Journal: Metabolic engineering

Article Title: Kinetically guided, ratiometric tuning of fatty acid biosynthesis

doi: 10.1016/j.ymben.2021.11.008

Figure Lengend Snippet: (A) Plasmid-borne systems for adjusting the intracellular concentrations of TesA, FabH, and FabB (pCDM4-TesA, pBad18-FabH, and pFD152-FabB, respectively). Genes: thioesterase 1 (tesA), β-ketoacyl-ACP synthase III (fabH), and β-ketoacyl-ACP synthase I (fabB). (B) Induction of TesA in a strain of E. coli harboring the TesA control system (ΔfadD + pCDM4-TesA). Production peaks at 1 μM IPTG. (C) Induction of FabH in a strain harboring the TesA and FabH control systems (ΔfadD + pCDM4-TesA + pBad18-FabH; 1 μM IPTG). Overexpression of FabH inhibits fatty acid synthesis and increases chain length. (D) Induction of dCas9 [FabB] (i.e., inducible dCas9 alongside a constitutively expressed sgRNA for fabB) in a strain harboring the TesA and FabB control systems (ΔfadD + pCDM4-TesA + pFD152-FabB; 1 μM IPTG). Transcriptional repression of FabB can reduce average chain length without altering total production. (E) Fatty acid profiles of the most productive cases from B and D. In B-E, plots of production depict the mean, SE, and associated measurements for n ≥ 3 biological replicates. Plots of length depict the mean for n ≥ 3 biological replicates (see Fig. S5 for estimates of average mole fraction and SE). For all plots, we measured fatty acids at 24 h after induction (30 °C).

Article Snippet: Plasmids and strains for fatty acid production We began the assembly of plasmid-based control systems by using templates supplied by colleagues from other labs and institutions: We received pCDM4 (Addgene #49796) from Mattheos Koffas; pBad18-TesA-R3M1 (TesA C12 ) and pBad18-TesA-R3M4 (TesA C8 ) from Brian Pfleger; and pFD152 (Addgene plasmid #125546) from David Bikard.

Techniques: Plasmid Preparation, Control, Over Expression

Journal: Metabolic engineering

Article Title: Kinetically guided, ratiometric tuning of fatty acid biosynthesis

doi: 10.1016/j.ymben.2021.11.008

Figure Lengend Snippet: (A) Plasmid-borne systems for adjusting the intracellular concentrations of TesA, FabF, and FabH (pBad18-TesA, pCDM4-FabF, and pFD152-FabH, respectively). Genes: thioesterase 1 (tesA), β-ketoacyl-ACP synthase II (fabF), and β-ketoacyl-ACP synthase III (fabH). (B) Induction of TesA in a strain of E. coli harboring the TesA control system (ΔfadD + pBad18-TesA). Production peaks at 0.1% (w/v) arabinose (Ara). (C) Induction of FabF in a strain harboring the TesA and FabF control systems (ΔfadD + pBad18-TesA + pCDM4-FabF; 0.1% arabinose). Rising concentrations of IPTG inhibit fatty acid synthesis and increase average chain length. (D) Induction of dCas9 [FabH] (i.e., inducible dCas9 alongside a constitutively expressed sgRNA for fabH) in a strain harboring the TesA, FabF, and FabH control systems (ΔfadD + pBad18-TesA + pCDM4-FabF + pFD152-FabH; 0.1% arabinose and 1 μM IPTG). Transcriptional repression of FabH does not influence either total production or chain length. (E) Fatty acid profiles of the most productive cases from B and D. In B-E, plots of production depict the mean, SE, and individual measurements for n ≥ 3 biological replicates. Plots of length depict the mean for n ≥ 3 biological replicates (see Fig. S14 for estimates of average mole fraction and SE). For all plots, we measured fatty acids at 24 h after induction (30 °C).

Article Snippet: Plasmids and strains for fatty acid production We began the assembly of plasmid-based control systems by using templates supplied by colleagues from other labs and institutions: We received pCDM4 (Addgene #49796) from Mattheos Koffas; pBad18-TesA-R3M1 (TesA C12 ) and pBad18-TesA-R3M4 (TesA C8 ) from Brian Pfleger; and pFD152 (Addgene plasmid #125546) from David Bikard.

Techniques: Plasmid Preparation, Control

Journal: Metabolic engineering

Article Title: Kinetically guided, ratiometric tuning of fatty acid biosynthesis

doi: 10.1016/j.ymben.2021.11.008

Figure Lengend Snippet: (A) Induction of TesAC12 in the ΔfadD strain harboring a TesAC12 control system (ΔfadD + pCDM4-TesAC12). Low levels of IPTG reduce average chain length without altering total production. (B) Induction of TesAC8 in the ΔfadD strain harboring a TesAC8 control system (ΔfadD + pCDM4-TesAC8). Low levels of IPTG can increase total production. Enzymes: TesA (thioesterase 1), TesAC12 (C12-specific mutant of TesA), TesAC8 (C8-specific mutant of TesA), and FabB (β-ketoacyl-ACP synthase I).C-D) Induction of dCas9 [FabB] in the presence of either (C) TesAC12 or (D) TesAC8 (ΔfadD + pCDM4-TesACx + pFD152-FabB, where x = 12 or 8; TesAC12: 1 μM IPTG, TesAC8: 10 μM IPTG). At low inducer concentrations, transcriptional repression of FabB reduces average chain length and causes a slight reduction in titer (e.g., 14–33% at 0.25 μg/mL aTC). In A-D, plots of total production depict the mean, SE, and individual measurements for n ≥ 3 biological replicates, and plots of length depict the mean for n ≥ 3 biological replicates (Fig. S17 includes the mean and SE of mole fractions). For all plots, we measured fatty acids at 24 h after induction (30 °C).

Article Snippet: Plasmids and strains for fatty acid production We began the assembly of plasmid-based control systems by using templates supplied by colleagues from other labs and institutions: We received pCDM4 (Addgene #49796) from Mattheos Koffas; pBad18-TesA-R3M1 (TesA C12 ) and pBad18-TesA-R3M4 (TesA C8 ) from Brian Pfleger; and pFD152 (Addgene plasmid #125546) from David Bikard.

Techniques: Control, Mutagenesis

Journal: Metabolic engineering

Article Title: Kinetically guided, ratiometric tuning of fatty acid biosynthesis

doi: 10.1016/j.ymben.2021.11.008

Figure Lengend Snippet: (A) The average chain length (bottom) and standard deviation (top) of fatty acids produced by strains of E. coli examined in this study (ΔfadD with control systems and inducer concentrations described Table S7). Compositional adjustments can shift average chain length from 11 to 16 carbons while maintaining total production levels at or above 1500 μM (>350 mg/L). Highlights: Highly productive strains that produce short (yellow, average = 11.0 ± 0.2 carbons) and long (blue, average = 16.1 ± 0.04) chains. (B) Fatty acid profiles for the strains highlighted in A. Control systems: Low CL (ΔfadD + pCDM4-TesA + pFD152-FabB; 1 μM IPTG and 0.25 μg/mL aTC) and high CL (ΔfadD + pBad18-TesA + pCDM4-FabF; 0.1% arabinose and 0 μM IPTG). Pie charts: normalized LFQ intensities of TesA, FabF, FabB, and FabH from quantitative proteomics performed on cell pellets from highlighted strains (unnormalized LFQ intensities appear in Fig. S20). For the low-CL strain, intracellular concentrations of TesA and FabF are high and low, respectively, relative to the high-CL strain. In the low-CL strain, the concentration of FabB is also low. FabH levels are similar for both strains. Enzymes: thioesterase 1 (TesA), β-ketoacyl-ACP synthase II (FabF), β-ketoacyl-ACP synthase I (FabB), and β-ketoacyl-ACP synthase III (FabH) (C–D) A comparison of fatty acid production by strains that produce large amounts of (C) C12 or (D) C16 fatty acids. Compositional adjustments can achieve titers of each chain length that are similar to those afforded by non-native thioesterases (bars with a black T), mutant thioesterases (bars with a red T) or mutant ACP (bars with a red A). Plots in A-B depict the mean, SE, and associated measurements for n ≥ 3 biological replicates. For this study, we measured fatty acids at 24 h after induction (30 °C). See Fig. S21 and Table S8 for detailed descriptions of the studies referenced in C-D. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Plasmids and strains for fatty acid production We began the assembly of plasmid-based control systems by using templates supplied by colleagues from other labs and institutions: We received pCDM4 (Addgene #49796) from Mattheos Koffas; pBad18-TesA-R3M1 (TesA C12 ) and pBad18-TesA-R3M4 (TesA C8 ) from Brian Pfleger; and pFD152 (Addgene plasmid #125546) from David Bikard.

Techniques: Standard Deviation, Produced, Control, Quantitative Proteomics, Concentration Assay, Comparison, Mutagenesis