Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine

Abstract

Background: The stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. These rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. There is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems.

Results: Here, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L(-1) day(-1) under growth conditions that did not rely on the use of high-salinity media.

Conclusions: The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the industrial workhorse C. glutamicum and thereby paved the way for further improvements in ectoine yield and biotechnological process optimization.

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 SpeI 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).

Figure 2

Cultivation profile of the heterologous ectoine producer strain C. glutamicum ECT-1. The C. glutamicum strain ECT-1 was cultivated in shake flasks at 30°C in a chemically defined medium. At the indicated time intervals, consumption of glucose and the extracellular accumulation of L-lysine, ectoine, and 5-hydroxyectoine were monitored. The data shown represent mean values and corresponding standard deviations from three biological replicates.

Figure 3

Influence of cultivation temperature on the growth and ectoine production performance of C. glutamicum ECT-1. Strain ECT-1 was grown in chemically defined medium with glucose on a miniaturized scale at the indicated growth temperatures. The specific growth rate μ, ectoine secretion (Ect_ex), and intracellular accumulation of ectoine (Ect_int) and hydroxyectoine (EctOH_int) were determined. Ectoines were quantified after 10h (27°C, 30°C, 35°C) and 20h (42°C) of cultivation. The data shown represent mean values and corresponding standard deviations from three biological replicates.

Figure 4

Production performance of the advanced ectoine-producer strain C. glutamicum ECT-2 during fed-batch fermentation. Cultivation profile of strain ECT-2 (A), and ectoine yield achieved in the different cultivation phases (B) are shown. The oxygen saturation in the fermenter was kept constant at 30% by variation of the stirrer velocity and the aeration rate. Automated feeding was initiated by a pO₂-based signal [53]. Glucose concentration was thereby kept below 5 g L^-1. The data shown represent mean values from two independent fermentation experiments.