Overproduction of 2,3-butanediol in recombinant Escherichia coli

• Main Authors: Zhi-Jie Yang, 楊智傑
• Other Authors: Hwan-You Chang
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/52555842458029579429

碩士 === 國立清華大學 === 生物技術研究所 === 87 === Acetoin and 2,3-butanediol are compounds of industrial importance that can be synthesized in many species of bacteria including Klebsiella, Bacillus, Serratia, and Pseudomonas. Among these microorganisms, K. pneumoniae was found to produce the highest yields of the diol. Our laboratory has cloned a DNA segment coding for the enzymes (a-acetolactate decarboxylase, a-acetolactate synthase, and acetoin (diacetyl) reductase) involved in the formation of 2,3-butanediol in K. pneumoniae. A number of culture conditions for the production of 2,3-butanediol and acetoin were tested in the recombinant Escherichia coli strains. Addition of 0.5% acetate to Luria-Bertani broth resulted in a significant increase of 2,3-butanediol synthesis by K. pneumoniae and the recombinant Escherichia coli. Inoculation of 800 ml and 200 ml of saturated bacterial culture to a final volume of 5 ml were found to be optimal for the production of 2,3-butanediol and acetoin, respectively. In addition, 6.0 was found to be the most suitable pH for the production of 2,3-butanediol and acetoin. We also found that, the bacteria grown without shaking produced more 2,3-butanediol. In contrast, shaking culture is more suitable for the production of acetoin. The highest productivity of 2,3-butanediol and acetoin in K. pneumoniae was 0.38 g/l and 0.21 g/l; 0.71 g/l and 1.46 g/l in the recombinant E. coli, that were obtained using an initial glucose concentration of 0.5% in LB. The highest 2,3-butanediol yield in E. coli of 24.46% (w/w, 3.99 g/l) against initially added glucose (2.0%) was obtained in a LB culture shaken for 6 hr at 37oC. When comparing the productivity of 2,3-butanediol in different E. coli strains, no apparent difference was observed. Finally, the production of 2,3-butanediol by E. coli carrying a plasmid with galU gene as a selectable marker is comparable with that with antibiotic selection.