Journal Article

FZJ-2022-02899

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Accelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation

Müller, C.FZJ* ; Bakkes, P. J.FZJ* ; Lenz, P.FZJ* ; Waffenschmidt, V. ; Helleckes, L. M.FZJ* ; Jaeger, K.-E.FZJ* ; Wiechert, W.FZJ* ; Knapp, A. ; Freudl, R.FZJ* ; Oldiges, M. (Corresponding author)FZJ*

2022
Springer New York

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Please use a persistent id in citations: http://hdl.handle.net/2128/31591  doi:10.1007/s00253-022-12017-7

Abstract: Secretion 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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Contributing Institute(s):

1. Biotechnologie (IBG-1)
2. Institut für Molekulare Enzymtechnologie (HHUD) (IMET)

Research Program(s):

1. 2172 - Utilization of renewable carbon and energy sources and engineering of ecosystem functions (POF4-217) (POF4-217)

Appears in the scientific report 2022

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Database coverage:
; ; ; BIOSIS Previews ; Biological Abstracts ; Clarivate Analytics Master Journal List ; Current Contents - Agriculture, Biology and Environmental Sciences ; Current Contents - Life Sciences ; DEAL Springer ; Ebsco Academic Search ; Essential Science Indicators ; IF >= 5 ; JCR ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

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