Tailored fatty acid synthesis via dynamic control of fatty acid elongation

Abstract

Medium-chain fatty acids (MCFAs, 4-12 carbons) are valuable as precursors to industrial chemicals and biofuels, but are not canonical products of microbial fatty acid synthesis. We engineered microbial production of the full range of even- and odd-chain-length MCFAs and found that MCFA production is limited by rapid, irreversible elongation of their acyl-ACP precursors. To address this limitation, we programmed an essential ketoacyl synthase to degrade in response to a chemical inducer, thereby slowing acyl-ACP elongation and redirecting flux from phospholipid synthesis to MCFA production. Our results show that induced protein degradation can be used to dynamically alter metabolic flux, and thereby increase the yield of a desired compound. The strategy reported herein should be widely useful in a range of metabolic engineering applications in which essential enzymes divert flux away from a desired product, as well as in the production of polyketides, bioplastics, and other recursively synthesized hydrocarbons for which chain-length control is desired.

Keywords: biochemicals; synthetic biology.

Fig. 1.

Engineering production of all even- and odd-length MCFAs in E. coli. (A) The KAS FabH elongates acetyl-CoA to form a four-carbon β-ketoacyl ACP, which is reduced to C4 acyl-ACP. In subsequent rounds of fatty acid synthesis, the KAS’s FabB and FabF elongate acyl-ACPs two carbons at a time to yield a range of even-chain–length fatty acyl-ACPs. Incorporation of propionyl-CoA in place of acetyl-CoA causes production of odd-chain acyl-ACPs; propionyl-CoA can be produced from propionate (prpE) or from expression of a genetic cassette that increases flux through the isoleucine pathway (thrA^frBC ilvA^fr) (15). Acyl-ACPs can be hydrolyzed to FFAs by an appropriate thioesterase (TE). Fatty acid degradation can be blocked by knocking out the β-oxidation enzymes fadD or fadE. (B) GC-MS analysis of FFA production by strain S001 [BL21*(DE3) ∆fadD] containing plasmid pEET (BfTES) (i), pTJF010 (CpFatB1) (ii), or pJT208 (UcFatB2) (iii) in M9 +0.5% glycerol alone (solid bars) or supplemented with 100 mM propionate (striped bars) 24 h after IPTG induction (n = 3, error bars = SEM). FFAs shown for the no-propionate experiments accounted for 67% (i), 85% (ii) and 72% (iii) of total FFAs (full chain-length profiles in Fig. S1). FFAs shown for the propionate experiments accounted for (i) 79%, (ii) 65%, and (iii) 84% of the total (Fig. S1). (C) Production of odd-chain FFAs by S001-pJT208-pCOLAthrA^frBCilvA^fr (n = 3, error bars = SEM). FFAs shown accounted for 81% of total. Asterisks in B and C indicate significantly increased odd-chain production (P < 0.05, one-tailed Student t test). (D) Final OD₅₉₅ of strains in this figure, with or without propionate supplementation (n = 3, error bars = SEM). Asterisks indicate decreased OD₅₉₅ compared with strain S001 alone (P < 0.05, one-tailed Student t test).

Fig. 2.

Chemical inhibition of fatty acid elongation can increase MCFA production. (A) Schematic diagram of fatty acid elongation. FabH performs the slow initial elongation step in fatty acid biosynthesis to generate C4-ACP; FabB and FabF more rapidly elongate the acyl-ACP 2 carbons at a time to produce 16–18 carbon acyl-ACPs, which are precursors to lipid synthesis and inhibit FabH more strongly than other acyl-ACPs (16, 17). Cerulenin inhibits FabB and FabF, but not FabH. (B) Total FFA produced by strain S001 expressing the indicated thioesterases over a range of cerulenin concentrations from 0 to 12.8 µg/mL, 24 h after IPTG induction, as measured by GC-MS (n = 3, error bars = SEM). Long-chain FFAs (C14–C18) accounted for ≤ 12 mg/L in any given measurement. Asterisks indicate the point at which mean fatty acid production is highest; the increase in production is significant for BfTES and CpFatB1 (P < 0.05, one-tailed Student t test), but not for UcFatB2. OD₅₉₅ data are provided in Fig. S2. (C) Filled circles: cerulenin concentration at which fatty acid production is maximal as a function of each thioesterase’s preferred chain length. C4 for BfTES (blue); C8 for CpFatB1 (green); C12 for UcFatB2 (orange). Filled triangles indicate the maximum fold-increase in FFA production over the no-cerulenin control (calculated as the ratio of maximal FFA production to that of the no-cerulenin control). (D) Ratio of the two most abundant FFAs produced by each thioesterase (shorter/longer), as a function of cerulenin concentration (n = 3, error bars = SEM). Single asterisks indicate that the given bar is significantly different from the no-cerulenin control with P < 0.05 (two-tailed Student t test). Double asterisks indicate that the given bar is significantly different from both other bars in the panel.

Fig. 3.

Engineering KAS for increased octanoic acid production. (A) Schematic indicating the elongation reactions carried out by ketoacyl synthases FabB and FabF. Wild-type FabB and FabF elongate acyl-ACPs with ≥ 4 carbons up to a length of 16–18 carbons. In our engineered system, FabBDeg can elongate up to 16–18 carbons but is degraded by the E. coli ClpXP system upon SspB expression. FabF* can only elongate up to 8 carbons. (B) StrepFabBDeg degradation in strain S007-SspBpET21b. Strain S007-SspBpET21b was induced (+IPTG, 1 mM) or not (−IPTG) at time 0 and StrepFabBDeg detected via Western blotting and densitometry at 0 and 8 h (n = 4, error bars = SEM). Loading was normalized to OD₆₀₀, and reported values are normalized to StrepFabBDeg levels at t = 0 h. StrepFabBDeg bands are shown below the bar graph; original blots are shown in Fig. S5. An asterisk indicates a significant decrease in StrepFabBDeg between the ± IPTG samples at 8 h (P < 0.05, one-tailed Student t test). (C) FFA production [as determined by the Free Fatty Acids, Half Micro Test (Roche)] and final culture OD₅₉₅ 44 h after induction, for strains expressing CpFatB1 and SspB in S002 [BL21*(DE3) ΔfadE] with the indicated modifications (n = 24; error bars = SEM). FFA production is represented as the percent increase over FFA production in the parent strain (S002). An asterisk indicates a significant increase in FFA production compared with S002 (P < 0.05 by one-tailed Student t test).

Fig. 4.

Optimizing octanoic acid yields via metabolic knockouts and SspB titration. (A) Total FFA production in BL21*(DE3) knockout strains. Each strain was grown in M9 + 0.5% glucose, CpFatB1 was induced from pTJF010 with 1 mM IPTG for 44 h, and total FFA measured with the Free Fatty Acids, Half Micro Test (Roche) (n = 3, error bars = SEM). Green, blue, and orange bars indicate single knockouts, double knockouts, and triple knockouts, respectively. Dashed lines indicate fatty acid production in the parent strains BL21*(DE3), BL21*(DE3) ∆fadD, and BL21*(DE3) ∆fadD ∆pta. An asterisk indicates FFA production significantly greater than the parent strain (P < 0.05 by one-tailed Student t test). OD₅₉₅ for these strains is shown in Fig. S6. (B) FFA production and OD₅₉₅ as a function of IPTG concentration (0, 0.125, 0.25, 0.5 or 1.0 mM). CpFatB1 and SspB expressed from pTJF038 and SspBpET21b respectively were cotitrated in S006 [BL21*(DE3)ΔfadD Δpta ΔlacY fabF* fabBDeg] (solid gray bars) with IPTG for 24 h in M9 + 0.5% glucose (n = 18, error bars = SEM). SspB alone was titrated with IPTG in S006 expressing SspB from SspBpET21b and CpFatB1 from aTc-inducible pJT255 induced with 400 ng/mL aTc (striped bars, n = 18, error bars = SEM). An asterisk indicates significantly different FFA production, or altered OD₅₉₅, versus the 0 mM IPTG control (P < 0.05 by two-tailed Student t test).