| L-isoleucine, which is one of eight essential amino acids of human body and also one of three branch-chain amino acids, plays an important role in human life as it special structure and function. It is the material to synthesize some human hormones and enzymes, and it can enhance protein integration and inhibit it from degradation. L-isoleucine finds a variety of application in food industry, biological medicine, cosmetics, structuring project, photochemistry, and electrochemistry. It is also be used as feed addictives and as functional nutrients to produce functional drink currently.In this paper, Brevibacterium lactofermentation strain, which could accumulate extracellular L-isoleucine largely, was used for L-isoleucine fermentation. A series of feasible strategies were carried out to further improve L-isoleucine yield, substrate conversion ratio, and productivity. The main research and results are as follows:(1) The effect of different dissolved oxygen—20%,30%,35% and 40% on L-isoleucine production of B. lactofermentation was investigated by applying DO-stat feeding strategy. The results demonstrated that L-isoleucine production was increased by 15.7% compared with batch culture when the dissolved oxygen was controlled at 20%, but the substrate conversion ratio was 12.2% which was lower than other conditions. In order to improve glucose utilization, quick fed-batch fermentation strategy was developed based on DO-stat feeding culture. It was indicated that by adding 150 mL fresh medium at 36 h and 52 h respectively, a relative higher L-isoleucine production (26.0 g/L) and substrate conversion ratio (13.0%) was obtained, which was increased by 7.0% and 6.56%. Then the effect of different initial glucose concentration on L-isoleucine production was studied which was also based on DO-stat feeding culture. Cell growth and glucose consumption was more rapid and DO-stat feeding culture was first started under the condition of initial glucose concentration at 80 g/L. The condition of initial glucose concentration at 140 g/L was last entry the feeding stage, L-isoleucine production was highest (26.2 g/L) but substrate conversion ratio was lowest. So we chose the initial glucose concentration at 100 g/L yet by comprehensive considering.(2) L-isoleucine production under different constant glucose concentration in the middle and late stage of fermentation process was investigated. It was found that different glucose concentration had significant impact on L-isoleucine synthesis. And L-isoleucine production was 26.8 g/L when glucose concentration was constant at 20 g/L, which was highest among all conditions. Two-stage glucose concentration control strategy, that glucose concentration controlled at 20 g/L between 24 h-48 h and 5.0 g/L between 48 h-68 h, was proposed by analyzing L-isoleucine specific production rate. The result was that cell concentration (OD) was about 42.5 in stable stage which was higher than batch culture. L-isoleucine production was 31.6 g/L, which was increased by 17.91% compared with the glucose concentration constant at 20 g/L. On the other hand, substrate conversion ratio and productivity was 13.8% and 0.465 g/(L-h).(3) The effect of biotin, VB1, methionine, and sodium citrate on L-isoleucine production was investigated in shake flask. The order of effect on L-isoleucine production was methionine< VB1< biotin< sodium citrate. And L-isoleucine synthesis was promoted obviously when sodium citrate concentration was 4.0 g/L, which was increased by 19.75% compared with the control without the addition of sodium citrate. The effect of 4.0 g/L sodium citrate on L-isoleucine production was further investigated in a 3 L bioreactor. The results demonstrated that sodium citrate had little impact on cell growth, but the consumption of dissolved oxygen and glucose was both decreased. The L-isoleucine synthesis rate was improved greatly and the production was 27.1 g/L which was increased by 16.31% compared with the control without addition of sodium citrate. Finally, L-isoleucine formation metabolic network by B. lactofermentation was constructed by applying metabolic engineering. The effect of sodium citrate on L-isoleucine metabolic flux was analyzed preliminaryly through metabolic flux analysis. And the result was that EMP pathway was decreased, while HMP pathway which can provide sufficient reduction power NADPH for L-isoleucine synthesis was increased under the control with addition of sodium citrate. |
