Constructing Of A Riboflavin High-producer Bacillus Subtilis And Optimization Of Riboflavin Production

Riboflavin(vitamin B2), is an essential vitamin required by all organisms. Riboflavin exists by flavin adenine dinucleotide(FAD) and flavin mononucleotide(FMN) in vivo, and involves in the transfer of hydrogen as a coenzyme of flavin enzymes. Nowadays, riboflavin has a broad application in the pharmaceutical, food, feed and other fields, and mainly produced by microbial fermentation. In our previous study, a genetically engineered strain Bacillus subtilis RF1 was constructed for riboflavin production.The Streptothricin acetyltransferase(encoded by gene sat) was inserted into expression vector p MA5 to create p MA5-sat. Then p MA5-sat was successfully transformed into B.subtilis RF1 selected with NTC, suggesting that p MA5-sat could replicate in B.subtilis RF1. Glucose-6-phosphate dehydrogenase(encoded by gene zwf) is one of key enzymes involved in pentose phosphate pathway, and gene zwf harbored in the new plasmid p MA5-sat was successfully transformed into B.subtilis RF1 to obtain recombinant strain B.subtilis RF1/p MA5-sat-zwf. The intracellular glucose-6-phosphate dehydrogenase activity of recombinant strain B. subtilis RF1/p MA5-sat-zwf was about 50-fold higher than that of strain B. subtilis RF1.Fermentation medium components were optimized through single factor and orthogonal experimental design with recombinant B.subtilis RF1/ p MA5-sat-zwf. The optimal medium compositions(g/L) were: glucose 40, yeast extract 7.5, ammonium citrate 4, potassium dihydrogen phosphate 1, dipotassium hydrogen phosphate 1, Mg SO4·7H2O 2, Mn C l2 0.04, Ca Cl2 0.06, Cu C l2 0.02. Using the optimal medium, riboflavin production reached 3.56 g/L by flasks. The optimum fermentation conditions: initial p H 7.0, culture temperature 40 ℃, inoculation size 4%, rotation speed is 220 rpm. Under the optimal conditions, riboflavin production of recombinant bacteria reached 3.65 g/L at 48 h. The recombinant B.subtilis RF1/ p MA5-sat-zwf cultivated in a 5 Lfermentor at a constant stirring speed of 600 rpm, riboflavin production reached 13.5 g/L at 60 h, which was increased by 32.3% than B.subtilis RF1.Dissolved oxygen is an important parameter for the fermentation process of riboflavin, and it is easy to be controled by changing agitation speed in fermentor. Based on different oxygen demand at different fermentation stage, a multi-stage speed control strategy to control dissolved oxygen level was proposed. At first 30 h, agitation speed was controlled at 600 rpm; at 30-34 h, agitation speed was controlled at 700 rpm; at 34-38 h, agitation speed was controlled at 800 rpm; subsequently agitation speed was controlled at 900 rpm. Using this strategy, riboflavin production reached 15.8 g/L in a 5 L tank, which was raised by 10.5% than a relatively constant speed.