Project description:Model-guided chassis strain design has the potential to accelerate cellfactory development. In this experiment genetic targets were identified in silico and implemented in vivo to design a yeast chassis strain for enhanced production of succinic, malic and fumaric acid. The phenotype of engineered chassis strains was further optimised through adaptive laboratory evolution. RNA-seq analysis of engineered yeast chassis strains, evolved strains and wild-type (CEN.PK background)was performed to determine the effect of engineered gene deletions and evolution on the transcriptome.
Project description:We developed an artificial genome evolution system, which we termed ‘TAQing’, by introducing multiple genomic DNA double-strand breaks using a heat-activatable endonuclease in mitotic yeast. The heat-activated endonuclease, TaqI, induced random DSBs, which resulted in diverse types of chromosomal rearrangements including translocations. Array comparative genomic hybridization (aCGH) analysis was performed with cell-fused Saccharomyces cerevisiae strains induced genome evolution by TAQing system. Some of copy number variations (CNVs) induced by massive genome rearrangements were detected in the TAQed yeast strains.
Project description:We constructed S. cerevisiae BY_DEH+ strain which is able to assimilate both 4-deoxy-L-erythro-5-hexoseulose uronate (DEH, a monouronic acid produced by digestion of alginate with exo-type alginate lyase) and mannitol from BY4742 strain and improved its ability to assimilate DEH through an adaptive evolution (Matsuoka et al. Sci. Rep. 2017, 7, 4206). To examine transcriptional responses of the yeast to DEH and mannitol, gene expressions of the evolved strain (BY_DEH++ strain) in DEH medium, mannitol medium, and glucose medum were analyzed. For revealing the mechanisms underlying the adaptive evolution, gene expressions of both BY_DEH+ strain and BY_DEH++ strain in both DEH medium and glucose medium were measured.
Project description:Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study how the mutational landscape varies across populations during adaptation, we performed experimental evolution on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining qPCR, array CGH, restriction digestion and CHEF gels, and whole genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. Over a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that are driven by the coexistence of independent beneficial mutations. Selective amplifications rapidly evolve under this selection pressure, in particular common inverted amplifications containing the sulfate transporter gene SUL1. Compared to single clones, detailed analysis of the populations uncovers a greater complexity whereby multiple subpopulations arise and compete despite a strong selection. The most common evolutionary adaptation to strong selection in these populations grown in sulfate limitation is determined by clonal interference, with adaptive variants both persisting and replacing one another.
