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Bacterial protein secretion


Transport of proteins across biological membranes is a crucial process in all kingdoms of life. In bacteria, the transport of proteins out of the cytosol is mediated mainly by two mechanistically different export pathways, the general secretion (Sec) pathway and the twin-arginine-translocation (Tat) pathway. Current research of our group focusses on the investigation of various mechanistic aspects of Tat-dependent protein translocation and the optimization of selected Gram-positive bacteria as host organisms for the secretory production of biotechnologically and pharmaceutically relevant heterologous proteins.

1. Genetic analysis of Tat-dependent protein translocation


The twin-arginine translocation (Tat)-system has the remarkable ability to translocate fully folded proteins across the bacterial cytoplasmic membrane. The signal peptides of Tat substrates, which possess a characteristic amino acid motif including two highly conserved consecutive arginine residues, mediate the targeting of the Tat substrates to the Tat translocase. Using a genetic approach that is based on a Tat-specific, selectable reporter protein, we are currently investigating the molecular interactions between Tat substrates and the TatBC substrate receptor complex which are required for the productive binding of the substrates to the translocase and, by this means, determine the overall efficiency of the transport process.

Abbildung Tat-Substrate InteractionCurrent model for the specific and high-affinity binding of Tat precursor proteins to an advanced-stage precursor binding pocket in the TatBC receptor complex. After initial recognition by the Tat translocase, the precursor is inserted in form of a hairpin loop, consisting of the signal peptide (blue) and the early mature region of the precursor (thick line), into an advanced-stage binding pocket, whose formation involves both TatB (yellow) and TatC (red). The amino acids of the Tat consensus motif are involved in the specificity of signal peptide binding and are also major contributing factors to the binding affinity between the inserted precursor and the binding pocket (red arrows). Additionally, also the h-region of Tat signal peptides significantly contributes to the overall binding affinity of Tat precursor proteins to the Tat translocase (green arrows). The productive high-affinity binding of the precursor to the advanced-stage binding pocket is proposed to be a prerequisite and an efficiency determinant for the following events that ultimately result in the membrane translocation of the mature protein.

Selected publications:
Ulfig, A.; Fröbel, J.; Lausberg, F.; Blümmel, A.-S.; Heide, A. K.; Müller, M.; Freudl, R. (2017) The h-region of twin-arginine signal peptides supports productive binding of bacterial Tat precursor proteins to the TatBC receptor complex. J. Biol. Chem. 292, 10865-10882. (http://dx.doi.org/10.1074/jbc.M117.788950)

Fröbel, J.; Rose, P.; Lausberg, F.; Blümmel, A.-S.; Freudl, R.; Müller, M. (2012) Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB. Nat. Commun. 3:1311. (http://dx.doi.org/10.1038/ncomms2308)

Lausberg, F.; Fleckenstein, S.; Kreutzenbeck, P.; Fröbel, J.; Rose, P.; Müller, M.; Freudl, R. (2012) Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli. PLoS ONE 7(6): e39867. (http://dx.doi.org/10.1371/journal.pone.0039867)

2. Heterologous protein secretion using Gram-positive host bacteria


The secretory production of biotechnologically relevant proteins is an attractive alternative to intracellular expression strategies since it greatly facilitates downstream processing and significantly reduces the production costs of a desired target protein. In this respect, Gram-positive bacteria (such as various Bacillus species) are especially interesting as host organisms since they usually lack an outer membrane and proteins that are transported across the cytoplasmic membrane via either the Sec or the Tat pathway can directly be released into the surrounding culture medium.

Like other members of the Corynebacterianeae, the industrial workhorse Corynebacterium glutamicum, although belonging to the group of Gram-positive bacteria, possesses an outer membrane-like structure composed of mycolic acids and other complex glycolipids. Despite this fact, in the recent years it became increasingly clear that C. glutamicum possesses an enormous potential as an alternative host organism for the secretory production of biotechnologically and pharmaceutically relevant proteins. Currently, the focus of our research lies on the development of various molecular strategies (such as the use of large signal peptide libraries for identifying the optimal signal peptide for a given target protein) that improve C. glutamicum for the secretory production of any desired heterologous target protein.

Abbildung Coryne Host plus Soxy Secretion Coomassie GelFor the secretion of heterologous proteins by C. glutamicum, the Sec or the Tat protein export route can be used (left). The Coomassie blue-stained SDS polyacrylamide gel on the right shows the Tat-dependent secretion of a normally cytosolic, FAD cofactor-containing sorbitol-xylitol oxidase (SoXy) from Streptomyces coelicolor into the C. glutamicum culture supernatant. Lane 1: molecular mass marker (kDa). The other lanes show the supernatant fractions of a negative control strain (lane 2) and of recombinant C. glutamicum strains secreting enzymtically-active SoXy via the Tat protein export route (lanes 3 and 4).


Abbildung Signal peptide used for cutinase secretion by C. glutamicumThe application of a signal peptide library consisting of 125 Bacillus subtilis-derived Sec signal peptides to various heterologous target proteins clearly shows that the nature of the signal peptide that is used to drive the Sec-dependent secretion (shown here for a cutinase from the fungus Fusarium solani pisi) strongly influences the respective yields of the desired target proteins in the C. glutamicum culture supernatant.

Selected publications:
Freudl, R. (2017) Beyond amino acids: Use of the Corynebacterium glutamicum cell factory for the secretion of heterologous proteins. J. Biotechnol. (epub ahead of print). (http://dx.doi.org/10.1016/j.jbiotec.2017.02.023)


Hemmerich, J.; Rohe, P.; Kleine, B.; Jurischka, S.; Wiechert, W.; Freudl, R.; Oldiges, M. (2016) Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum. Microb. Cell Fact. 15:208. (http://dx.doi.org/10.1186/s12934-016-0604-6)


Scheele, S.; Oertel, D.; Bongaerts, J.; Evers, S.; Hellmuth, H.; Maurer, K.-H.; Bott, M.; Freudl, R. (2013) Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol-xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum. Microb. Biotechnol. 6, 202-206. (http://dx.doi.org/10.1111/1751-7915.12005)


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