Fig. 2 | Nature Communications

Fig. 2

From: Industrial brewing yeast engineered for the production of primary flavor determinants in hopped beer

Fig. 2

Iterative improvement of strain design towards targeted monoterpene production levels. a Schematic of design–build–test–learn cycle. Design: Constructs were designed by combining yeast toolkit parts (i.e., promoters, terminators, linkers, etc.) with monoterpene biosynthesis pathway genes. Build: Methodology for integrating constructs into brewer’s yeast. Note that, for simplicity, only a single allelic copy of the ADE2 locus is diagrammed. The ADE2Δ strain was co-transformed with a Cas9/sgRNA plasmid and repair template, which targeted a double-stranded break (DSB) in the ADE2 3′ sequence. Test: Data were collected using LC/MS, HPLC, and GC/MS. Learn: Correlation analyses informed design principles. Mathematical models were used to evaluate the extent to which design principles improved strain search efficiency. Variables corresponding to measured protein levels are highlighted. b, c Transformation plate illustrating colorimetric screening method. ADE2 encodes an enzymatic step in purine biosynthesis and its deletion results in the accumulation of a metabolite with red pigment when grown on media containing intermediate adenine concentration. Because the repair template contains the ADE2 gene, templated DSB repair results in a white colony phenotype. Because brewer’s yeasts have multiple allelic copies of ADE2, stable integration requires repair at multiple ADE2Δ genomic loci. White colonies streaked from transformation plates result in either white colony color (b) or variegated colony color (c); white colony color corresponds to homozygous integration; variegated colony color corresponds to heterozygous integration, illustrating genetic instability of heterozygous allele containing a large DNA construct. d Illustration of assembly steps from parts (promoters/genes) to gene cassettes, to repair templates for first iteration strains. Assemblies are simplified for clarity—for detailed description see Supplementary Fig. 3. e Relative promoter strengths with corresponding protein and product abundances and sugar consumption (attenuation). Strains are sorted by total monoterpene production

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