Figure 1

Metabolic engineering strategy for heterologous production of ectoine and 5-hydroxyectoine in Corynebacterium glutamicum from the building block L-aspartate-β- semialdehyde. L-aspartate-β-semialdehyde is synthesized through the concerted actions of the aspartokinase (Ask; EC: 2.7.2.4) and aspartate-semialdehyde-dehydrogenase (Asd; EC: 1.2.1.11). It is then converted into the compatible solutes ectoine and 5-hydroxyectoine, respectively, by the L-2,4-diaminobutyrate transaminase (EctB; EC: 2.6.1.76) to form L-2,4-diaminobutyrate, a metabolite that is then acetylated by the 2,4-diaminobutyrate acetyltransferase (EctA; EC: 2.3.1.178) to produce N-γ-acetyl-2,4-diaminobutyrate, which is subsequently cyclized via a water elimination reaction by the ectoine synthase (EctC; EC: 4.2.1.108), to yield ectoine. Ectoine can then serve as the substrate for the formation of 5-hydroxyectoine through the activity of ectoine hydroxylase (EctD; EC: 1.14.11). Heterologous production in C. glutamicum was mediated via the codon-optimized ectABCD gene cluster based on that present in P. stutzeri A1501. The synthetic gene cluster was designed to be constitutively expressed from the promoter for the tuf gene from C. glutamicum. For genome-based integration via double-recombination event, the construct was equipped with flanking regions of about 560 bp DNA sequences derived from the upstream and downstream regions of the ddh gene. Recognition sites for the restriction enzyme Spe I were added to facilitate cloning of this DNA fragment into the vector pClik_int_sacB (A). The ddh gene, encoding diaminopimelate dehydrogenase, was chosen as integration site to minimize competing carbon flux towards lysine. Tracer studies with 3-¹³C glucose identified this biosynthetic branch as major contributor to the overall lysine flux under conditions with high ammonium availability which is readily present under industrial-scale production conditions (B).