP
Alfred Puhler - One of the best experts on this subject based on the ideXlab platform.
-
Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110
Applied Microbiology and Biotechnology, 2020Co-Authors: Lena Schaffert, Julian Droste, Marcus Persicke, Alfred Puhler, Susanne Schneiker-bekel, Jessica Gierhake, Winfried Rosen, Jörn KalinowskiAbstract: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 ∆ cgt , 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.
-
essentiality of the maltase amle in maltose utilization and its transcriptional regulation by the repressor amlr in the acarbose producing bacterium Actinoplanes sp se50 110
Frontiers in Microbiology, 2019Co-Authors: Lena Schaffert, Susanne Schneikerbekel, Julian Droste, David Brandt, Marcus Persicke, Alfred Puhler, Tobias Busche, Saskiamichelle Dymek, Jörn KalinowskiAbstract:Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. Bioinformatic analysis located a putative maltase gene amlE (ACSP50_2474, previously named malL (Wendler et al., 2015a)), in an operon with an upstream PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. Targeted gene deletion mutants of amlE and amlR were constructed by use of CRISPR/Cas9 technology. By growth experiments and functional assays of ∆amlE, we could show that AmlE is essential for maltose utilization in Actinoplanes sp. SE50/110. Neither a gene encoding a maltose phosphorylase (MalP) nor MalP enzyme activity were detected in the wild type. By this, the maltose/maltodextrin system appears to be fundamentally different from other described prokaryotic systems. By sequence similarity analysis and functional assays from the species Streptomyces lividans TK23, S. coelicolor A3(2) and S. glaucescens GLA.O, first hints for a widespread lack of MalP and presence of AmlE in the class Actinobacteria were given. Transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ∆amlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ∆amlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes. We ascribe these effects to ACSP50_2473, which is localized downstream of amlE and presumably involved in the metabolism of the second messenger c-di-GMP. It can be assumed, that maltose does not only represent the most important carbon source of Actinoplanes sp. SE50/110, but that its metabolism is coupled to the nucleotide messenger system of c-di-GMP.
-
The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110
'Springer Science and Business Media LLC', 2017Co-Authors: Timo Wolf, Julian Droste, Alfred Puhler, Susanne Schneiker-bekel, Tetiana Gren, Vera Ortseifen, Till Zemke, Jörn KalinowskiAbstract:Abstract Background Acarbose 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. Results The 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. Conclusions This 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
-
RESEARCH ARTICLE Open Access The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110
2016Co-Authors: Patrick Schwientek, Jörn Kalinowski, Udo F Wehmeier, Rafael Szczepanowski, Christian Rückert, Andreas Klein, Klaus Selber, Jens Stoye, Alfred PuhlerAbstract:Background: Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. Results: Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32 % and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence
-
complete genome sequence of the actinobacterium Actinoplanes friuliensis hag 010964 producer of the lipopeptide antibiotic friulimycin
Journal of Biotechnology, 2014Co-Authors: Christian Rückert, Alfred Puhler, D Schwartz, Rafael Szczepanowski, Andreas Albersmeier, Alexander Goesmann, Nicole Fischer, Anne Steinkamper, R Biener, Jörn KalinowskiAbstract:Actinoplanes friuliensis HAG 010964 (DSM 7358) was isolated from a soil sample from the Friuli region in Italy and characterized as a producer of the antibiotic friulimycin. The complete genome sequence includes genomic information of secondary metabolite biosynthesis and of its lifestyle. Genbank/EMBL/DDBJ Accession Nr: CP006272 (chromosome).
Pühler Alfred - One of the best experts on this subject based on the ideXlab platform.
-
pSETT4, an Improved φC31-Based Integrative Vector System for Actinoplanes sp. SE50/110
'American Society for Microbiology', 2020Co-Authors: Schaffert Lena, Jacob Lucas, Schneiker-bekel Susanne, Persicke Marcus, März Camilla, Rückert Christian, Pühler Alfred, Kalinowski JörnAbstract:Schaffert L, Jacob L, Schneiker-Bekel S, et al. pSETT4, an Improved φC31-Based Integrative Vector System for Actinoplanes sp. SE50/110. Microbiology Resource Announcements. 2020;9(39): e00596-20.ABSTRACT The pSETT4 vector integrates into the Actinoplanes sp. SE50/110 chromosome via the bacteriophage φC31 integrase and allows cloning of a gene of interest by Golden Gate assembly (BsaI). T4 terminators surround the expression cassette to isolate the transcriptional unit and to prevent antisense transcription. The system can be used in other Actinomycetales by exchanging the promoter.
-
A maltose-regulated large genomic region is activated by the transcriptional regulator MalT in Actinoplanes sp. SE50/110
'Springer Science and Business Media LLC', 2020Co-Authors: Droste Julian, Schaffert Lena, Schneiker-bekel Susanne, Pühler Alfred, Wolf Timo, Kulisch Martin, Kalinowski JörnAbstract:Droste J, Kulisch M, Wolf T, et al. A maltose-regulated large genomic region is activated by the transcriptional regulator MalT in Actinoplanes sp. SE50/110. Applied microbiology and biotechnology. 2020;104:9283–9294.Actinoplanes sp. SE50/110 is the industrially relevant producer of acarbose, which is used in the treatment of diabetes mellitus. Recent studies elucidated the expression dynamics in Actinoplanes sp. SE50/110 during growth. From these data, we obtained a large genomic region (ACSP50_3900 to ACSP50_3950) containing 51 genes, of which 39 are transcribed in the same manner. These co-regulated genes were found to be stronger transcribed on maltose compared with glucose as a carbon source. The transcriptional regulator MalT was identified as an activator of this maltose-regulated large genomic region (MRLGR). Since most of the genes are poorly annotated, the function of this region is farther unclear. However, comprehensive BLAST analyses indicate similarities to enzymes involved in amino acid metabolism. We determined a conserved binding motif of MalT overlapping the -35 promoter region of 17 transcription start sites inside the MRLGR. The corresponding sequence motif 5'-TCATCC-5nt-GGATGA-3' displays high similarities to reported MalT binding sites in Escherichia coli and Klebsiella pneumoniae, in which MalT is the activator of mal genes. A malT deletion and an overexpression mutant were constructed. Differential transcriptome analyses revealed an activating effect of MalT on 40 of the 51 genes. Surprisingly, no gene of the maltose metabolism is affected. In contrast to many other bacteria, MalT is not the activator of mal genes in Actinoplanes sp. SE50/110. Finally, the MRLGR was found partly in other closely related bacteria of the family Micromonosporaceae. Even the conserved MalT binding site was found upstream of several genes inside of the corresponding regions. KEY POINTS : MalT is the maltose-dependent activator of a large genomic region in ACSP50_WT. The consensus binding motif is similar to MalT binding sites in other bacteria. MalT is not the regulator of genes involved in maltose metabolism in ACSP50_WT
-
The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase.
'Springer Science and Business Media LLC', 2020Co-Authors: Droste Julian, Schaffert Lena, Schneiker-bekel Susanne, Persicke Marcus, Pühler Alfred, Ortseifen Vera, Kalinowski JörnAbstract:Droste J, Ortseifen V, Schaffert L, et al. The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase. BMC genomics. 2020;21: 818.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 an expected metabolic switch from primary to secondary metabolism during transition phase, we observed a continuously decreasing transcript abundance of all acarbose biosynthetic genes from the early growth phase until stationary phase, 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
-
Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110
'Springer Science and Business Media LLC', 2020Co-Authors: Schaffert Lena, Schneiker-bekel Susanne, Persicke Marcus, Pühler Alfred, Gierhake Jessica, Droste Julian, Rosen Winfried, Kalinowski JörnAbstract:Schaffert L, Schneiker-Bekel S, Gierhake J, et al. Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110. Applied Microbiology and Biotechnology. 2020;104:5395–5408.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 ∆cgt, 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
-
Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110
'Springer Science and Business Media LLC', 2019Co-Authors: Schaffert Lena, Schneiker-bekel Susanne, Persicke Marcus, März Camilla, Droste Julian, Rosen Winfried, Busche Tobias, Brandt David, Burkhardt Lisa, Pühler AlfredAbstract:Schaffert L, März C, Burkhardt L, et al. Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110. Microbial Cell Factories. 2019;18(1): 114.Background Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC. Results The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5′-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC-overexpression mutant. According to LC–MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis. Conclusion Development of an overexpression system for Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives
Lena Schaffert - One of the best experts on this subject based on the ideXlab platform.
-
Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110
Applied Microbiology and Biotechnology, 2020Co-Authors: Lena Schaffert, Julian Droste, Marcus Persicke, Alfred Puhler, Susanne Schneiker-bekel, Jessica Gierhake, Winfried Rosen, Jörn KalinowskiAbstract: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 ∆ cgt , 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.
-
essentiality of the maltase amle in maltose utilization and its transcriptional regulation by the repressor amlr in the acarbose producing bacterium Actinoplanes sp se50 110
Frontiers in Microbiology, 2019Co-Authors: Lena Schaffert, Susanne Schneikerbekel, Julian Droste, David Brandt, Marcus Persicke, Alfred Puhler, Tobias Busche, Saskiamichelle Dymek, Jörn KalinowskiAbstract:Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. Bioinformatic analysis located a putative maltase gene amlE (ACSP50_2474, previously named malL (Wendler et al., 2015a)), in an operon with an upstream PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. Targeted gene deletion mutants of amlE and amlR were constructed by use of CRISPR/Cas9 technology. By growth experiments and functional assays of ∆amlE, we could show that AmlE is essential for maltose utilization in Actinoplanes sp. SE50/110. Neither a gene encoding a maltose phosphorylase (MalP) nor MalP enzyme activity were detected in the wild type. By this, the maltose/maltodextrin system appears to be fundamentally different from other described prokaryotic systems. By sequence similarity analysis and functional assays from the species Streptomyces lividans TK23, S. coelicolor A3(2) and S. glaucescens GLA.O, first hints for a widespread lack of MalP and presence of AmlE in the class Actinobacteria were given. Transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ∆amlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ∆amlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes. We ascribe these effects to ACSP50_2473, which is localized downstream of amlE and presumably involved in the metabolism of the second messenger c-di-GMP. It can be assumed, that maltose does not only represent the most important carbon source of Actinoplanes sp. SE50/110, but that its metabolism is coupled to the nucleotide messenger system of c-di-GMP.
