Molecular cloning, expression, and characterization of acyltransferase from Pseudomonas protegens

The formation of C-C bonds by using CoA independent acyltransferases may have significant impact for novel methods for biotechnology. We report the identification of Pseudomonas strains with CoA-independent acyltransferase activity as well as the heterologous expression of the enzyme in E. coli. The cloning strategies and selected expression studies are discussed. The recombinant acyltransferases were characterized with regard to thermal and storage stability, pH,- and co-solvent tolerance. Moreover, the impact of bivalent metals, inhibitors, and other additives was tested. Careful selection of expression and working conditions led to obtain recombinant acyltransferase form Pseudomonas protegens with up to 11 U mL−1 activity. Electronic supplementary material The online version of this article (10.1007/s00253-018-9052-z) contains supplementary material, which is available to authorized users.


Plasmid construction of recombinant ATases
Primer sequences and plasmids used in this study are listed in SI 5

Protein sequence-alignment of PhlA, PhlC and PhlB
The phlACB operon from P. protegens DSM19095 and P. brassicacearum DSM13227 was amplified from the genomic DNA using primer sequences which were identified in a BLAST-search (Table S3).

Cloning of PbATaseWT (pEG330) and PpATaseWT (pEG331)
PCR-amplification of the wild-type phlACB operon. The ATase encoding phlACB operon (approx. 2770 bp) was amplified from the genomic DNA of P. protegens or P. brassicacearum. The genomic DNA was isolated according to the manufacture's protocol (PureLink® Genomic DNA Minikit, Thermo Fischer). The following primers were used (restriction site underlined): The PCR reaction mixture consisted of the following components:

Cloning of PpATaseCH (pEG332)
The genes phlA, phlC and phlB originating from P. protegens were codon-harmonized by manually matching the codon-frequency of Pseudomonas to E. coli. Ribosomal binding sites (RBS) were introduced at the 5'-end of each phl gene. The optimized phl* genes were ordered as gene strings (gBlocks®, IDT). Cloning of the gene strings into the pASK-IBA3plus expression vector was accomplished by Gibson assembly and overlap extension PCR (OExPCR).
The following amounts were applied (Table S4): The assembly was performed according to the manufacture's protocol followed by direct transformation into E.

IBA3-REV: 5'-CGCAGTAGCGGTAAACG-3'
The PCR reaction mixture consisted of the following components: Selected clones were restreaked onto LB plates containing 100 µg mL -1 ampicillin for selection and the isolated plasmids were sequenced. Misassembled DNA stretches within phlA* and phlC* of the Gibson assembled plasmid pEG332_C20 ( Figure S6, a) were corrected by OEx PCR to establish the correct phlACB* construct (pEG332).

SI 12
The PCR reaction mixture consisted of the following components: The entire reaction mixture was loaded onto an agarose gel and the desired gene product phlAC*_OE (approx. 1986 bp) was gel-purified ( Figure S7).

SI 14
Restriction and ligation. The misassembled DNA stretch in pEG332_C20 was removed by restriction digest (EcoRI). The remaining backbone pEG332_C20 was recovered and gel-purified. Ligation with phlAC*_OE finally established the correct phlACB* construct (pEG332).

Cloning of PpATaseCH (pCAS1)
Restriction and ligation. Cloning of the codon-harmonized phlACB* construct from pEG332 into the T7regulated pCAS1 expression vector was accomplished by restriction digest with EcoRI and BamHI (1 µg DNA).
The fragments were gel-purified prior to ligation.
The triple ligation (1:1:1 ratio) consisted of the following components: The reaction was incubated overnight at 4 °C. 5 µL of the ligation mix was transformed into E. coli DH5α and streaked onto LB plates containing 100 µg mL -1 ampicillin for selection. Isolated plasmids of randomly picked clones were controlled by restriction digest and sequencing.

Modified procedure to test the impact of bivalent metals
The chloride salt of Ca 2+ , Mg 2+ , Zn 2+ , Cu 2+ , Co 2+ , Mn 2+ , Sr 2+ or Ni 2+ (5.0 or 8.0 mM final concentration) was added to the reaction mixture containing ATase and substrate ( Figure S9). The bioacetylation of 8 with DAPG was performed as described in the manuscript. The influence of various temperatures on expression visibility are shown in Figure S10.