Etablierung und Optimierung der sekretorischen Gewinnung thermostabiler Lipasen in Gram-positiven Bakterien

Thermostable lipases like the lipase GTL from $\textit{Geobacillus thermoleovorans}$ IHI-91 offer great potential for biotechnological applications, as they can be used instead of chemical catalysts for the conversion of long chain fatty acids under high temperatures. A secretory production of these biotechnological, relevant enzymes in Grampositve bacteria seems advantageous because the costs of downstream processing are low, since proteins can be purified directly from the culture supernatant. However, recombinant proteins are often secreted inefficiently in these heterologous host organisms, because they are not adapted to the secretory pathways in these bacteria. The secretory production of the lipase GTL, which was only produced intracellularly so far, was analyzed by making use of a toolbox principle, i.e. combining different Gram-positive host organisms, secretory pathways, and signal sequences with the lipase GTL. In $\textit{Staphylococcus carnosus}$, the lipase GTL was secreted with significant enzyme yields both Sec-dependently with its authentic signal sequence and Tat-dependently as a fusion with the signal sequence of the Escherichia coli TMAO-Reductase (TorA). The activity of the lipase GTL in the supernatant was 4 times higher when it was secreted via the Sec-pathway in an unfolded conformation (2,6 U/ml) than when it was translocated via the Tat-pathway in a fully folded conformation (0,7 U/ml). In order to compare the Tat-dependent secretion of the lipase GTL in different Gram-positive bacteria, the lipase GTL was expressed as a fusion with the $\textit{E.coli}$ TorA-signal sequence also in $\textit{Corynebacterium glutamicum}$ and in $\textit{Bacillus subtilis}$. While the expression of the precursor TorA$^{SP}$ GTL in $\textit{B.subtilis}$ disturbed the cell integrity and caused cell lysis, the lipase GTL was secreted with a significant activity of about 1,25 U/ml via the TatABC-system of $\textit{C.glutamicum}$. Strikingly the overexpression of the lipase GTL in $\textit{C.glutamicum}$ caused a substrate saturation of the Tat-translocase, consequently active enzyme species accumulated in the cytosol of $\textit{C.glutamicum}$. As substrate saturation is a typical bottleneck of Tat-dependent secretion, it was chosen as a case study for testing the ability of three independent strategies to relieve this bottleneck. The objective of the first strategy was to adjust the unrelated $\textit{E.coli}$ TorA-signal sequence to the Tat-pathway in $\textit{C.glutamicum}$ in such a way that the lipase GTL is secreted with a higher efficiency into the supernatant. Therefore, about 11.000 variants of the TorA-signal sequence were generated by random mutagenesis and fused to the lipase GTL. However, no $\textit{C.glutamicum}$ transformants with significantly increased enzyme activities in the corresponding culture supernatants could be detected in a subsequent high throughput screening. Nevertheless, using this approach, several determinants were identified which are important for the functionality of the $\textit{E.coli}$ TorA-signal sequence. The aim of the second strategy was to increase the translocation effiency of lipase GTL by overexpression of homologous and heterologous Tat-components. While coexpression of $\textit{E.coli}$ Tat-components failed to produce functional Tat-translocases in $\textit{C.glutamicum}$, the overexpression of the homologous $\textit{C.glutamicum}$ TatA and TatC proteins increased the activity of lipase GTL in the supernatant more than 3 times. This demonstrated that the number of Tat-components was a limitation for Tat-translocation under overexpression conditions of the hybrid precursor protein TorA$^{SP}$GTL. As a third strategy, it was examined whether the coexpression of the heterologous $\textit{E.coli}$ chaperone TorD, which specificially binds to the TorA-signal sequence, will increase the Tat-dependent secretion in $\textit{C.glutamicum}$. This chaperone coordinates the maturation of TorAprecursor proteins during Tat-proofreading in $\textit{E.coli}$. In contrast to $\textit{E.coli}$, where co-overexpression of TorD results in a more efficient membrane translocation of TorA-precursor proteins, the coexpression of TorD in $\textit{C.glutamicum}$ caused surprisingly an almost complete export block of TorA-precursor proteins. A possible reason for this finding is that a special release factor for TorD, that is present in $\textit{E.coli}$, is missing in $\textit{C.glutamicum}$. Since such a release factor is probably a component of the $\textit{E.coli}$ Tat-translocase, different $\textit{E.coli}$ Tat-components were expressed together with the chaperone TorD and the model substrate TorASPGFP in $\textit{C.glutamicum}$ wild-type cells. As a result, it could be shown that secretion of the model substrate was only possible, if the $\textit{E.coli}$ TatABC-translocase was concommitantly coexpressed together with TorD in $\textit{C.glutamicum}$. From this result it can be concluded, that the $\textit{E.coli}$ TatABC-translocase is directly involved in the release of the TorD chaperone from the TorA-signal sequence