Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium

Biochemical Engineering, Technical University Braunschweig, GBF/TU-BCE, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Biotechnology and Bioengineering (Impact Factor: 4.13). 03/2007; 96(4):780-94. DOI: 10.1002/bit.21167
Source: PubMed


Production and secretion of a 28,172 Da hydrolase from Thermobifida fusca (TFH) in Bacillus megaterium MS941 and WH323 was investigated in shake flask and pH controlled bioreactors. Successful production of heterologous TFH was achieved by adapting the original tfh gene to the optimal codon usage of B. megaterium. A codon adaption index close to one was reached. The codon optimized tfh was cloned into an open reading frame with DNA sequence for the N-terminal signal peptide of B. megaterium lipase A and a C-terminal His(6)-tag, all under the control of a xylose inducible promoter. Successful TFH production and secretion were observed using batch reactor cultivations with complex medium. Expression of the tfh gene from the P(xylA) promoter and secretion of produced TFH were compared in detail to batch reactor cultivations with semi-defined growth medium. For the first time, significant TFH secretion was achieved using a semi-defined medium in glucose limited fed batch cultivations yielding 10-fold higher cell densities compared to LB medium cultivation. Comparable volumetric TFH activities were obtained for both cultivation strategies. Surprisingly, measured specific TFH activities exhibited drastic discrepancies between preparations from LB and semi-defined medium grown B. megaterium. TFH recovery by Ni-chelate affinity chromatography resulted in higher purification factors when LB medium was used. These results indicated that secreted TFH is favorably produced by batch cultures of B. megaterium WH323 in LB medium.

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    • "E. coli BL21- Gold(DE3) pET- 20b(+) α- hemolysin secretion pathway Tfu_0883 (Su et al., 2012) Bacillus megaterium MS941 / WH323 pYYBm LipA of B. megaterium TfH C-terminal (Fürch et al., 2007; Yang et al., 2007 "
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    Advances in applied microbiology 08/2014; 89:267-305. DOI:10.1016/B978-0-12-800259-9.00007-X · 2.74 Impact Factor
    • "During the last years, B. megaterium was developed for the high yield production, secretion, and purification of recombinant proteins by improving different elements of the used plasmid systems. This strategies included the introduction of signal peptides for secretion of recombinant proteins (Biedendieck et al. 2007a; Malten et al. 2006; Stammen et al. 2010a), the utilization of affinity tags for protein purification (Biedendieck et al. 2007c; Malten et al. 2006), the optimization of the established xylose-inducible promoter system (Korneli et al. 2013a; Stammen et al. 2010a), the development of new inducible promoter systems (Biedendieck et al. 2007b; Gamer et al. 2009; Stammen et al. 2010b), and the use of codon adapted genes (Bäumchen et al. 2007; Yang et al. 2007). In this last context, the adaptation of the codon usage to the host B. megaterium has to occur individually for each gene in a time-and cost-intensive manner. "
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    • "The thermophiles Thermobifida fusca and T. alba have been isolated from composts and are potent degraders of polyesters such as aliphatic-co-aromatic polyesters (Kleeberg et al. 1998; Hu et al. 2008; Sinsereekul et al. 2010). The genes for polyesterhydrolyzing enzymes from T. fusca and T. alba have been cloned and determined to encode serine hydrolases (Kleeberg et al. 2005; Dresler et al. 2006; Yang et al. 2007: Chen et al. 2008; Hu et al. 2010) that belong to the lipase/esterase family (Arpigny and Jäger 1999). The complete genome of T. fusca YX has been disclosed (Lykidis et al. 2007), and the genome includes two tandem genes that encode extracellular lipases. "
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