Molecular Cloning

Six synthetic genes were designed and codon-optimized for L. lactis with the GeneSmart tool from GeneScript adding a six-histidine tag at the N-terminus of every precursor prochlorosin. All of the synthetic genes were provided cloned in pUC57 plasmid (Amp^R) by GeneScript and cloned into E. coli TOP-10 for their propagation and isolation.

For the cloning of all synthetic genes in their respective plasmid, primers were designed for USER ligation.⁵⁵ The SyncM enzyme encoding gene was cloned in the pTLR-plasmid, using the primers pTLR-user-RV and FW to amplify the backbone and Pnis-user-Fw and LanM-user-RV to amplify SyncM together with the P_nis promoter. Further, each SyncA was cloned in pNZ8048. For this, the backbone pNZ8048 was amplified, using pNZery-user-fw and pNZ-user-rv (Table 3). Differently, the syncA genes were grouped in five different pUC57 plasmids (Table 3). Each pUC57 with a different group of syncA’ genes was first digested with XbaI and HindIII located between each gene, yielding single-cloned syncA’s. Subsequently, they were amplified with USER primers. The same reverse (ProcA-rv) for every reaction and different forward (SyncA2-USER-fw and Proc33-USER-fw) primers were used.

PCR and digestion products were purified using the PCR cleaning kit (MN, Duren, Germany). Subsequently, a ligation reaction was performed according to the manufacturer’s instruction, using the USER enyme⁵⁵ (New England Biolabs, Massachusetts) with a 1:1 ratio of fragment and backbone. Ligations were dialyzed against ultrapure water and transformed into electrocompetent cells of L. lactis NZ9000 pTLR-SyncM using a Bio-Rad Gene Pulser (Bio-Rad, Richmond, CA). Preparation of L. lactis NZ9000 electrocompetent cells and transformation was performed following Holo and Nes.⁵⁶ Finally, all plasmid constructs were confirmed by DNA sequencing (Macrogen Europe, Amsterdam, The Netherlands).

Once all sequences were confirmed, the plasmids containing precursor peptides were isolated and then transformed into the electrocompetent L. lactisNZ9000 pTLR_SyncM strain.