Classification of three corynebacterial strains isolated from a small paddock in North Rhine-Westphalia: proposal of Corynebacterium kalinowskii sp. nov., Corynebacterium comes sp. nov. and Corynebacterium occultum sp. nov.

Three novel corynebacterial species were isolated from soil sampled at a paddock in Vilsendorf, North Rhine-Westphalia, Germany. The strains were coccoid or irregular rod-shaped, catalase-positive and pale white to yellow-orange in colour. By whole genome sequencing and comparison of the 16S rRNA genes as well as the whole genome structure, it was shown that all three strains represent novel species of the family Corynebacteriaceae, order Corynebacteriales, class Actinobacteria. This project describes the isolation, identification, sequencing, and phenotypic characterization of the three novel Corynebacterium species. We propose the names Corynebacterium kalinowskii sp. nov. (DSM 110639T=LMG 31801T), Corynebacterium comes sp. nov. (DSM 110640T=LMG 31802T), and Corynebacterium occultum sp. nov. (DSM 110642T=LMG 31803T).

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

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.

Comparative transcription profiling of two fermentation cultures of Xanthomonas campestris pv. campestris B100 sampled in the growth and in the stationary phase.

The ɣ-proteobacterium Xanthomonas campestris pv. campestris (Xcc) is the producer of the biopolymer xanthan, a polysaccharide which is used as a thickener in numerous industrial applications. In this study, we present a global transcriptome profiling of two Xcc strain B100 cultures obtained from fermentation during the growth phase and the subsequent stationary phase associated with xanthan biosynthesis. During the xanthan production phase, highly abundant transcripts belonged to genes encoding for small RNAs, glycogen biosynthesis, and xanthan export. A total of 1850 (40%) genes were differentially transcribed during the stationary phase where 924 were transcriptionally up-regulated and 926 genes were down-regulated. An overview of differentially transcribed genes includes a significant down-regulation of genes involved in transcription, translation, and amino acid biosynthesis pathways. A group of up-regulated genes was involved in cellular response against oxidative stress, such as those coding for superoxide dismutase and catalase. Genes encoding enzymes involved in nucleotide sugar precursor synthesis of xanthan biosynthesis, such as xanA, galU, and ugd, exhibited a transcription pattern that did not change during the growth and stationary phase. Regarding the transcription pattern of the gum gene cluster that govern xanthan biosynthesis, a significant up-regulation of the genes gumB, gumC, and gumD was observed, while the transcript pools of the genes gumG, gumH, gumI, and gumJ were reduced and those of genes gumE, gumF, gumK, gumL, and gumM remained un-changed during the stationary phase compared to the growth phase. The obtained data represents the first analysis of gene expression patterns under xanthan production conditions and provides the bases for future studies aiming at enhancing xanthan yield.

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

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.

Comprehensive Proteome Profiling of a Xanthomonas campestris pv. Campestris B100 Culture Grown in Minimal Medium with a Specific Focus on Nutrient Consumption and Xanthan Biosynthesis.

Xanthan, a bacterial polysaccharide, is widespread in industrial applications, particularly as a food additive. However, little is known about the process of xanthan synthesis on the proteome level, even though Xanthomonas campestris is frequently used for xanthan fermentation. A label-free LC-MS/MS method was employed to study the protein changes during xanthan fermentation in minimal medium. According to the reference database, 2416 proteins were identified, representing 54.75 % of the proteome. The study examined changes in protein abundances concerning the growth phase and xanthan productivity. Throughout the experiment, changes in nitrate concentration appeared to affect the abundance of most proteins involved in nitrogen metabolism, except Gdh and GlnA. Proteins involved in sugar nucleotide metabolism stay unchanged across all growth phases. Apart from GumD, GumB, and GumC, the gum proteins showed no significant changes throughout the experiment. GumD, the first enzyme in the assembly of the xanthan-repeating unit, peaked during the early stationary phase but decreased during the late stationary phase. GumB and GumC, which are involved in exporting xanthan, increased significantly during the stationary phase. This study suggests that a potential bottleneck for xanthan productivity does not reside in the abundance of proteins directly involved in the synthesis pathways.

Analysis of Gum proteins involved in xanthan biosynthesis throughout multiple cell fractions in a "single-tube".

Xanthomonas is a phytopathogenic bacterium and of industrial interest due to its capability to produce xanthan, used as a thickener and emulsifier in the food and non-food industry. Until now, proteome analyses of Xcc lacking a detailed view on the proteins involved in xanthan biosynthesis. The proteins involved in the biosynthesis of this polysaccharide are located near, in or at the cell membrane. This study aims to establish a robust and rapid protocol for a comprehensive proteome analysis of Xcc strains, without the need to isolate different cell fractions. Therefore, a method for the analysis of the whole cell proteome was compared to the isolation of specific fractions regarding the total number of identified proteins, the overlap, and the differences between the approaches. The whole cell proteome analysis with extended peptide separation methods resulted in more than 3254 identified proteins covering 73.1% of the whole proteome. The protocol was used to study xanthan production in a label-free quantification approach. Expression profiles of 8 Gum proteins were compared between the stationary and logarithmic growth phase. Differential expression levels within the operon structure indicate a complex regulatory mechanism for xanthan biosynthesis. Data are available via ProteomeXchange with identifier PXD027261. SIGNIFICANCE: Bacteria are metabolite factories with a wide variety of natural products. Thus, proteome analyses play a crucial role to understand the biological processes within a cell behind the biosynthesis of those metabolites. Proteins involved in the biosynthesis of secreted products are often organised on, in or around the membrane allowing metabolite channelling. Experiments targeting those biosynthesis pathways on protein level often require the analysis of multiple cell fractions like cytosolic, inner, and outer membrane. This is time consuming and demands different protocols. The protocol presented here is a rapid and robust solution to study biosynthetic pathways of biological or biotechnological interest in a single approach on protein level, where gene products are partitioned across multiple cell fractions. The use of a single method also simplifies the comparison of different experiments, for example, production vs. nonproduction conditions.

Two Flagellar mutants of Xanthomonas campestris are characterized by enhanced xanthan production and higher xanthan viscosity.

Xanthomonas campestris strains are used world-wide for the production of the industrially important exopolysaccharide xanthan. The high industrial relevance of xanthan can be explained by its extraordinary qualities as rheological control agent in aqueous systems and by its stabilizing properties in suspensions and emulsions. The phytopathogen Xanthomonas campestris is a motile bacterium with one polar flagellum. The flagellum is a cost intensive structure, in terms of energy and building block consumption. Based on the assumption that inhibition of the flagellar biosynthesis and related proton driven motility might be beneficial for the xanthan production in Xcc, two genes (fliC and fliM) were mutated to inhibit the motility. Both mutants Xcc JBL007 fliC- and Xcc JBL007 fliM- showed an increased xanthan production. Remarkably, the produced xanthan from both mutants showed enhanced rheological properties. While the chemical composition of the produced xanthan of the initial and both mutant strains did not change, notable differences in persistence length could be measured via atomic force microscopy. Results presented in this study demonstrate the possibility to further improve the xanthan production by Xcc through rational strain design.

Microfluidics for Biotechnology: Bridging Gaps to Foster Microfluidic Applications.

Microfluidics and novel lab-on-a-chip applications have the potential to boost biotechnological research in ways that are not possible using traditional methods. Although microfluidic tools were increasingly used for different applications within biotechnology in recent years, a systematic and routine use in academic and industrial labs is still not established. For many years, absent innovative, ground-breaking and "out-of-the-box" applications have been made responsible for the missing drive to integrate microfluidic technologies into fundamental and applied biotechnological research. In this review, we highlight microfluidics' offers and compare them to the most important demands of the biotechnologists. Furthermore, a detailed analysis in the state-of-the-art use of microfluidics within biotechnology was conducted exemplarily for four emerging biotechnological fields that can substantially benefit from the application of microfluidic systems, namely the phenotypic screening of cells, the analysis of microbial population heterogeneity, organ-on-a-chip approaches and the characterisation of synthetic co-cultures. The analysis resulted in a discussion of potential "gaps" that can be responsible for the rare integration of microfluidics into biotechnological studies. Our analysis revealed six major gaps, concerning the lack of interdisciplinary communication, mutual knowledge and motivation, methodological compatibility, technological readiness and missing commercialisation, which need to be bridged in the future. We conclude that connecting microfluidics and biotechnology is not an impossible challenge and made seven suggestions to bridge the gaps between those disciplines. This lays the foundation for routine integration of microfluidic systems into biotechnology research procedures.

Regulatory associations between the metabolism of sulfur-containing amino acids and xanthan biosynthesis in Xanthomonas campestris pv. campestris B100.

The γ-proteobacterium Xanthomonas campestris pv. campestris (Xcc) B100 synthesizes the exopolysaccharide xanthan, a commercially relevant thickening agent produced commonly by industrial scale fermentation. This work was inspired by the observation that methionine is an inhibitor of xanthan formation in growth experiments. Therefore, the global effects of methionine supplementation were characterized through cultivation experiments, genome-wide microarray hybridizations and qRT-PCR. Specific pull down of DNA-binding proteins by using the intergenic regions upstream of xanA, gumB and gumD led to the identification of six transcriptional regulators, among them the LysR-family transcriptional regulator CysB. An insertion mutant of this gene was analyzed by growth experiments, microarray experiments and qRT-PCR. Based on our experimental data, we developed a model that describes the methionine-dependent co-regulation of xanthan and sulfur-containing compounds in Xanthomonas. These data substantially contribute to better understand the impact of methionine as a compound in xanthan production media used in industrial fermentations.

The complete genome sequence of the actinobacterium Streptomyces glaucescens GLA.O (DSM 40922) carrying gene clusters for the biosynthesis of tetracenomycin C, 5`-hydroxy streptomycin, and acarbose.

The secondary metabolite acarbose is used worldwide in the clinical treatment of diabetes mellitus type 2 patients. Acarbose is a - glucosidase inhibitor and supports patients to control their blood glucose as well as their serum insulin levels. The secondary metabolite is produced by strains of the class Actinobacteria, in particular from Actinoplanes sp. SE50/110, which is a progenitor of today`s production strains. Moreover, secondary metabolite clusters could also be identified in Streptomyces coelicoflavus ZG0656 as well as Streptomyces glaucescens GLA.O. In this study, the genome S. glaucescens GLA.O with focus on the acarbose biosynthesis cluster (gac-cluster) was analyzed. First, the tetracenomycin C and the 5`-hydroxy streptomycin gene clusters could be described completely. Then the gac gene region in S. glaucescens GLA.O is compared to the other known biosynthesis gene cluster. In comparison to Actinoplanes sp. SE50/110 the gac-cluster showed structural variances, like the missing homolog of the glycosyltransferase AcbD in the whole genome of S. glaucescens GLA.O. Due to the lack of the glycosyltransferase, it was of particular interest whether additional acarviose metabolites other than acarbose could be formed. For detection of acarviose metabolites biosynthesis the supernatant of S. glaucescens GLA.O grown in starch supplemented complex media was harvested at 72 and 96 hours. Although a homolog of the known glycosyltransferase is absent, the LC-MS-supported analysis revealed that a spectrum of acarviose metabolites was formed.

Genome improvement of the acarbose producer Actinoplanes sp. SE50/110 and annotation refinement based on RNA-seq analysis.

Actinoplanes sp. SE50/110 is the natural producer of acarbose, which is used in the treatment of diabetes mellitus type II. However, until now the transcriptional organization and regulation of the acarbose biosynthesis are only understood rudimentarily. The genome sequence of Actinoplanes sp. SE50/110 was known before, but was resequenced in this study to remove assembly artifacts and incorrect base callings. The annotation of the genome was refined in a multi-step approach, including modern bioinformatic pipelines, transcriptome and proteome data. A whole transcriptome RNA-seq library as well as an RNA-seq library enriched for primary 5'-ends were used for the detection of transcription start sites, to correct tRNA predictions, to identify novel transcripts like small RNAs and to improve the annotation through the correction of falsely annotated translation start sites. The transcriptome data sets were also applied to identify 31 cis-regulatory RNA structures, such as riboswitches or RNA thermometers as well as three leaderless transcribed short peptides found in putative attenuators upstream of genes for amino acid biosynthesis. The transcriptional organization of the acarbose biosynthetic gene cluster was elucidated in detail and fourteen novel biosynthetic gene clusters were suggested. The accurate genome sequence and precise annotation of the Actinoplanes sp. SE50/110 genome will be the foundation for future genetic engineering and systems biology studies.

Corynebacterium glutamicum ggtB encodes a functional γ-glutamyl transpeptidase with γ-glutamyl dipeptide synthetic and hydrolytic activity.

In this work the role of γ-glutamyl transpeptidase in the metabolism of γ-glutamyl dipeptides produced by Corynebacterium glutamicum ATCC 13032 was studied. The enzyme is encoded by the gene ggtB (cg1090) and synthesized as a 657 amino acids long preprotein. Gamma-glutamyl transpeptidase activity was found to be associated with intact cells of C. glutamicum and was abolished upon deletion of ggtB. Bioinformatic analysis indicated that the enzyme is a lipoprotein and is attached to the outer side of the cytoplasmic membrane. Biochemical parameters of recombinant GgtB were determined using the chromogenic substrate γ-glutamyl-p-nitroanilide. Highest activity of the enzyme was measured in sodium bicarbonate buffer at pH 9.6 and 45°C. The KM value was 123µM. GgtB catalyzed the concentration-dependent synthesis and hydrolysis of γ-glutamyl dipeptides and showed strong glutaminase activity. The intracellular concentrations of five γ-glutamyl dipeptides (γ-Glu-Glu, γ-Glu-Gln, γ-Glu-Val, γ-Glu-Leu, γ-Glu-Met) were determined by HPLC-MS and ranged from 0.15 to 0.4mg/g CDW after exponential growth in minimal media. Although deletion and overexpression of ggtB had significant effects on intracellular dipeptide concentrations, it was neither essential for biosynthesis nor catabolism of these dipeptides in vivo.

An integrated metagenome and -proteome analysis of the microbial community residing in a biogas production plant.

To study the metaproteome of a biogas-producing microbial community, fermentation samples were taken from an agricultural biogas plant for microbial cell and protein extraction and corresponding metagenome analyses. Based on metagenome sequence data, taxonomic community profiling was performed to elucidate the composition of bacterial and archaeal sub-communities. The community's cytosolic metaproteome was represented in a 2D-PAGE approach. Metaproteome databases for protein identification were compiled based on the assembled metagenome sequence dataset for the biogas plant analyzed and non-corresponding biogas metagenomes. Protein identification results revealed that the corresponding biogas protein database facilitated the highest identification rate followed by other biogas-specific databases, whereas common public databases yielded insufficient identification rates. Proteins of the biogas microbiome identified as highly abundant were assigned to the pathways involved in methanogenesis, transport and carbon metabolism. Moreover, the integrated metagenome/-proteome approach enabled the examination of genetic-context information for genes encoding identified proteins by studying neighboring genes on the corresponding contig. Exemplarily, this approach led to the identification of a Methanoculleus sp. contig encoding 16 methanogenesis-related gene products, three of which were also detected as abundant proteins within the community's metaproteome. Thus, metagenome contigs provide additional information on the genetic environment of identified abundant proteins.

Genetic engineering in Actinoplanes sp. SE50/110 - development of an intergeneric conjugation system for the introduction of actinophage-based integrative vectors.

The α-glucosidase inhibitor acarbose is used for treatment of diabetes mellitus type II, and is manufactured industrially with overproducing derivatives of Actinoplanes sp. SE50/110, reportedly obtained by conventional mutagenesis. Despite of high industrial significance, only limited information exists regarding acarbose metabolism, function and regulation of these processes, due to the absence of proper genetic engineering methods and tools developed for this strain. Here, a basic toolkit for genetic engineering of Actinoplanes sp. SE50/110 was developed, comprising a standardized protocol for a DNA transfer through Escherichia coli-Actinoplanes intergeneric conjugation and applied for the transfer of ϕC31, ϕBT1 and VWB actinophage-based integrative vectors. Integration sites, occurring once per genome for all vectors, were sequenced and characterized for the first time in Actinoplanes sp. SE50/110. Notably, in case of ϕC31 based vector pSET152, the integration site is highly conserved, while for ϕBT1 and the VWB based vectors pRT801 and pSOK804, respectively, no sequence similarities to those in other bacteria were detected. The studied plasmids were proven to be stable and neutral with respect to strain morphology and acarbose production, enabling future use for genetic manipulations of Actinoplanes sp. SE50/110. To further broaden the spectrum of available tools, a GUS reporter system, based on the pSET152 derived vector, was also established in Actinoplanes sp. SE50/110.

Comparative proteome analysis of Actinoplanes sp. SE50/110 grown with maltose or glucose shows minor differences for acarbose biosynthesis proteins but major differences for saccharide transporters.

Actinoplanes sp. SE50/110 is known for the production of the α-glucosidase inhibitor and anti-diabetic drug acarbose. Acarbose (acarviosyl-maltose) is produced as the major product when the bacterium is grown in medium with maltose, while acarviosyl-glucose is the major product when glucose is the sole carbon source in the medium. In this study, a state-of-the-art proteomics approach was applied combining subcellular fractionation, in vivo metabolic labeling and shotgun mass spectrometry to analyze differences in the proteome of Actinoplanes sp. SE50/110 cultures grown in minimal medium containing either maltose or glucose as the sole carbon source. To study proteins in distinct subcellular locations, a cytosolic, an enriched membrane, a membrane shaving and an extracellular fraction were included in the analysis. Altogether, quantitative proteome data was obtained for 2497 proteins representing about 30% of the ca. 8270 predicted proteins of Actinoplanes sp. SE50/110. When comparing protein quantities of maltose- to glucose-grown cultures, differences were observed for saccharide transport and metabolism proteins, whereas differences for acarbose biosynthesis gene cluster proteins were almost absent. The maltose-inducible α-glucosidase/maltase MalL as well as the ABC-type saccharide transporters AglEFG, MalEFG and MstEAF had significantly higher quantities in the maltose growth condition. The only highly abundant saccharide transporter in the glucose condition was the monosaccharide transporter MstEAF, which may indicate that MstEAF is the major glucose importer. Taken all findings together, the previously observed formation of acarviosyl-maltose and acarviosyl-glucose is more closely connected to the transport of saccharides than to a differential expression of the acarbose gene cluster. BIOLOGICAL SIGNIFICANCE: Diabetes is a global pandemic accounting for about 11% of the worldwide healthcare expenditures (>600 billion US dollars) and is projected to affect 592 million people by 2035 (www.idf.org). Whether Actinoplanes sp. SE50/110 produces type 2 diabetes drug acarbose (acarviosyl-maltose) or another acarviose metabolite such as acarviosyl-glucose as the major product depends on the offered carbon source. The differences observed in this proteome in this study suggest that the differences in the formation of acarviosyl-maltose and acarviosyl-glucose are more closely connected to the transport of saccharides than to a differential expression of the acarbose gene cluster. In addition, the present study provides a comprehensive overview of the proteome of Actinoplanes sp. SE50/110.

Complete genome sequence of the actinobacterium Streptomyces glaucescens GLA.O (DSM 40922) consisting of a linear chromosome and one linear plasmid.

Here we report the complete and finished genome sequence of Streptomyces glaucescens GLA.O (DSM 40922), a natural producer of the alpha-glucosidase inhibitor acarbose, which is used in the treatment of type-2 diabetes mellitus. The genome of S. glaucescens GLA.O consists of two replicons, the chromosome with a size of 7,453,200bp and a G+C content of 73.0% as well as a plasmid named pSglau1 with a size of 170,574bp and a G+C content of 69.06%.

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

Acarbose is an α-glucosidase inhibitor produced by Actinoplanes sp. SE50/110 that is medically important due to its application in the treatment of type2 diabetes. In this work, a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out to determine the location of proteins of the acarbose (acb) and the putative pyochelin (pch) biosynthesis gene cluster. Therefore, a comprehensive state-of-the-art proteomics approach combining subcellular fractionation, shotgun proteomics and spectral counting to assess the relative abundance of proteins within fractions was applied. The analysis of four different proteome fractions (cytosolic, enriched membrane, membrane shaving and extracellular fraction) resulted in the identification of 1582 of the 8270 predicted proteins. All 22 Acb-proteins and 21 of the 23 Pch-proteins were detected. Predicted membrane-associated, integral membrane or extracellular proteins of the pch and the acb gene cluster were found among the most abundant proteins in corresponding fractions. Intracellular biosynthetic proteins of both gene clusters were not only detected in the cytosolic, but also in the enriched membrane fraction, indicating that the biosynthesis of acarbose and putative pyochelin metabolites takes place at the inner membrane. BIOLOGICAL SIGNIFICANCE: Actinoplanes sp. SE50/110 is a natural producer of the α-glucosidase inhibitor acarbose, a bacterial secondary metabolite that is used as a drug for the treatment of type 2 diabetes, a disease which is a global pandemic that currently affects 387 million people and accounts for 11% of worldwide healthcare expenditures (www.idf.org). The work presented here is the first comprehensive investigation of protein localization and abundance in Actinoplanes sp. SE50/110 and provides an extensive source of information for the selection of genes for future mutational analysis and other hypothesis driven experiments. The conclusion that acarbose or pyochelin family siderophores are synthesized at the inner side of the cytoplasmic membrane determined from this work, indicates that studying corresponding intermediates will be challenging. In addition to previous studies on the genome and transcriptome, the work presented here demonstrates that the next omic level, the proteome, is now accessible for detailed physiological analysis of Actinoplanes sp. SE50/110, as well as mutants derived from this and related species.

Carbon source dependent biosynthesis of acarviose metabolites in Actinoplanes sp. SE50/110.

In this work the biosynthesis of the type 2 diabetes mellitus therapeutic acarviosyl-maltose (acarbose) and related acarviose metabolites produced by Actinoplanes sp. SE50/110 was studied in liquid minimal medium supplemented with the defined carbon sources maltose, glucose, galactose or mixtures of maltose/glucose and maltose/galactose. Quantifying acarviosyl-maltose by HPLC and UV detection revealed that only cultures grown in maltose-containing minimal media produced acarviosyl-maltose in significant amounts. A qualitative analysis of the cytosolic and extracellular proteome for the presence of proteins from the acarbose biosynthesis gene cluster showed that these were not only synthesized in maltose-containing media, but also in media with glucose or galactose as the sole carbon source. A LC-MS-based detection method was applied to test the hypothesis that different acarviose metabolites are produced in media with maltose, glucose or galactose. The analysis revealed that a spectrum of acarviose metabolites (acarviose with 1-4 glucose equivalent units) was formed under all tested conditions. As expected, in maltose-containing minimal media acarviosyl-maltose was produced as the major component exceeding the remaining minor components by 2-3 orders of magnitude. In minimal medium supplemented with glucose acarviosyl-glucose was the major component, while in minimal medium with galactose no major component was present. Based on the results presented, a model for the intracellular biosynthesis of major and minor acarviose metabolites was developed.