Evaluation of promoter sequences for the secretory production of a Clostridium thermocellum cellulase in Paenibacillus polymyxa

Autor:	Simon Heinze, Kerstin Zimmermann, Wolfgang Liebl, Vladimir V. Zverlov, Wolfgang H. Schwarz, Stephanie Heinzlmeir, Christina Ludwig, Petra Kornberger
Rok vydání:	2018
Předmět:	0301 basic medicine Expression vector biology Hypothetical protein Promoter General Medicine Bacillus subtilis biology.organism_classification Applied Microbiology and Biotechnology 03 medical and health sciences 030104 developmental biology Biochemistry Clostridium thermocellum Heterologous expression Paenibacillus polymyxa Biotechnology Bacillus megaterium
Zdroj:	Applied Microbiology and Biotechnology. 102:10147-10159
ISSN:	1432-0614 0175-7598
DOI:	10.1007/s00253-018-9369-7
Popis:	Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.
Databáze:	OpenAIRE
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