Journal: bioRxiv

Article Title: Biosynthesis of the Antibiotic Nonribosomal Peptide Penicillin in Baker’s Yeast

doi: 10.1101/075325

Figure Lengend Snippet: (a) Construction and sequencing of a library with alternative promoters for pcbC , pclA and penDE . Ten different promoters of four categories (strong, medium, weak or inducible) were randomly used to drive expression of each of the genes responsible for conversion of ACV to benzylpenicillin. Plasmid libraries of single genes with different promoters were initially constructed and then pooled to randomly assemble a multigene plasmid library. Single gene and multigene libraries were subjected to MinION nanopore sequencing. The multigene library was transformed into ACV producer Sc.A2 and transformants producing benzylpenicillin were Sanger sequenced to reveal identity of promoters. (b) Concentrations of benzylpenicillin secreted into the supernatant by the ten yeast strains selected from the promoter screen. The best producing strain makes ~5 ng/mL of benzylpenicillin as determined by LCMS. Under each bar, the category of the promoter in front of each gene is shown. Error bars represent standard error of the mean from three biological replicates. (c) Comparison of the percentage of promoters from each of the four categories for each pathway gene during Golden Gate library construction (single and multiple gene steps) and among the ten benzylpenicillin producing yeast strains as determined by DNA sequencing.

Article Snippet: A fresh MinION R7 Flow Cell Mk I (FLO-MIN104, Oxford Nanopore Technologies) was loaded into a MinION MK I (MIN-MAP002, Oxford Nanopore Technologies) and primed using a Nanopore Sequencing Kit according to the manufacturer’s instructions.

Techniques: Sequencing, Expressing, Plasmid Preparation, Construct, Nanopore Sequencing, Transformation Assay, Comparison, DNA Sequencing