title: High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli.
abstract: BACKGROUND
  Recently, the compatible solute 1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) has attracted considerable interest due to its great potential as a protecting agent. To overcome the drawbacks of high salinity in the traditional bioprocess of ectoine using halophilic bacteria, various attempts have been made to engineer ectoine biosynthesis in nonhalophilic bacteria. Unfortunately, the yields of ectoine in these producers are still low and hardly meet the demands of large scale production. In this paper, the whole-cell biocatalytic process using aspartate and glycerol as substrates was tried for high production of ectoine in nonhalophilic bacteria.
  
  
  RESULTS
  The ectoine genes ectABC from the halophilic bacterium Halomonas elongata were successfully introduced into Escherichia coli K-12 strain BW25113 under the arabinose-inducible promoter. To our delight, a large amount of ectoine was synthesized and excreted into the medium during the course of whole-cell biocatalysis, when using aspartate and glycerol as the direct substrates. At the low cell density of 5 OD/mL in flask, under the optimal conditions (100 mM sodium phosphate buffer (pH 7.0), 100 mM sodium aspartate, 100 mM KCl and 100 mM glycerol), the concentration of extracellular ectoine was increased to 2.67 mg/mL. At the high cell density of 20 OD/mL in fermentor, a maximum titre of 25.1 g/L ectoine was achieved in 24 h. Meanwhile, the biomass productivity of ectoine is as high as 4048 mg per gram dry cell weight (g DCW)(-1), which is the highest value ever reported. Furthermore, it was demonstrated that the same batch of cells could be used for at least three rounds. Finally, a total yield of 63.4 g ectoine was obtained using one litre cells.
  
  
  CONCLUSIONS
  Using aspartate and glycerol as the direct substrates, high production of ectoine was achieved by the whole-cell biocatalysis in recombinant E. coli. Multiple rounds of whole-cell biocatalysis were established to further improve the production of ectoine. Our study herein provided a feasible biosynthesis process of ectoine with potential applications in large-scale industrial production.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. heyz@im.ac.cn.|University of Chinese Academy of Sciences, Beijing, 100049, China. heyz@im.ac.cn.
authors.forename: Yong-Zhi
authors.initials: YZ
authors.lastname: He
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. Jiaogong0203@hotmail.com.
authors.forename: Jiao
authors.initials: J
authors.lastname: Gong
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. Yuhy62@aliyun.com.
authors.forename: Hai-Ying
authors.initials: HY
authors.lastname: Yu
authors.affiliation: Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. taoyong@im.ac.cn.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tao
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. Zhangshan1304@gmail.com.
authors.forename: Shan
authors.initials: S
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. dongzy@im.ac.cn.
authors.forename: Zhi-Yang
authors.initials: ZY
authors.lastname: Dong
mesh_terms: D000599:Amino Acids, Diamino / Q000096:biosynthesis*; D000613:Aminobutyrates / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D002851:Chromatography, High Pressure Liquid; D048168:Escherichia coli K12 / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D005990:Glycerol / Q000378:metabolism*; D020632:Halomonas / Q000201:enzymology / Q000235:genetics; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D009876:Operon / Q000235:genetics; D015203:Reproducibility of Results; D000637:Transaminases / Q000235:genetics / Q000378:metabolism
chemical_list: D000599:Amino Acids, Diamino; D000613:Aminobutyrates; D001426:Bacterial Proteins; C005959:2,4-diaminobutyric acid; D001224:Aspartic Acid; C045628:ectoine; D000637:Transaminases; D006836:Hydro-Lyases; C106231:ectoine synthase; D005990:Glycerol