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    STUDIA BIOLOGIA - Ediţia nr.1 din 2019  
         
  Articol:   HETEROLOGOUS LOW-SALT PRODUCTION OF ECTOINE AND HYDROXYECTOINE: IS ESCHERICHIA COLI THE ANSWER?.

Autori:  LUKAS BETHLEHEM, KATHARINA DOMENIKA MORITZ, MORITZ LUCIUS SÜMMERMANN, PETER SCHEIN, ERWIN ARNO GALINSKI. 		 
       
         
  Rezumat:  The compatible solute ectoine is the most successful biotechnological product of halophilic Bacteria. It is at present still produced industrially with the halophilic Halomonas elongate (bitop-AG, Dortmund, Germany). Because of an ever increasing demand and the inherent draw-back of separating the product from a salt-containing fermentation broth, heterologous production has become an issue. However, numerous attempts using either Escherichia coli or Corynebacterium glutamicum as a chassis have remained unsatisfying. We have for the first time applied the ectoine/hydroxyectoine biosynthetic gene cluster from a non-halophilic donor, namely the acidophilic Acidiphilium cryptum, expecting prime performance of its enzymes even at low salinity. In comparison with other heterologous processes published so far, we achieved an unchallenged maximal specific production rate of 345 mg ectoine/(g dcw x h) in shaking-flasks with glycerol as carbon source. Biosynthesis of the product, which was excreted into the medium, was so efficient that its specific production amounted to 2.9 g/g biomass. Using:13C-flux analysis with a novel inhouse software solution, we were able to identify PEP-carboxylase as the key reaction for anaplerotic carbon flux into the product. Further quantitative calculations revealed that we achieve 66% of the theoretical maximal yield. In order to comprehend potential limitations in the scale-up process, a systems approach will be presented including plasmid copy number, mRNA (pPCR) and protein abundance (mass spectrometry) determinations as well as metabolomic studies (GC-MS).

Keywords: Acidiphilium cryptum, compatible solute, Escherichia coli, heterologous production, hydroxyectoine/ectoine. 		 
         
     
         
         
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