Fig. 5

CRISPRi-mediated improvement of P3G production through increased SAM availability. a MetJ promoter (P _metJ ) region architecture with anti-metJ CRISPR elements indicated (spacers in red, PAMs in yellow). Three promoters drive metJ expression (+1 sites in green and −35 and −10 sites for P _metJ2 and P _metJ3 in blue), so CRISPR spacers were designed to target the downstream promoter P _metJ3 in order to simultaneously repress transcription from all three promoters, collectively indicated as P _metJ , b schematic representation of the regulatory cascade engendered by CRISPRi-mediated deregulation of the metJ regulon. Repression of MetJ de-represses the metJ regulon, which is composed of at least twelve genes and nine promoters involved in methionine and SAM biosynthesis, including MetR (red). De-repression of MetR prompts activation of methionine synthase (metH/metE in green), the final step in methionine biosynthesis immediately preceding SAM synthase. The overall effect of MetJ silencing is unregulated, increased flux through the methionine and SAM biosynthetic pathways, c both anti-metJ CRISPR spacers achieved twofold increase in P3G titer and approximately 2.5-fold increase in C3G to P3G conversion (d) relative to the non-targeting, scrambled CRISPR negative control spacer (NT), e sampling time course demonstrates that C3G (red circles) and P3G (blue circles) degradation occurs in culture and that optimization is required to reduce the detrimental effect of degradation on final titers, c, d utilize maximum titers obtained from optimal sampling conditions (blue arrows). Induction of slightly slower growing MetJ-repression strains (middle and right) was delayed by 1 h (black arrows) to ensure OD₆₀₀ (gray squares) at induction was identical to NT strain (left), specifically corresponding to pre-determined optimal mid-log phase induction at OD₆₀₀ ~5.1–5.4