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· RBS sequences were designed to finetune the expression of EctA, EctB and EctC.
· Heterologous aspartate kinase and aspartate-semialdehyde dehydrogenase were introduced to enhance ectoine precursor supply.
· A high yield of 60.7 g L-1 of ectoine with a conversion rate of 0.25 g/g from glucose was achieved.
Ectoine is a natural macromolecule protector and synthesized by some extremophiles. It provides protections against radiation-mediated oxidative damages and is widely used as a bioactive ingredient in pharmaceutics and cosmetics. To meet its growing commercial demands, we engineered Escherichia coli strains for the high-yield production of ectoine. The ectABC gene cluster from the native ectoine producer Halomonas elongata was introduced into different Escherichia coli (E. Coil) strains via plasmids and 0.8 g L-1 of ectoine was produced in flask cultures by engineered E. coli BL21 (DE3). Subsequently, we designed the ribosome-binding sites of the gene cluster to fine-tune the expressions of genes ectA, ectB, and ectC, which increased the ectoine yield to 1.6 g L-1. After further combinatorial overexpression of Corynebacterium glutamicum aspartate kinase mutant (G1A, C932T) and the H. elongate aspartate-semialdehyde dehydrogenase to increase the supply of the precursor, the titer of ectoine reached to 5.5 g L-1 in flask cultures. Finally, the engineered strain produced 60.7 g L-1 ectoine in fed-batch cultures with a conversion rate of 0.25 g/g glucose.
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