Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2,3-butanediol

• Published in: Metabolic engineering Vol. 23; pp. 22 - 33
• Main Authors: Xu, Youqiang, Chu, Haipei, Gao, Chao, Tao, Fei, Zhou, Zikang, Li, Kun, Li, Lixiang, Ma, Cuiqing, Xu, Ping
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01-05-2014
• Subjects: 2,3-Butanediol; Bacillus licheniformis; Bacillus subtilis; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Biofuel; Biofuels; Biomass; Butylene Glycols - metabolism; Enterobacter cloacae; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Klebsiella pneumoniae; Metabolic engineering; Multigene Family; Promoter; Promoter Regions, Genetic; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Serratia marcescens; Promoter; Metabolic engineering; Biofuel; Fermentation; Escherichia coli; 2,3-Butanediol
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2014.02.004

The production of biofuels by recombinant Escherichia coli is restricted by the toxicity of the products. 2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical with low toxicity to microbes, could be a promising alternative for biofuel production. However, the yield and productivity of 2,3-BD produced by recombinant E. coli strains are not sufficient for industrial scale fermentation. In this work, the production of 2,3-BD by recombinant E. coli strains was optimized by applying a systematic approach. 2,3-BD biosynthesis gene clusters were cloned from several native 2,3-BD producers, including Bacillus subtilis, Bacillus licheniformis, Klebsiella pneumoniae, Serratia marcescens, and Enterobacter cloacae, inserted into the expression vector pET28a, and compared for 2,3-BD synthesis. The recombinant strain E. coli BL21/pETPT7-EcABC, carrying the 2,3-BD pathway gene cluster from Enterobacter cloacae, showed the best ability to synthesize 2,3-BD. Thereafter, expression of the most efficient gene cluster was optimized by using different promoters, including PT7, Ptac, Pc, and Pabc. E. coli BL21/pET-RABC with Pabc as promoter was superior in 2,3-BD synthesis. On the basis of the results of biomass and extracellular metabolite profiling analyses, fermentation conditions, including pH, agitation speed, and aeration rate, were optimized for the efficient production of 2,3-BD. After fed-batch fermentation under the optimized conditions, 73.8g/L of 2,3-BD was produced by using E. coli BL21/pET-RABC within 62h. The values of both yield and productivity of 2,3-BD obtained with the optimized biological system are the highest ever achieved with an engineered E. coli strain. In addition to the 2,3-BD production, the systematic approach might also be used in the production of other important chemicals through recombinant E. coli strains.
•2,3-BD was selected as the target biofuel produced through recombinant E. coli.•Gene clusters in several native strains were compared for 2,3-BD production.•Expression of the most efficient gene cluster was optimized using different promoters.•Fermentative conditions were optimized by extracellular metabolite profile analysis.•2,3-BD production was highly acquired by systematically engineered E. coli.