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000908892 1001_ $$0P:(DE-Juel1)177776$$aMüller, Carolin$$b0$$ufzj
000908892 245__ $$aAccelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation
000908892 260__ $$aNew York$$bSpringer$$c2022
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000908892 520__ $$aSecretion of bacterial proteins into the culture medium simplifies downstream processing by avoiding cell disruption for target protein purification. However, a suitable signal peptide for efficient secretion needs to be identified, and currently, there are no tools available to predict optimal combinations of signal peptides and target proteins. The selection of such a combination is influenced by several factors, including protein biosynthesis efficiency and cultivation conditions, which both can have a significant impact on secretion performance. As a result, a large number of combinations must be tested. Therefore, we have developed automated workflows allowing for targeted strain construction and secretion screening using two platforms. Key advantages of this experimental setup include lowered hands-on time and increased throughput. In this study, the automated workflows were established for the heterologous production of Fusarium solani f. sp. pisi cutinase in Corynebacterium glutamicum. The target protein was monitored in culture supernatants via enzymatic activity and split GFP assay. Varying spacer lengths between the Shine-Dalgarno sequence and the start codon of Bacillus subtilis signal peptides were tested. Consistent with previous work on the secretory cutinase production in B. subtilis, a ribosome binding site with extended spacer length to up to 12 nt, which likely slows down translation initiation, does not necessarily lead to poorer cutinase secretion by C. glutamicum. The best performing signal peptides for cutinase secretion with a standard spacer length were identified in a signal peptide screening. Additional insights into the secretion process were gained by monitoring secretion stress using the C. glutamicum K9 biosensor strain.
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000908892 7001_ $$0P:(DE-Juel1)176296$$aBakkes, Patrick J.$$b1$$ufzj
000908892 7001_ $$0P:(DE-Juel1)171683$$aLenz, Patrick$$b2$$ufzj
000908892 7001_ $$0P:(DE-Juel1)186097$$aWaffenschmidt, Vera$$b3
000908892 7001_ $$0P:(DE-Juel1)178687$$aHelleckes, Laura M.$$b4$$ufzj
000908892 7001_ $$0P:(DE-Juel1)131457$$aJaeger, Karl-Erich$$b5$$ufzj
000908892 7001_ $$0P:(DE-Juel1)129076$$aWiechert, Wolfgang$$b6$$ufzj
000908892 7001_ $$0P:(DE-Juel1)131469$$aKnapp, Andreas$$b7
000908892 7001_ $$0P:(DE-Juel1)128960$$aFreudl, Roland$$b8$$ufzj
000908892 7001_ $$0P:(DE-Juel1)129053$$aOldiges, Marco$$b9$$eCorresponding author
000908892 773__ $$0PERI:(DE-600)1464336-4$$a10.1007/s00253-022-12017-7$$gVol. 106, no. 12, p. 4481 - 4497$$n12$$p4481 - 4497$$tApplied microbiology and biotechnology$$v106$$x0171-1741$$y2022
000908892 8564_ $$uhttps://juser.fz-juelich.de/record/908892/files/M%C3%BCller2022_Article_AcceleratedStrainConstructionA.pdf$$yOpenAccess
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