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Expression of chaperone-dependent lipases in Bacillus subtilis by fusing the chaperone to Bacillus PrsA chaperone signal peptide Disclosure Number: IPCOM000246688D
Publication Date: 2016-Jun-28
Document File: 3 page(s) / 50K

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Expression of chaperone-dependent lipases in Bacillus subtilis by fusing the chaperone to Bacillus PrsA chaperone signal peptide.

Some bacterial lipases depend on presence of foldases/chaperones that belong to PFAM family PF03280. Since lipolytic chaperones are found in Gram-negative bacteria, their expression in Gram- positive hosts is usually troublesome or leads to low expression yields. It is not trivial to express lipases in, for example, Gram-positive Bacillus subtilis, a commonly used expression host for industrial enzymes. In Bacillus, amylases can be processed by the PrsA extracellular chaperone which in Bacillus is anchored to the outer membrane of the cell. Both a lipase and a suitable chaperone may be expressed in the same Bacillus host using different chromosomal integration sites, for example: the pectate lyase pel locus for the lipase and the amylase amy locus for the chaperone. The PrsA signal peptide sequence MKKIAIAAITATSVLALSAC gene was operably linked to a PF03280 lipolytic chaperone on the N-terminus, replacing its native signal sequence. The cysteine residue becomes linked with diacelyglycerol thus connecting PrsA to the outer leaflet of the cytoplasmic Bacillus membrane. The following examples describe how a chaperone-dependent lipase was successfully expressed in Bacillus subtilis by anchoring the chaperone to the outer cell membrane through fusing the chaperone to PrsA signal peptide.

Example 1: Cloning of the Gallaecimonas pentaromativorans lipase

The codon-optimized synthetic lipase-encoding gene of SEQ ID NO:1 (see below) was synthesized and purchased commercially.

The lipase-gene was cloned into a Bacillus subtilis expression vector as described in WO 12/025577. The DNA encoding the mature lipase peptide was cloned in-frame to a Bacillus clausii secretion signal (BcSP) with the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA (the encoding DNA sequence is shown in bold in SEQ ID NO:1), originating from the protease AprH of B. clausii.

The lipase expression plasmid (BcSP-lipase) was transformed into a Bacillus subtilis expression host. The lipase BcSP-fusion genes were integrated by homologous recombination into the into the pectate lyase (pel) locus of the Bacillus subtilis host cell genome upon transformation.

The lipase gene construct was expressed under the control of the promoter system described in Sloma et al. 2000, Development of marker-free strains of Bacillus subtilis capable of secreting high levels of industrial enzymes, Journal of Industrial Microbiology & Biotechnology, 25, 204-212. The gene coding for chloramphenicol acetyltransferase was used as maker (as described in (Diderichsen et al., 1993, Plasmid 30: 312-315)). Transformants were selected on LB media agar supplemented with 6 microgram of chloramphenicol per ml. One recombinant Bacillus subtilis clone containing the respective lipase expression construct was selected and verified.


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Example 2: Cloning of Ga...