Dataset Information

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

ABSTRACT: Due to the high capacity of their secretion machinery 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 a few Bacillus species are used for enzyme production at the industrial scale. In this work, we introduce with 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 efficient heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A was increased by optimizing the expression medium and testing various promoter sequences on the expression plasmid pBACOV. To identify promising new promoter sequences from the genome of P. polymyxa itself, quantitative mass spectrometry was used to analyze the secretome. The most strongly secreted host proteins were identified and the promoters regulating the expression of the corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from B. subtilis and B. megaterium. The best result was achieved with the promoter of the hypothetical protein PPOLYM_03468 from P. polymyxa, which in combination with the improved expression medium enabled the production of 5,475 U/l 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Paenibacillus Polymyxa

SUBMITTER: Christina Ludwig

LAB HEAD: Christina Ludwig

PROVIDER: PXD009882 | Pride | 2018-09-05

REPOSITORIES: Pride

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Publications

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

Heinze Simon S Zimmermann Kerstin K Ludwig Christina C Heinzlmeir Stephanie S Schwarz Wolfgang H WH Zverlov Vladimir V VV Liebl Wolfgang W Kornberger Petra P

Applied microbiology and biotechnology 20180926 23

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 eas ...[more]

PMID: 30259100

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Project description:The Gram-positive soil bacterium Corynebacterium glutamicum is widely used in industrial fermentative processes for the production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose is taken up into the C. glutamicum cell by the phosphotransferase system PTS which can be replaced and/or enhanced by a permease and a glucokinase. Heterologous expression of the gene for the high-affinity glucose permease from Streptomyces coelicolor and of the Bacillus subitilis glucokinase gene fully compensated for the absence of the PTS in ï??hpr strains and strains grew as fast with glucose as C. glutamicum wild type. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and the glucokinase from Bacillus subtilis were overproduced using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid, a 40% higher L-lysine titer and a 30% higher volumetric productivity as compared to GRLys1(pEKEx3) resulted. The non-proteinogenic amino acid pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the L-lysine producing strain, the L-Lysine dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were expressed as synthetic operon. This enabled C. glutamicum to L-PA with a yield of 0.49 Â± 0.03 gg-1 and a volumetric productivity of 0.04 Â± 0.00 gL-1h-1.To the best of our knowledge, this is the first fermentative process for the production of L-PA. Two conditions tested, 200 mM NaCl Vs 200 mM pipecolic supplemented in the culture medium, control experiments done with the addition of 200mM of NaCl. Four technical replicates.

Dataset Information

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

Publications

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

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