In silico identification of gene targets to enhance C12 fatty acid production in Escherichia coli

Abstract

The global interest in fatty acids is steadily rising due to their wealth of industrial potential ranging from cosmetics to biofuels. Unfortunately, certain fatty acids, such as monounsaturated lauric acid with a carbon atom chain length of twelve (C12 fatty acids), cannot be produced cost and energy-efficiently using conventional methods. Biosynthesis using microorganisms can overcome this drawback. However, rewiring a microbe's metabolome for increased production remains challenging. To overcome this, sophisticated genome-wide metabolic network models have become available. These models predict the effect of genetic perturbations on the metabolism, thereby serving as a guide for metabolic pathways optimization. In this work, we used constraint-based modeling in combination with the algorithm Optknock to identify gene deletions in Escherichia coli that improve C12 fatty acid production. Nine gene targets were identified that, when deleted, were predicted to increase C12 fatty acid titers. Targets play a role in anaplerotic reactions, amino acid synthesis, carbon metabolism, and cofactor-balancing. Subsequently, we constructed the corresponding (combinatorial) deletion mutants to validate the in silico predictions in vivo. Our highest producer (ΔmaeB Δndk ΔpykA) reaches a titer of 6.7 mg/L, corresponding to a 7.5-fold increase in C12 fatty acid production. This study demonstrates that model-guided metabolic engineering is a useful tool to improve C12 fatty acid production. KEY POINTS: •Escherichia coli as a promising biofactory for unsaturated C12 fatty acids. •Optknock to identify non-obvious gene deletions for increased C12 fatty acids. •7.5-fold higher C12 fatty acid production achieved by deleting maeB, ndk, and pykA.

Keywords: Escherichia coli; C12 fatty acids; Lauric acid; Model-guided metabolic engineering; Oleochemicals; Optknock.

Fig. 1

Schematic overview of the fatty acid biosynthesis in E. coli. Enzymes are indicated in red and pathway sections in green. The fatty acid biosynthesis is initiated by converting acetyl-CoA to malonyl-CoA. Subsequently, the latter is functionalized by replacing CoA with an ACP and further converted into acetoacetyl-ACP, which enters the elongation as the first ketoacyl-ACP. The elongation consists of four iterative steps, where each completed cycle adds two carbon atoms to the acyl chain. In detail, ketoacyl-ACP is reduced to hydroxyacyl-ACP, which is hydrated to enoyl-ACP and further reduced to acyl-ACP. This intermediate can either be elongated further by completing another cycle of the fatty acid biosynthesis cycle, or it can be terminated by an acyl-ACP thioesterase, which removes the ACP and releases the free fatty acid. To produce unsaturated fatty acids, the hydroxyacyl-ACP intermediate is dehydrated and isomerized into a cis-enoyl-ACP intermediate to retain the double bond. Thereafter, the ACP-coupled unsaturated fatty acid can be either released by a thioesterase or it can continue one or more cycles similarly to their saturated analogues. CoA coenzyme A, ACP acyl carrier protein

Fig. 2

Schematic overview of computational approaches for increasing the FA120 ACPHi (BTE) flux. A Impact of FA120 ACPHi on growth. The lower bound of FA120 ACPHi was increased in a stepwise manner and the biomass flux was determined for each step. B Identifying deletion targets based on maximizing FA120 ACPHi with GK1 representing the E. coli gene gmk, CHORS aroC, PSCVT aroA, and SHKK aroL. C Utilization of Optknock to identify gene deletions for optimized FA120 ACPHi and biomass flux with ACS representing the E. coli gene acs, NDPK4 adk and ndk, PYK6 pykA, LDH-D ldhA, DRPA deoC, PYK2 pykA, CHORS aroC, NDPK2 adk and ndk, ME2 maeB and PYK3 pykA. Predicted fluxes are listed in Table 2

Fig. 3

Optimizing BTE expression in E. coli for optimal C12 fatty acid production. A Total fatty acid and B C12 fatty acid production in the BTE-overexpressing E. coli strain induced with 0–1000 ng/mL anhydrotetracycline and grown for 24 h in LB. C, D Wild-type E. coli (wt) and E. coli expressing BTE (BTE) were grown for 24 h in LB medium with an inducer concentration of 100 ng/mL and with and without 10 g/L glucose. C Total fatty acids and D C12 fatty acids (in mg/L) in the wild-type (wt) and BTE-overexpressing (BTE) strains in glucose-poor and -rich conditions. Mean values and SEM (standard error of the mean) are shown (n ≥ 3)

Fig. 4

Effect of the predicted (combinatorial) gene knock-outs on the production of C12 fatty acids in E. coli, cultivated in LB. E. coli strains (indicated on the x-axis) expressing BTE from an anhydrotetracycline-inducible promoter were grown for 24 h in LB medium containing 100 ng/mL of inducer. A Total fatty acid production. B C12 fatty acid production. Mean values and SEM (standard error of the mean) are shown (n ≥ 3). Asterisks indicate significant differences calculated with ANOVA in saturated and unsaturated C12 fatty acids compared to E. coli BW25113 expressing BTE, indicated as BTE (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 5

Effect of the predicted (combinatorial) gene knock-outs on the production of C12 fatty acids in E. coli, cultivated in 10 g/L glucose-enriched LB. E. coli strains (indicated on the x-axis) expressing BTE from an anhydrotetracycline-inducible promoter were grown for 24 h in LB medium containing 10 g/L glucose and 100 ng/mL of inducer. A Total fatty acid production. B C12 fatty acid production. Mean values and SEM (standard error of the mean) are shown (n ≥ 3). Asterisks indicate significant differences calculated with ANOVA in saturated and unsaturated C12 fatty acids compared to E. coli BW25113 expressing BTE, indicated as BTE (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)