Abstract
Ectoine, an osmotic pressure-compensated solute, is used in the food, agriculture, medicine, and cosmetics industries due to its ability to protect macromolecules. In this study, an ectoine-producing variant of Escherichia coli, ET08, was genetically constructed by introducing the ectABC gene cluster and eliminating metabolic pathways involving lysine and pyruvate. Medium optimization enhanced ectoine production from 1.87 to 10.2 g/L. Analysis of the transcriptional levels revealed that supplementation with ammonium sulfate enhanced the metabolic flux towards the biosynthesis of ectoine. Furthermore, by optimizing the copy number of ectA, ectB, and ectC, the recombinant E. coli ET11 (ectA:ectB:ectC = 1:2:1) produced 12.9 g/L ectoine in the shake flask and 53.2 g/L ectoine in a fed-batch fermenter, representing the highest ectoine titer produced by E. coli, which has great industrial prospects.
Highlights
As a compatible solute, ectoine (4S-2-methyl-1,4,5,6-tetrahydro-4-pyrimidinecarboxylic acid) is commonly found in halophilic and halotolerant microorganisms and was first discovered in Ectothiorhodospira halochloris by Galinski et al (1985)
Similar gene clusters involved in ectoine biosynthesis were disclosed in Marinococcus halophilus (Louis and Galinski, 1997), Halobacillus dabanensis D-8T (Zhao et al, 2006), Methylomicrobium alcaliphilum 20Z (Reshetnikov et al, 2006), and Nesterenkonia halobia DSM20541 (Zhang et al, 2008)
By optimizing nutritional element and analyzing the transcription levels, we could conclude that ammonium sulfate, as the optimal amino donor, has a positive effect on ectoine synthesis
Summary
Ectoine (4S-2-methyl-1,4,5,6-tetrahydro-4-pyrimidinecarboxylic acid) is commonly found in halophilic and halotolerant microorganisms and was first discovered in Ectothiorhodospira halochloris by Galinski et al (1985). Ectoine has been synthesized from the precursor L-aspartate-β-semialdehyde (ASA) with L-2,4diaminobutyrate transaminase (EctB), 2,4-diaminobutyrate acetyltransferase (EctA), and ectoine synthase (EctC) as catalysts (Göller et al, 1998; Calderon et al, 2004; Schwibbert et al, 2011; Li et al, 2017). These three enzymes are encoded by genes which are typically organized in the ectABC gene cluster, and may comprise the ectD gene (Bursy et al, 2008). Similar gene clusters involved in ectoine biosynthesis were disclosed in Marinococcus halophilus (Louis and Galinski, 1997), Halobacillus dabanensis D-8T (Zhao et al, 2006), Methylomicrobium alcaliphilum 20Z (Reshetnikov et al, 2006), and Nesterenkonia halobia DSM20541 (Zhang et al, 2008)
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