Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract： Vitamin K2 (VK2) is a series of menaquinone compounds with isoprene side chains, which are represented by MK-n depending on the length of the side chains. Highly active VK2 is mainly synthesized by microorganisms and has the physiological function of preventing and treating diseases such as osteoporosis, hemorrhage, liver cirrhosis and Parkinson's disease. Flavobacterium is an important production strain and can synthesize a variety of VK2 homologs including MK4, MK5 and MK6. We found that by regulating the fermentation temperature, the type and yield of VK2 homologs synthesized by Flavobacterium can be controlled. In the range of 20~37℃, Flavobacterium sp. M1-14 grows best at 25℃, the biomass reaches 8.8 g/L, but the fermentation product is completely MK6, the yield is 13.9 mg/L, and the unit cell yield is 1.6 mg/L g. When the fermentation temperature is higher than 30°C, Flavobacterium can synthesize MK4, MK5 and MK6 at the same time. At 37°C, the yields of MK4 and MK5 are the highest, which are 1.6 mg/L and 1.7 mg/L, respectively, and the total amount of VK2 is 12.5 mg/L. At this time, the biomass was only 5.5g/L, and the unit cell yield was 2.3mg/g. In view of the difference in the optimum temperature for the growth of Flavobacterium cells and the synthesis of VK2 homologs, variable temperature fermentation was considered to increase biomass and VK2 production. After optimization of multiple factors, we developed a two-stage temperature change strategy with low temperature first and then high temperature, that is, fermentation at 25°C for 48 hours, and then fermented at 37°C for 96 hours, the VK2 yield reached 20.9 mg/L (among which MK4 was 2.1 mg). /L, MK5 is 2.3 mg/L, MK6 is 16.5 mg/L), the biomass is 8.8 g/L, and the unit cell yield is 2.4 mg/g. Then, on the 30L fermenter, we investigated the oxygen demand of fermentation at different temperatures by controlling the ventilation rate and rotation speed. It was found that at 25℃ and 37℃, the optimum kLa for VK2 synthesis by Flavobacterium fermentation was 360 h-1 and 60 h-1, respectively. A two-stage variable kLa control strategy was developed for the changes in the oxygen demand of bacteria during variable temperature fermentation. After optimization, the kLa was 360 h-1 in the first 24 hours of variable temperature fermentation, and the kLa was 60 h-1 in the next 120 hours. was 22.5 mg/L), which was 107% higher than the initial value, the biomass was 15.5 g/L, and the unit cell yield was 1.9 mg/g. The staged fermentation regulation strategy of changing temperature and kLa can change the type of homologues of Flavobacterium synthesizing VK2, significantly increase the production of VK2, and lay an optimized foundation for realizing the industrialization of VK2 bioproduction.
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