A secretion biosensor for monitoring Sec-dependent protein export in Corynebacterium glutamicum

Abstract

Background: In recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable. Results: High level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments. Conclusions: We have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.

Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide

“…Figure 2 , Panel A is a schematic of the S. aureus signal peptide showing the consensus sequence of the Ala-X-Ala box as well as the three associated domains. The C-terminal of the signal peptide contains the signal peptidase cleavage site, which has a conserved “(−3, −1) amino acids” often as Ala-X-Ala [ 12 ].. The designed secretory signal peptide includes three domains.…”

“…In the first method, a methionine amino acid is necessarily added to the amino terminus as the starting codon for protein expression. Given that this methionine has been shown to stimulate the immune system against heterologous proteins, and since this is very important for pharmaceutical proteins, additional methods must be used to remove this methionine such as the use of different peptidases which imposes an additional step on the system [ 12 ]. Expression of the intercellular form can lead to the formation of inclusion bodies that has no functions [ 13 ].…”

“…In addition, the secretion of heterologous protein in E. coli is an effective strategy to help reduce the contamination with host cell proteins and thus improve the quality of the recombinant products [ 11 ]. In addition, if the product is secreted into the culture medium, cell lysis is no longer necessary to recover the product and the product is obtained from the culture medium without disrupting the cell to release cytoplasmic protein contaminants [ 12 ]. This strategy is also preferred because it greatly reduces proteolytic activity in the culture medium that occurs immediately after cell lysis with the release of proteases.…”

“…Various strategies have been used to increase the extracellular secretion of hGH in E. coli strain BL21(DE3), including the use of physical and chemical methods such as osmotic shock, freeze-thaw cycles, lysozyme treatment, and chloroform shock, each of which has its own problems. In the present study, the recombinant hGH was expressed and purified with a very low level of endotoxin contamination and without N-terminal methionine in E. coli [ 12 ]. For this purpose, signal peptide from Staphylococcus aureus protein A (SpA) was modified, redesigned based on the Sec secretory system and linked to the coding region of the mature form of the HGH.…”

Optimization of Expression, Purification and Secretion of Functional Recombinant Human Growth Hormone in Prokaryotic Hosts Using Modified Staphylococcal Protein A Signal Peptide

“…Signal peptide sequences in the N-terminal region of proteins lead these proteins to the Sec system and eventually secrete them out of the cell [21]. These signal peptides have a three-dimensional structure with a positively charged N region, a hydrophobic H region, and a C region in which Ala-X-Ala motif is identi ed and cleaved by the cellular signal peptidase enzymes [21,22].…”