Deletion of the otc cluster from the genome of the ATCC 10970 strain was carried out using an iterative marker excision system (Additional file 1: Figure S15) [43]. For construction of the plasmid for deletion of the otc gene cluster, the BAC vector R3A4 from a BAC library (unpublished) was initially identified, which contained a 37 kb fragment with the entire otc gene cluster and the up and down region of the otc cluster. The antibiotic cassette (e.g., hygromycin, erythromycin) was flanked with the P-GG and B-CC sites for ΦC31 integrase, and amplified by PCR with primers HSU-OTC34 and HSU-OTC35 (Additional file 1: Table S1). Using the PCR-based λ-red recombination technique [44], the amplified hyg-ery cassette (Additional file1: Table S3) was used to delete the otc cluster on BAC R3A4 containing the entire otc gene cluster from the otrB gene to the otrA gene, on the other side of the otc gene cluster. The resulting BAC with the entire cluster substituted with the hyg-ery resistance cassette was introduced into ATCC 10970 by conjugation [39], and was selected for erythromycin resistance. Double crossover mutants were selected using blue-white [45]. Finally, the antibiotic cassette was excised from the chromosome of the selected double-crossover mutants by expression of the ΦC31 integrase from the pUWLint31 plasmid [43]. Cluster deletion was confirmed by PCR, using the HSU-OTC41 and HSU-OTC46 primers (Additional file 1: Table S1), followed by sequencing of the PCR-amplified fragment.

M4018 is a medium OTC-producer strain that was kindly provided by Prof. I.S. Hunter (University Strathclyde, Glasgow, UK). M4018 is a prototrophic strain that was used for commercial production of OTC by Pfizer [46]. The 15883S strain is an M4018 lineage strain derived by spontaneous deletion of the otc gene cluster during a strain improvement program [20].

The strategy for deletion of the otc cluster in HP0508 (kindly provided by Prof. M. Guo, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology. Shanghai, China ) was based on homology recombination. The pAB13 plasmid was pKC1139 [39] derived plasmid with a thermo-sensitive replicon carrying 2.6 kb ‘up’ homology and 2.6 kb ‘down’ homology, which was used to delete the entire otc gene cluster, including the genes from oxyA and oxyT. Regulatory genes (otcR, oxyTA1, otcG) and genes responsible for resistance (otrA, otrB) were maintained in the genome. Homologies for the otc cluster deletion were amplified using the OTC_UP and OTC_DOWN primers, as listed in Additional file 1: Table S1. The up homology was cut with the XbaI and DraI restriction enzymes, and the down homology was cut with the DraI and EcoRi restriction enzymes. Both of the homologs were simultaneously cloned into the pAB13 plasmid opened with XbaI and EcoRI. The nonmethylated plasmid was introduced into the HP0508 strain by transformation [40], and transformants were further selected on MS agar supplemented with erythromycin (30 µg/mL). The cultures were then subcultivated at 37 °C, five times without selection pressure (antibiotic) in TSB, and from the third day onward, plated for serial dilutions in MS plates. After 5 days of incubation, the colonies from the MS plates were patched onto MS plates without antibiotic and MS plates containing erythromycin. The primary recombinants were still resistant to erythromycin, while the secondary recombinants had lost their erythromycin resistance. The erythromycin-sensitive colonies were further confirmed by colony PCR amplification using the HSU-OTC41 and HSU-OTC46 primers (Additional file 1: Table S1).

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