Vector construction and creation of positive transformants

Mramy1and Mramy2 (encoding alpha-amylases) were amplified via polymerase chain reaction (PCR) from the cDNA and total DNA of M. ruber CICC41233 as the template. The primers 440333-Hind III-F and 440333-SacI-R were used to amplify Mramy1, and 324551-Hind III-F and 324551-SacI-R were used to amplify Mramy2 (Supplementary Table S1). Then, Mramy1 and Mramy2 were sequenced by BGI, analyzed using BLAST on NCBI database, and aligned with the sequence from M. ruber NRRL1597. The plasmid pNeo0380 (Long et al. 2018b) and the gene fragments Mramy1 and Mramy2 were digested with HindIII and SacI, respectively. Then, the digested gene fragments Mramy1 and Mramy2 were ligated to the digested plasmid pNeo0380 to construct the binary expression vector pNeo0380-440333 and pNeo0380-324551, respectively. The vectors pNeo0380-440333 and pNeo0380-324551 were transferred into M. ruber CICC41233 via A. tumefaciens EHA105.The positive strains were identified using PCR.