Verlag des Forschungszentrums Jülich
JUEL-4125
Vollstedt, Angela
Sekretorische Gewinnung von Enzymen aus dem thermoalkaliphilen Bakterium Anaerobranca gottschalkii im mesophilen Wirt Staphylococcus carnosus
132 S., 2004
Secretory production of enzymes from the thermoalkaliphilic bacterium Anaerobranca gottschalkii
in the mesophilic host Staphylococcus carnosus
Enzymes from extremophilic microorganisms offer great potential as biocatalysts for industrial applications.
However, no optimal expression systems exist for the production of such "extremozymes" to date. In
contrast to the intracellular production of enzymes, secretory protein production with mesophilic Grampositive
bacteria such as Staphylococcus carnosus offers the advantage of the proteins being secreted directly
into the culture supernatant. Contrary to most other Gram-positive bacteria, S. carnosus is a particularly
suitable production host as it does not secrete any proteases into the culture supernatant that may
compromise the yields of secreted heterologous proteins. Expression systems were established for the
secretory production of extremozymes derived from the thermoalkaliphilic bacterium Anaerobranca
gottschalkii in the host S. carnosus.
In principle, two secretion pathways exist in Gram-positive bacteria which may be used for the secretion of
heterologous proteins, for which the general Sec pathway is particularly well established. Sec-dependent
secretion of heterologous proteins, which necessitates translocated proteins to assume their native
conformation outside the cytosol, is often inefficient as these proteins frequently fail to fold rapidly enough in
the cell envelope to avoid degradation by cell wall-associated proteases. The recently identified Tat pathway,
for which biotechnological applications in Gram-positive bacteria have not been described so far, is unique in
secreting fully folded proteins from the cell. The use of the Tat pathway, in which folding of the substrates
occurs prior to their export, would avoid many of the problems associated with the Sec-dependent secretion
of heterologous proteins and allow the secretory production of such proteins in bacteria for which this has so
far not been feasible.
It was investigated whether S. carnosus possesses a Tat pathway and whether this is capable of secreting
foreign proteins from the cell. Fusion of two model proteins, an intracellular branching enzyme and an
extracellular CGTase from A. gottschalkii, with the signal sequence of the E. coli Tat substrate TorA resulted
in the secretion of both enzymes in an active conformation into the culture supernatant of S. carnosus.
Inactivation of the putative Tat translocase receptor, TatC, inhibited the secretion of both enzymes in
S. carnosus, providing proof for the first time that S. carnosus possesses a functional Tat pathway, which can
be used for the secretion of heterologous protein.
The biotechnological applicability of the Tat pathway was subsequently evaluated by comparing the Tat- and
Sec-dependent secretion of branching enzyme and CGTase respectively. It was shown that a Sec-dependent
production of both enzymes in S. carnosus was not feasible. The direct fusion of each enzyme with the signal
sequence of a Sec-dependent lipase from S. hyicus did not result in the secretion of either enzyme via the Sec
pathway, which was only made possible by the further addition of a propeptide known to enhance the
secretion of heterologous proteins. Nonetheless, the use of the propeptide was also shown to be unsuitable
for the secretory production of the enzymes, despite the secretion of large amounts of the propeptide fusions.
The presence of the propeptide interfered with the correct folding of the branching enzyme, leading to the
secretion of inactive protein into the culture supernatant, while the pro-CGTase fusion was largely degraded
by cell wall-associated proteases. Sec-dependent secretion of the CGTase with its cognate signal sequence lead
to only low yields of secreted enzyme.
Compared with the secretion of both branching enzyme and CGTase via the Sec pathway, their Tatdependent
secretion each resulted in the highest yields of active enzyme in the culture supernatant, making
this pathway a highly promising alternative to Sec-dependent protein production and the method of choice
for the secretory production of these enzymes in S. carnosus. The results indicate that it is advantageous for
heterologous proteins to assume their native conformation with the aid of cytosolic chaperones prior to
secretion. However, the biotechnological potential of the Tat pathway is limited by the cell wall's sieve effect.
Secretion of the CGTase via the Tat pathway resulted in the accumulation of the majority of the translocated
enzyme in the cell wall compartment, being released into the supernatant only by cell wall turnover. The use
of the Tat pathway for the secretory production of heterologous proteins in Gram-positive bacteria is
therefore primarily suitable for smaller proteins that are capable of diffusing into the culture supernatant
through the pores of the cell wall.
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