Improvement Of Acarbose-producing Strain By Genome Shuffling

Acarbose,industrially produced by Actinoplanes sp.,is a potent?-glucosidase inhibitor.It has a strong inhibitory effect towards intestinal sucrase,maltase,dextrin enzymes,and can reduce blood glucose levels.Because of its excellent pharmacokinetic and high safety,it has been widely used in the treatment of type II diabetes.This research is aimed to improve the acarbose productivity of the acarbose-producing bacterium Actinoplanes utahensis ZJB-08196 by genome shuffling.Firstly,the effect of factors including glycine concentration,mycelium age,lysozyme concentration,treating time,temperature and mycelial concentration on protoplast formation and regeneration was investigated.Moreover,the effect of the addition of L-proline,bovine serum albumin(BSA),polyvinylpyrrolidone(PVP)into regeneration medium on protoplast regeneration was further examined.The results indicated that protoplasts preparation and regeneration were achieved with following method.The 56 h-old seed were inoculated into mycelia medium with 0.4%glycine and cultured for 22 h.Then the mycelia were collected,and treated by 10 mg/mL lysozyme at 35 ? for 4 h and then spread on the regeneration-agar(regeneration medium supplemented with 4 g/L L-proline and 0.2 g/L PVP)plates.The maximum protoplast regeneration rate reached 15.14%.The protoplast can be preserved at-20 ? for a short time.The protoplasts of Actinoplanes utahensis ZJB-08196 were treated with UV,microwave and combination of UV-microwave,respectively.Two high-yield mutants UN-52 and WN-44 were obtained.The acarbose production of mutants UN-52 and WN-44 reached 5210.1 mg/L and 5110.0 mg/L,and increased by 13.7%and 12.8%compared with the orignal strains,respectively.The condition for protoplast fusion was optimized.The results showed that the highest fusion frequency was obtained when protoplasts were fused by 40%PEG6000 for 5 min.Mutants UN-52 and WN-44 were uesd as the parental strains for genome shuffling.After three rounds of genome shuffling,F61,a mutant with acarbose production of 5672.8 mg/L was obtained.The acarbose production of F61 increased by 9.8%and 15.7%compared with the parental strains,and increased 23.8%than the original strain ZJB-08196.Subculture experiment indicated that F61 was genetically stable.