| Glucosamine(GlcN) and its acetylated derivative, N-acetylglucosamine(GlcN Ac), have been widely applied in nutraceuticals, cosmetics and pharmaceutical industries. GlcN and GlcNAc are currently produced by acid or enzymatic hydrolysis of chitin extracted from shrimp shells and crab. As an environmentally friendly method, microbial fermentation by filamentous fungi or Escherichia coli becomes an alternative method for producing GlcN and GlcNAc. In our previous work, a recombinant Bacillus subtilis strain for production of GlcNAc was constructed. In this work, to enhance the production of GlcNAc and avoid the problem about cell lysis, nonessential genes of five regions were knocked out. Next, we chose the optimal strain for further investigation to enhance GlcNAc production. Main contents of research is as follows:(1) Firstly, we knocked out nonessential genes of five regions including skin, PBSX, prophage1, prophage3 and pks. Then we compared the effect of different nonessential genes knockout on cell growth, GlcNAc synthesis and the consumption of glucose in the same condition. Compared with the starting strain(BSGN6), cell growth of PBSX knockout strain(BSGN6-2) remained constant. The maximum GlcNAc production of BSGN6-2 increased to 2.90 g·L-1, which improved 16.9%, the average glucose consumption rate of BSGN6-2 was 1.15 g·L-1·h-1, which reduced 31.1%. GlcNAc yield on glucose was 0.078 mol·mol-1, which improved 16.4% compared with BSGN6.(2) Next, according to the growth curve of BSGN6-2 which was made in this study(0-4 h lag phase; 4-14 h exponential phase; 14-22 h stationary phase; 22-30 h decline phase), the effect of different factors on GlcNAc production and cell growth were investigated for shake flask fermentation. The optimal inoculum age was 8 h. The opitimum medium(g·L-1) were as following: corn steep liquor 30, glucose 40, yeast extract 20, CaCO3 5, MgSO4 3, KH2PO4 12.5, K2HPO4 2.5. Then the effect of inoculum amount, initial pH, temperature, shaking speed and loaded volume were investigated. GlcNAc production reached 7.51 g·L-1 in the following conditions: 4%(v/v) inoculum amount, initial pH 7.4, 37 oC, 300 r·min-1, 50/500 mL·mL-1, and we found that dissolve oxygen plays a significant role on improving GlcNAc production. After optimization, the GlcNAc yield was 3.20 times before and layed the foundation for optimizing on 3 L fermentor.(3) In 3 L fermentor, we first studied the influences of different intial glucose concentration(40 g·L-1, 60 g·L-1, 80 g·L-1, 100 g·L-1) in batch culture. We found that the maximum GlcNAc production in batch culture reached 8.57 g·L-1 when initial glucose concentration was 60 g·L-1. Then we studied the effects of different glucose feeding strategies including pulse fed-batch culture, exponential fed-batch culture, constant rate fed-batch culture and glucose control(5 g·L-1, 10 g·L-1, 15 g·L-1) fed-batch culture on GlcNAc production and cell growth. The results showed that GlcNAc yield in glucose control(5 g·L-1) culture was 3.10 times that in batch culture, which reached 26.58 g·L-1. Next, we investigated the effect of different DO levels(20%, 30%, 40% and 50%) on GlcN Ac production. Finally, a step-wise DO control strategy(0-7 h, 30%; 7-15 h, 50%; 15-50 h, 40%; 50-72 h, 30%) with the optimal glucose control strategy was introduced, the yield of GlcN Ac reached 35.77 g·L-1, which was 4.17 times the yield in batch culture without DO control. |
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