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Our research mainly focuses on the model microorganisms Gluconobacter oxydans and Corynebacterium glutamicum. G. oxydans is an acetic acid bacterium (AAB) and used for the conversion of renewable carbon sources to industrially important chemicals by regioselective and stereoselective periplasmatic oxidations of sugars, sugar alcohols, sugar acids, alcohols and other substrates.

Strains of C. glutamicum are used in industrial biotechnology for amino acid production and in metabolic engineering approaches including new methods such as metabolite sensors and new biosynthesis pathways for improving and developing strains for various purposes including the access to new alternative carbon sources.

Our work includes studies on the AAB G. oxydans, for example to develop and establish regulatable and tunable expression systems in ABB and studies on C. glutamicum for basic and applied research including small non-coding RNAs and the development of chassis strains for production of new valuable products. We use Next Generation Sequencing including RNAseq and ChIP/ChAPseq approaches, DNA microarrays, LC/MS- based proteomics, HPLC analysis and further methods to study and analyse wild-type, engineered or mutagenized strains with respect to SNPs, genomic alterations, genome stability and global RNA, protein level changes, as well as metabolites.

For example, our expert Dipl. Ing. Astrid Wirtz develops, tests and implements methods for the analysis of low molecular weight substances by liquid chromatography (1290 Infinity I, 1260 Infinity II, 1200/1100 Agilent), performs bottom-up nano/microLC-coupled mass spectrometry (TTOF 6600, Sciex), and provides advice and guidance in such analytical methods. Feel free to contact Astrid and discuss your points with her.

Handling and analysis of all of these microbial (omics) data is pivotal and requires application of diverse software including bioinformatics tools and databases as well as specifically developed software tools and databases to obtain comprehensive genome-based data views and knowledge. Building and establishing reference genome-based knowledge databases for microbial genome, omics, phenotypic, regulation and related (meta) data is highly required.

Publications via ORCID

Gluconobacter oxydans

The α-proteobacterium G. oxydans is a Gram-negative acetic acid bacterium used for a broad range of industrial applications due to its ability to oxidize a great variety of carbohydrates in the periplasm. In biotechnological applications and basic research, timed production of proteins in bacterial cell culture for various purposes by inducible or regulatable expression of target genes is a standard method well established in many bacteria except in acetic acid bacteria.

See also our review:

Fricke P.M., Klemm A., Bott M., Polen T. (2021)
On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases.
Appl Microbiol Biotechnol 105(9):3423-3456
doi: 10.1007/s00253-021-11269-z

Also for G. oxydans, no systems were available that allowed regulatable target gene expression. Recently, we found that the L-arabinose-inducible ParaBAD promoter and the transcriptional regulator AraC from E. coli MC4100 perform very well in G. oxydans with a pBBR1MCS-5 backbone allowing up to 480-fold induction with good tunability from 0.1 to 1% L-arabinose, demonstrating superior functionality of an AraC-ParaBAD system in G. oxydans. This represents the first system for regulatable target gene expression in G. oxydans, which could potentially be used in other AAB.

Graphic Regulatable expression plasmid

Fricke P.M., Link T., Gätgens J., Sonntag C., Otto M., Bott M., Polen T. (2020)
A tunable l-arabinose-inducible expression plasmid for the acetic acid bacterium Gluconobacter oxydans.
Appl Microbiol Biotechnol 104(21):9267-9282
doi:10.1007/s00253-020-10905-4

Furthermore, recently we found with a pBBR1MCS-5-based TetR-Ptet system that inducible target gene expression based solely on de-repression of the heterologous target promoter can also perform extremely well in G. oxydans. With the pBBR1MCS-5-based plasmid, the anhydrotetracycline (ATc)-inducible promoter Ptet derived from the E. coli transposon Tn10 exhibited excellently tunable expression performance in an inducer concentration-dependent manner with maximal induction ratios up to more than 3,500-fold. This was due to extremely low basal reporter expression in the absence of an inducer, thus well repressed Ptet, and Ptet being very strong in G. oxydans.

Plasmid-based mNeonGreen fluorescence reporter protein expression was gradually inducible by increasing ATc concentrations and the induction was highly homogeneous according to FACS results:

Picture of FACS results

Fricke P.M., Lürkens M., Hünnefeld M., Sonntag C.K., Bott M., Davari M.D., Polen T. (2021)
Highly tunable TetR-dependent target gene expression in the acetic acid bacterium Gluconobacter oxydans.
Appl Microbiol Biotechnol 105(18):6835-6852
doi:10.1007/s00253-021-11473-x

In G. oxydans, the high periplasmic oxidation of substrates combined with a low carbon flux into the cytoplasmic metabolism having no genes encoding for phosphofructokinase, succinyl-CoA synthetase, and succinate dehydrogenase typically results in a low biomass yield which limits a broader range of industrial applications. Recently, heterologous genes were chromosomally integrated and expressed to complete the TCA cycle resulting in about 60% increased biomass yield (PMID 28484812).

Enforcing the cytoplasmic glucose catabolism in G. oxydans in such a way may cause suppressor mutations and structural variants affecting the genome stability. To check this we conducted a hybrid approach for genome sequencing by combining Oxford Nanopore’s MinION® sequencing for long reads and Illumina’s technology for short accurate reads. The genome sequence of the engineered G. oxydans strain was stable at least up to 70 generations of strain handling including process time in a controlled bioreactor. The long read data also revealed a novel 1420 bp transposon-flanked and ORF-containing sequence which was hitherto unknown in the G. oxydans 621H reference. Further analysis and genome sequencing showed that this region is also already present in G. oxydans 621H wild-type strains.

 

Schema of read mappingSchema of read mapping and de novo assembly at the GOX_RS13230-GOX_RS13235 locus. Illumina reads mapped to the G. oxydans 621H reference sequence indicate a structural variant (upper panel) that was resolved by de novo assembly using long nanopore reads spanning a novel 1420 bp transposon-flanked sequence insertion (lower panel). As a result of sequence analysis and ORF search GOX_RS13232 and GOX_RS13233 were added to the updated annotation of G. oxydans 621H.

Our data of G. oxydans 621H wild-type DNA from different resources also revealed in 73 annotated coding sequences about 91 uniform nucleotide differences including InDels. The results were used to improve the high quality genome reference for G. oxydans 621H which is available via ENA accession PRJEB18739.

Sequencing of primary transcriptomes of G. oxydans 621H revealed 2449 TSSs, which were classified according to their genomic context followed by identification of promoter and ribosome binding site motifs, analysis of 5´-UTRs including validation of predicted cis-regulatory elements and correction of start codons. 1144 (41%) of all genes were found to be expressed monocistronically, whereas 1634 genes were organized in 571 operons. Together, TSSs and whole transcriptome data revealed 18 novel intergenic, 328 intragenic, and 313 antisense transcripts.



TSS Data

The estimated mRNA half-lives in G. oxydans overall ranged mainly from 3 min to 25 min with a global mean of 5.7 min. The transcripts encoding GroES and GroEL required for proper protein folding ranked at the top among transcripts exhibiting both long half-lives and high abundance. The F-type H+-ATP synthase transcripts involved in energy metabolism ranked among the transcripts with the shortest mRNA half-lives. Also, RNAseq analysis revealed low expression levels for genes of the incomplete TCA cycle and also the mRNA half-lives of several of those were short and below the global mean.


mRNA decay analysis


The mRNA decay analysis also revealed an apparent instability of full-length 23S rRNA. Further analysis of the ribosome-associated rRNA revealed a 23S rRNA fragmentation pattern exhibiting new cleavage regions in 23S rRNAs which were previously not known.


Kranz A., Vogel A., Degner U., Kiefler I., Bott M., Usadel B., Polen T. (2017)
High precision genome sequencing of engineered Gluconobacter oxydans 621H by combining long nanopore and short accurate Illumina reads.
J Biotechnol pii: S0168-1656(17)30170-0
doi: 10.1016/j.jbiotec.2017.04.016

Kranz A., Busche T., Vogel A., Usadel B., Kalinowski J., Bott M., Polen T. (2018)
RNAseq analysis of alpha-proteobacterium Gluconobacter oxydans 621H.
BMC Genomics 19(1):24
doi: 10.1186/s12864-017-4415-x

Kranz A., Steinmann A., Degner U., Mengus-Kaya A., Matamouros S., Bott M.,
Polen T. (2018)
Global mRNA decay and 23S rRNA fragmentation in Gluconobacter oxydans 621H.
BMC Genomics 19(1):753
doi: 10.1186/s12864-018-5111-1

Small 6C RNA from Corynebacterium glutamicum

Small RNAs (sRNAs) have been detected in all three domains of life in unexpectedly large numbers. Such transcripts typically do not encode proteins and are therefore also referred to as non-coding RNAs. sRNAs serve various regulatory functions mediated by diverse mechanisms which also include the action of cis- and trans-acting sRNAs antisense to target RNAs and sRNAs binding to proteins.

The 6C RNA family is a widespread class of sRNAs conserved in Actinobacteria including the genera Mycobacterium, Corynebacterium, Frankia, Nocardia and Streptomyces, respectively. This RNA family, originally discovered by bioinformatics in S. coelicolor, has been termed 6C due to at least 6 conserved cytosine residues found in each of two loop regions of the conserved 80 nt stem-loops structure. The 6C RNA from C. glutamicum exhibits 8 consecutive cytosine residues in each of the two conserved loop regions.


Graphic of 6C RNAPredicted secondary structure of the 6C RNA from C. glutamicum which exhibits 8 consecutive cytosine residues in each of the two conserved loop regions.



We found the 6C RNA appears to be highly stable in C. glutamicum with a level of approximately 180 to 240 molcules per cell during exponential growth. DNA affinity purification of proteins using the promoter region of the 6C RNA gene revealed a binding of the LexA protein, the master regulator of the SOS response. Generally, SOS genes are repressed by LexA to varying degrees that depend on the position and exact sequence of the SOS box and the strength of the promoter. In C. glutamicum, the LexA-binding site is near to the -35 region about 50 bp upstream of the transcriptional 6C RNA start. The expression of 6C RNA is increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA.

The deletion of the 6C RNA gene cgb_03605 results in a higher sensitivity of C. glutamicum toward MMC and UV radiation. This can be reversed by plasmid-based expression of the 6C RNA.

Graphic of a 6C RNA deletion strainA 6C RNA deletion strain shows higher sensitivity toward UV radiation which can be reversed by plasmid-based complementation.

Together, the results show that 6C RNA is involved in the DNA damage response in C. glutamicum and may also be involved in other processes. Further studies are required to reveal the detailed molecular roles and mode of actions.


Pahlke J., Dostálová H., Holátko J., Degner U., Bott M., Pátek M., Polen T. (2016)
The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.
RNA Biol 13(9):848-60
doi: 10.1080/15476286.2016.1205776

Peptidyl-prolyl cis/trans isomerases

Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids.

We found that in C. glutamicum, the PPIase FkpA confers a temperature-dependent growth advantage in terms of biomass yield. FkpA is a single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase exhibiting typical PPIase activity with artificial substrates and also chaperone activity. FkpA is encoded by fkpA directly downstream of gltA encoding citrate synthase (CS). This gene cluster is conserved and also present in other Actinobacteria.

Graphic of Gene cluster gltA-fkpAGene cluster gltA-fkpA is conserved and also present in other Actinobacteria.

Although FkpA delays the aggregation of CS (often used as a model substrate) and has a positive effect on the activity of CS and its temperature range in vitro, in vivo the growth advantage appears to be unrelated to CS since overexpression of CS did not increase the biomass yield of a fkpA deletion mutant. Thus, other substrates than CS are responsible for the growth difference. Comparative transcriptome analysis revealed genes which exhibit mRNA level changes upon mild heat stress in the absence of FkpA. However, further studies including proteomic analysis could reveal consequences of the absence of FkpA resulting in the decreased biomass yield of C. glutamicum.

Kallscheuer N., Bott M., van Ooyen J., Polen T. (2015)
Single-domain peptidyl-prolyl cis/trans isomerase FkpA from Corynebacterium glutamicum improves the biomass yield at increased growth temperatures.
Appl Environ Microbiol 81(22): 7839-7850
doi: 10.1128/AEM.02113-15