Accelerated Growth of Corynebacterium glutamicum by Up-Regulating Stress- Responsive Genes Based on Transcriptome Analysis of a Fast-Doubling Evolved Strain.

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 (td=1.15 h) than the parental type strain (PT, td=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 (td=1.82 h) than its control (td=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 (encoding a cold-shock protein) and NCgl2435 (encoding a putative peptidyl-tRNA hydrolase) enabled the PT to double 22% and 32% faster than its control, respectively (empty vector: td=1.93 h, CspA: td=1.58 h, and Pth: td=1.44 h). Based on the results, the factors boosting growth rate in C. gluctamicum were further discussed in the viewpoints of cellular energy state, oxidative stress management, and translation.

Current Status and Applications of Adaptive Laboratory Evolution in Industrial Microorganisms.

Adaptive laboratory evolution (ALE) is an evolutionary engineering approach in artificial conditions that improves organisms through the imitation of natural evolution. Due to the development of multi-level omics technologies in recent decades, ALE can be performed for various purposes at the laboratory level. This review delineates the basics of the experimental design of ALE based on several ALE studies of industrial microbial strains and updates current strategies combined with progressed metabolic engineering, in silico modeling and automation to maximize the evolution efficiency. Moreover, the review sheds light on the applicability of ALE as a strain development approach that complies with non-recombinant preferences in various food industries. Overall, recent progress in the utilization of ALE for strain development leading to successful industrialization is discussed.

Investigation of 3-aryl-pyrimido[5,4-e][1,2,4]triazine-5,7-diones as small molecule antagonists of ß-catenin/TCF transcription.

Nearly all colorectal cancers (CRCs) and varied subsets of other cancers have somatic mutations leading to ß-catenin stabilization and increased ß-catenin/TCF transcriptional activity. Inhibition of stabilized ß-catenin in CRC cell lines arrests their growth and highlights the potential of this mechanism for novel cancer therapeutics. We have pursued efforts to develop small molecules that inhibit ß-catenin/TCF transcriptional activity. We used xanthothricin, a known ß-catenin/TCF antagonist of microbial origin, as a lead compound to synthesize related analogues with drug-like features such as low molecular weight and good metabolic stability. We studied a panel of six candidate Wnt/ß-catenin/Tcf-regulated genes and found that two of them (Axin2, Lgr5) were reproducibly activated (9-10 fold) in rat intestinal epithelial cells (IEC-6) following ß-catenin stabilization by Wnt-3a ligand treatment. Two previously reported ß-catenin/TCF antagonists (calphostin C, xanthothricin) and XAV939 (tankyrase antagonist) inhibited Wnt-activated genes in a dose-dependent fashion. We found that four of our compounds also potently inhibited Wnt-mediated activation in the panel of target genes. We investigated the mechanism of action for one of these (8c) and demonstrated these novel small molecules inhibit ß-catenin transcriptional activity by degrading ß-catenin via a proteasome-dependent, but GSK3ß-, APC-, AXIN2- and ßTrCP-independent, pathway. The data indicate the compounds act at the level of ß-catenin to inhibit Wnt/ß-catenin/TCF function and highlight a robust strategy for assessing the activity of ß-catenin/TCF antagonists.