Screening Of High-yield ε-poly-L-lysine Producing Strains Through Ribosome Engineering And Optimizing The PH Fermentation Strategy

ε-Poly-L-lysine(ε-PL) was an amino acid-homopolymer which consists of 25-35 L-lysine, and those monomer were linked by the amide bond between α-carboxyl groups and ε-amino groups. ε-PL is a very effective food preservatives: thermal stability, high solubility, biodegradable, widely bacteriostatic. Now, many countrys(China, Amercia and Japan) approved ε-PL to be used as food preservatives. This study attempted to screen of a high-yie ld ε-PL producing strain through ribosome engineering on the basic of strain S. albulus AS3-14(ε-PL production 2.5 g·L-1). At last, we got a strain S. albulus WG-608, and optimized it’s p H control fermentation stragtegy. The mian results were described as follows:1) Ribosme engineering technology were used to improve the capacity of S. albulus AS3-14 to produce ε-PL. Str, Gen and Rif resistant Strains were obtained from the original S. albulus AS3-14 with the presence of mutagen(Str, Gen and Rif). A Str high concentratio n resistant strain S. albulus WG-601 was obtained on the basis of a Str resistant strain S. albulus WG-600. Then, a double drug-resistant mutant S. albulus WG-608 was obtained on the basis of single drug-resistant mutant S. albulus WG-601 with the presence of mutagen of Rif, which of the ε-PL production was improved to 3.7 g·L-1, 48% higher than the parent strain.2) The difference between strain S. albulus WG-608 and S. albulus AS3-14 were compared by means of the membrance fatty acid composition analysis, cell staining, comparison of strain resistence, and 5 L fed-batch fermentation. In addition, by comparing the key enzymes of S. albulus WG-608 and S. albulus AS3-14, we found that the increasing key enzymes’ activity of center metabolic pathways may accounted for the enhanced ε-PL productivity of S. albulus WG-608.3) We optimized the pH and time of pre-acid-shock, the time of acidic p H shock, and confirmed the final fermentation strategy: pre-acid-shock adaption at p H 5.5 to al evia te the damage that caused by the followed p H shock, and then acidic p H shock at 3.0 for 10 h(including p H decline from 4.0 to 3.0) to positively regulate mycelia metabolic activit y, finally restoring p H to 4.0 to provide optimal condition for ε-PL production. This strategy was tested in a 5 L fermentator: After 192 h of fed-batch fermentation, the ε-PL production and productivity reach 72.1 g·L-1 and 9.0 g×L-1×d-1,1.7-fold high than parent strains S. albulus AS3-14 under original p H shock fermentation strategy.