Abstract
Corynebacterium glutamicum is a commonly applied host for the industrial production of amino acids. While valued for its robustness, it is somewhat inferior to competing strains such as Escherichia coli because of the relatively low growth rate of 0.40 h−1 in synthetic, industrial media. Accordingly, adaptive laboratory evolution (ALE) experiments were performed in continuous cultivation mode to select for a growth-improved host. To ensure industrial attractiveness, this ALE study aimed at a reduction of dependency on costly growth-boosting additives such as protocatechuate (PCA) or complex media supplements. Consequently, double selection pressures were installed consisting of a steady increase in growth rate demands and a parallel reduction of complex medium fractions. Selection yielded C. glutamicum EVO5 achieving 0.54 h−1 and 1.03 gGlc gCDW−1 h−1 in minimal medium without abovementioned supplements. Sequencing revealed 10 prominent mutations, three of them in key regulator genes.
Highlights
AND MOTIVATIONMicrobial bioprocesses play a major role in the production of biofuels, foods, food ingredients, feeds, cosmetics, and pharmaceuticals (Straathof, 2013)
A total of five consecutive continuous bioreactor processes were conducted with the goal of increasing the growth rate of C. glutamicum with an evolutionary approach thereby selecting for the fastest-growing subpopulation
Minimal medium CGXII supplemented with 1% (w/v) glucose, omitting the standard CGXII component PCA (Keilhauer et al, 1993; Kind et al, 2010), and varying amounts of yeast extract (YE) was used as growth medium
Summary
Microbial bioprocesses play a major role in the production of biofuels, foods, food ingredients, feeds, cosmetics, and pharmaceuticals (Straathof, 2013). To ensure their economic competitiveness, processes are developed such that their performance is ideal under large-scale production conditions by either optimizing the producing biocatalysts, i.e., pro-/eukaryotic organisms or cellular parts (for example enzymes), or the surrounding process conditions. The latter comprises elements such as technical process setup, production strategy and corresponding process parameters, and the cultivation medium. Metabolic engineering strategies, optionally combined with omics techniques (Becker and Wittmann, 2018), are applied to influence cell-specific attributes, e.g., growth rate, substrate spectrum, and uptake rate, or production rate, boosting productivity (Löffler et al, 2016; Nielsen and Keasling, 2016; Michalowski et al, 2017) or reducing cultivation media costs
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