ABSTRACT
Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in Saccharomyces cerevisiae under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.
Subject(s)
Chromosomes, Artificial, Yeast/chemistry , Genome, Fungal , Saccharomyces cerevisiae/genetics , Synthetic Biology/methods , Bacterial Proteins , CRISPR-Associated Protein 9 , Chromosomes, Artificial, Yeast/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Endonucleases , Gene Editing , Gene Rearrangement , Meiosis , Models, Genetic , Saccharomyces cerevisiae/cytology , Transformation, GeneticABSTRACT
Cellular redox status and oxygen availability influence the product formation. Herein, decreasing agitation speed or adding vitamin C (Vc) achieved the 2,3-BDL yield of 0.40 g g(-1) or 0.39 g g(-1)glucose under batch fermentation, respectively. To our knowledge, this is the highest 2,3-BDL yield reported so far for Paenibacillus polymyxa without adding acetic acid. The NADH/NAD(+) ratio and 2,3-BDL titer could be increased significantly by reducing the agitation speed or adding Vc, indicating that the enhancement of 2,3-BDL is closely associated with the adjustment of NADH/NAD(+) ratio. Especially, Vc addition elevated the 2,3-BDL titer from 43.66 g L(-1) to 71.71 g L(-1) within 54 h under fed-batch fermentation. This is the highest titer of 2,3-BDL so far reported for P. polymyxa from glucose fermentation. This work provides a new strategy to improve 2,3-BDL production and helps us to understand the responses of P. polymyxa to extracellular oxidoreduction potential.
