The plasmids and primers used in this study are listed in Tables S1 and S2 . The ectABC gene cluster from Halomonas elongata was synthesized by GeneralBio (Chuzhou, China) after codon optimization (Table S3 ). Primers were used to amplify the pathway fragments. The vector fragment was amplified using plasmid pBARGPE1-hygro (Miaoling, Wuhan, China) as the template, with primers vector-F/R. The purified gene fragments ectA, ectB, and ectC were assembled with the vector fragment using the Hieff Clone Plus Multi One Step Cloning Kit (YEASEN, Shanghai, China), resulting in plasmid pBARGPE-hygro-ectABC (P1), pBARGPE-hygro-ectACB (P2), pBARGPE-hygro-ectBAC (P3), pBARGPE-hygro-ectBCA (P4), pBARGPE-hygro-ectCAB (P5), and pBARGPE-hygro-ectCBA (P6). The three genes in different orders were organized into an operon expressed from the respective plasmids.
The construction of the M. purpureus ectoine-producing strain (MppECT) was performed as described by Shi et al. [21 (link)]. The pBARGPE-hygro-ectABC plasmid was incubated with 100 µL of M. purpureus ATCC 16365 WT strain protoplasts to construct the ectoine-producing strain (MppECT). The expression of the ectABC gene cluster was verified by diagnostic PCR. The transformant colonies were used as templates of diagnostic PCR.
The construction of the M. purpureus ectoine-producing strain (MppECT) was performed as described by Shi et al. [21 (link)]. The pBARGPE-hygro-ectABC plasmid was incubated with 100 µL of M. purpureus ATCC 16365 WT strain protoplasts to construct the ectoine-producing strain (MppECT). The expression of the ectABC gene cluster was verified by diagnostic PCR. The transformant colonies were used as templates of diagnostic PCR.
Free full text: Click here
