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. 2022 Jan 25:10:824859.
doi: 10.3389/fbioe.2022.824859. eCollection 2022.

Metabolic Engineering of Escherichia coli for Ectoine Production With a Fermentation Strategy of Supplementing the Amino Donor

Hao Zhang  1   2 , Zhong Liang  1   2 , Ming Zhao  1   2 , Yanqin Ma  1   2 , Zhengshan Luo  1   2 , Sha Li  1   2 , Hong Xu  1   2   3
Affiliations

Metabolic Engineering of Escherichia coli for Ectoine Production With a Fermentation Strategy of Supplementing the Amino Donor

Hao Zhang et al. Front Bioeng Biotechnol. .

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.

Keywords: Escherichia coli; amino donor; ectoine; medium optimization; metabolic engineering.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Related metabolic pathways for the synthesis of ectoine in engineered E. coli. The genes marked in green indicate the key genes of biosynthetic ectoine introduced into the engineered E. coli. The genes marked in red indicate deletion of the corresponding gene. Abbreviations: PEP, phosphoenolpyruvate.
FIGURE 2
FIGURE 2
Comparison of ectoine titer, glucose consumption, and DCW in recombinant E. coli strains. Values represent the mean ± SD. Statistical analysis was performed by Duncan’s test (p < 0.05). Different lowercase letters indicate significant differences.
FIGURE 3
FIGURE 3
Effects of (A) different organic nitrogen sources, (B) the yeast extract mixed with inorganic nitrogen, (C) different ammonium chloride concentrations, (D) different ammonium sulfate concentrations, (E) different sodium glutamate concentrations on ectoine production, glucose consumption, and cell growth in E. coli ET08. Values represent the mean ± SD. Statistical analysis was performed by Duncan’s test (p < 0.05). Different lowercase letters indicate significant differences.
FIGURE 4
FIGURE 4
Transcription levels of the key genes in ammonium metabolic pathways and ectoine synthesis. The level of transcription was calculated relative to transcription of the control (0 mM ammonium sulfate), which were defined as 1. Values represent the mean ± SD. Different lowercase letters indicate significant differences at p < 0.05.
FIGURE 5
FIGURE 5
Ectoine production in metabolically engineered E. coli with different copy numbers of ectA, ectB, ectC. (A) Ectoine production; (B) Glucose consumption; (C) Cell growth; (D) Transcription levels of gene ectA, ectB, ectC. Values represent the mean ± SD. Statistical analysis was performed by Duncan’s test (p < 0.05). Different lowercase letters indicate significant differences.
FIGURE 6
FIGURE 6
Fed-batch fermentation of ET11 in a 7.5 L bioreactor.
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