As microorganisms are repeatedly subcultured in the laboratories, the lag phase is shortened and the growth rate is increased compared to the initial culture. This is the result of "struggling for existence", as Darwin stated. During the process of competing in an environment with limited resource, adaptive mutations are passed down, which bring about changes in the whole population. In this way, unintended evolution occurs over a short period. Adaptive laboratory evolution (ALE) is a narrow-experimental evolution that mimics this natural phenomenon in laboratory and derives the desired phenotype. It is possible to change the environment by applying artificial selection pressure and obtain the ameliorated organism generated by the accumulation of beneficial mutations via natural selection [1,2]. ALE was first used by the evolutionary scientist Dallinger in the seven-year hightemperature adaptation experiment [3] and has since been applied to studies on various organisms, from microalgae, mammalian cells, and viruses, to standard model organisms such as E. coli and yeast [4][5][6][7][8][9].Efficient rational engineering of cellular metabolism is possible only if comprehensive knowledge of metabolic pathways is acquired; however, this aspect is elusive even in the most well-characterized model organism, E. coli. As coenzymes such as ATP, NADH, and NADPH are used in multiple metabolic reactions in common, complex interactions between metabolic reactions frequently impede strain improvement, resulting in different metabolic outcomes (e.g., sub-optimal growth, lower product concentration) [6,10]. Besides, practical difficulties arise when the genetic manipulation itself is complicated by polyploidy, gene essentiality, etc. ALE can overcome these limitations because it does not require prior knowledge of the genotype-phenotype relationship and is easy to implement practically. In particular, it is advantageous for inducing a counter-intuitive phenotype spanning numerous intracellular pathways such as diverse stress tolerances and rapid growth in specific environments. ALE has been used as a powerful complement to metabolic engineering by subsequently re-optimizing the cellular fitness of crippled recombinants [11,12]. In addition to the strain improvement, the omics approach for the analysis of causative mutations in growth-improved strains is used for expanding intracellular regulatory networks by revealing underlying mechanisms that regulate cell metabolism [6,[13][14][15][16]. For example, transcriptome analysis of evolved E. coli with minimal genome showed that increasing the Entner-Doudoroff pathway flux, which enables efficient glucose utilization and increased intracellular reducing power, contributed to rapid growth [12]. Thus, ALE provides a straightforward reverse engineering approach that can overcome the shortcomings of existing rational metabolic engineering [2,17].Industrial microorganisms have long been exploited as key producers in almost every field, including food, pharmaceuticals, and other value-add...

The reproduction of microorganisms is defined as the increase in their number while maintaining the same cellular components. The doubling time of these cells varies widely depending on environmental factors and genetic factors inherent in each species. Vibrio natriegens, one of the fastest doubling microorganism, has been reported to double in less than 10 min in BHIN medium [1]. V. natriegens has been studied widely in order to understand this desirable characteristic of rapid cell division for industrial applications. The following genetic factors have been identified to contribute to rapid reproduction: 1) two chromosomes that can replicate independently and rapidly [2]; 2) a large number of rRNA operons (12 sets) that can perform translation efficiently; 3) localization of genes involved in transcription and translation near the bacterial origin of replication (oriC) to facilitate assembly of ribosomes; 4) rich in genes related to respiratory pathways that help in rapid cell growth [3]. Several following studies focus on the ways to leverage this species as a host for molecular biology and biotechnology applications [4,5].Corynebacterium glutamicum has been a useful industrial strain [6] for the production of biochemicals and recombinant proteins [7]. The doubling time of C. glutamicum in a defined CGXII medium without protocatechuate is known to be more than 2 h (corresponding growth rate = 0.34 h -1 ) [8]. Given that E. coli doubles every 0.9 h in glucose minimal medium [9], C. glutamicum grows much slower, which is disadvantageous for industrial use. To overcome this shortcoming and expand its applicability, adaptive laboratory evolution for accelerating the growth rate of C. glutamicum can be considered. Adaptive laboratory evolution has been used to Corynebacterium glutamicum, an important industrial strain, has a relatively slower reproduction rate. To acquire a growth-boosted C. glutamicum, a descendant strain was isolated from a continuous culture after 600 generations. The isolated descendant C. glutamicum, JH41 strain, was able to double 58% faster (t d =1.15 h) than the parental type strain (PT, t d =1.82 h). To understand the factors boosting reproduction, the transcriptomes of JH41 and PT strains were compared. The mRNAs involved in respiration and TCA cycle were upregulated. The intracellular ATP of the JH41 strain was 50% greater than the PT strain. The upregulation of NCgl1610 operon (a putative dyp-type heme peroxidase, a putative copper chaperone, and a putative copper importer) that presumed to role in the assembly and redox control of cytochrome c oxidase was found in the JH41 transcriptome. Plasmid-driven expression of the operon enabled the PT strain to double 19% faster (t d =1.82 h) than its control (t d =2.17 h) with 14% greater activity of cytochrome c oxidase and 27% greater intracellular ATP under the oxidative stress conditions. Upregulations of genes those might enhance translation fitness were also found in the JH41 transcriptome. Plasmid-driven expressions of NCgl0171 (encodin...