Abstract: This work aims to obtain a new strain of high-yield Surfactin via Bacillus natto and Bacillus siamansi interspecies protoplast fusion. The protoplasts of B. natto and B. siamansi were prepared using B. natto and B. siamansi as parent strains,and the protoplasts were fused by PEG-mediated parental inactivation labeling. A new strain producing high yield Surfactin was obtained after the potential fusions identified by PCR,high-throughput screening by CPC-BTB method,HPLC rescreening and stability verification of fusion strains. The results showed as such：the optimal enzymatic hydrolysis concentration of protoplasts of B. natto and B. siamansi were 0.25 mg/mL and 0.2 mg/mL,respectively;the optimal enzymatic hydrolysis time was 25 min and 20 min,respectively;and protoplast yield were 83.1% and 84.3%,respectively. The conditions of protoplast fusion were as follows：B. natto was inactivated by UV for 80 min,B. siamansi was inactivated at 85℃ for 40 min,and the fusion system was 50% PEG6000 at 38℃ for 15 min. A stable fusion strain F8 was obtained,and it produced a 33.3% increase in Surfactin production compared to sole B. natto and a 60% increase by sole B. siamansi.

Key words: protoplast fusion, surfactin, parental inactivation, CPC-BTB reaction