Repairing growth of a genome-reduced strain using adaptive laboratory evolution and targeted gene knock-in
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ABSTRACT: We profiled growth of an evolved version of the genome reduced E. coli strain DGF-298 in various growth conditions. We used RNA-seq to compare this strain's growth to the parent wild-type strain. We also profiled the transcriptional effect of adding in a removed gene predicted by models to have a positive impact on growth (aldA).
Project description:We carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, frdA) involved in acetyl-CoA consumption, allowing the efficient utilization of acetate as its sole carbon and energy source. The transcriptomes according to the medium status (M9 aceate, M9 glucose) of the evolved strain (SBA01) and its parent strain (DSM01) were compared using RNA-seq.
Project description:We used RNA-seq to profile E. coli K-12 MG1655 strains subjected to adaptive laboratory evolution after chorismate synthase knockouts. Either isochorismate synthase (menF) or isochorismate synthase AND chorismate lyase (ubiC) was deleted from a strain of E. coli K-12 MG1655 that had already been previously adapted for growth on glucose minimal media. RNA-seq profiles of the original glucose-adapted strain, the 2 deletion strains, and 4 laboratory-evolved strains from each deletion are included in duplicate. ubiC catalyzes the first committed step of ubiquinone synthesis, an important molecule for the electron transport chain. Thus, these experiments allowed assessment of cellular adaptations to restore energy metabolism capability.
Project description:We attempted to improve the resistance of taxadiene-producing yeast strain to oxidative stress to develop a more robust yeast cell factory for improved Taxol® drug oxyenated taxanes precursors production from taxadiene. To this end, we evolved a yeast strain on H2O2-containing defined growth medium, supplemented with galactose as carbon source to induce the heterologous taxadiene biosynthesis pathway genes in that strain. The oxidative stress re-induction effect on the expression profiles of the superior evolved yeast strain (E_LRS5) was then studied before (steady state I) and during its continous use (steady state II) in galactose-limited chemostats, in parallel with the parent strain (LRS5).
Project description:Rhamnolipids (RL) are well-studied biosurfactants naturally produced by pathogenic strains of P. aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however CH4 provides an intriguing alternative as a substrate for RL production because it is low-cost and may mitigate greenhouse gas emissions. Here we demonstrate RL production from CH4 by Methylotuvimicrobium alcaliphilum 20Z. RLs were inhibitory to M. alcaliphilum growth at low concentrations (<0.05 g/L), so adaptive evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/L of RLs. Metabolomics and proteomics of the adapted strain grown on CH4 in the absence of RLs revealed metabolic changes increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared to the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 M of RLs, a 600-fold increase compared to the parent strain. This work has promise for developing technologies to produce fatty acid-derived bioporducts, including biosurfactants, from CH4.
Project description:Synthetic biology aims to design and construct bacterial genomes harboring the minimum number of genes required for self-replicable life. However, the genome-reduced bacteria often show impaired growth under laboratory conditions that cannot be understood based on the removed genes. The unexpected phenotypes highlight our limited understanding of bacterial genomes. Here, we deploy adaptive laboratory evolution (ALE) to re-optimize growth performance of a genome-reduced strain. The basis for suboptimal growth is the imbalanced metabolism that is rewired during ALE. The metabolic rewiring is globally orchestrated by mutations in rpoD altering promoter binding of RNA polymerase. Lastly, the evolved strain has no translational buffering capacity, enabling effective translation of abundant mRNAs. Multi-omic analysis of the evolved strain reveals transcriptome- and translatome-wide remodeling that orchestrate metabolism and growth. These results reveal that failure of prediction may not be associated with understanding individual genes, but rather from insufficient understanding of the strain's systems biology.
Project description:We have performed adaptive laboratory evolution of E. coli pdhR gene deletion strain to examine the adaptive strategies of E. coli.
Project description:Gene number of the fission yeast Schizosaccharomyces pombe is the lowest in free-living eukaryotes. We further reduced the gene number to examine a minimal gene set required for growth. The genome-reduced strain, named ASP3894, has four chromosomal deletion regions: 168.4 kb deletion in the left arm of chromosome 1 (ALT), 155.4 kb deletion in the right arm of chromosome 1 (ART), 211.7 kb deletion in the left arm of chromosome 2 (BLT) and 121.6 kb deletion in the right arm of chromosome 2 (BRT). As a result of the 657.3 kb genome reduction, 224 genes of approximately 5100 total genes were reduced. ASP3894 transcriptional profiling was compared with a parent strain, ASP3880, that is a non-genome-reduced strain to examine the influence of genome reduction.
Project description:Although fluorine is abundant in the earth’s crust, it is scarcely found in biomolecules. Adaptive laboratory evolution (ALE) experiments were conducted to introduce organofluorine into living microorganisms. By cultivating Escherichia coli with fluorinated indole analogs, microbial cells evolved that relinquished their dependence on indole and are instead capable of utilizing either 6-fluoroindole (6Fi) or 7-fluoroindole (7Fi) for growth (TrpS-catalyzed in-situ conversion of fluoroindole into fluorotryptophan (FTrp) and TrpRS-catalyzed incorporation of FTrp in context of protein biosynthesis). Consistent and complete adaptation of microbial populations was achieved and the quantitative proteome-wide replacement of Trp by either 6FTrp or 7FTrp was confirmed by nano-LC-MSMS. The dataset comprises the ancestral strain TUB00, two positive controls W-TUB165 and Ind-TUB165 (adapted to grow on tryptophan and indole), three 6Fi adapted lineages 6TUB128-OC, 6TUB165-MB4, 6TUB165-MB3 and two 7Fi adapted lineages 7TUB165-OC and 7TUB165-MB.