Disruption of membrane transport systems
4.5. Discussion
In this study, screening of a library of transposon insertion mutants led to the identification of three genes that mediate FFA overproduction in E. coli. Deletion of envR, gusC, or mdlA resulted in increased total FFA production (1.4-, 1.8-, or 1.2-fold, respectively) without a detrimental effect on cell growth. Interestingly, all three proteins encoded by these genes are involved in membrane transport systems, suggesting that engineering of membrane transport proteins may be crucial for increasing FFA production. However, further work is needed to investigate the precise relationship between increased FFA production and inactivation of these genes.
Our study shows that the combination of transposon mutagenesis with the use of a fatty acid biosensor is a powerful strategy for the isolation of strains exhibiting increased FFA production. In a previous study, a library of ~2,800 transposon mutants was screened for mutants that produced high levels of FFAs using a Nile red-based high-throughput screening method[103]. Inactivation of fadB, encoding enoyl-CoA hydratase, was observed to cause a 20% increase in FFA titer compared with the control strain. However, transposon mutations in envR, gusC, and mdlA were not identified in the
production than deletion of fadB. The Nile red-based screening method also showed a high false discovery rate, and the library was not large enough for complete coverage of the E. coli genome.
These considerations suggest that our strategy, which utilizes a fatty acid biosensor-based screening method and a larger library, may be more effective for the isolation of mutants that produce high levels of FFAs.
The transcriptional regulator EnvR represses expression of the multidrug efflux pump gene acrAB[111]. AcrAB is known to be a drug/H+ antiporter that confers resistance to antibiotics, detergents, organic compounds, and free fatty acids[111-113]. Engineering of the efflux pump previously increased tolerance or production of several compounds including free fatty acids[113, 119-121]. The observation that deletion of envR and overexpression of AcrAB yielded similar increases in total FFA levels and the proportion of extracellular FFAs (Fig. 4.1) suggests that deletion of envR might lead to high FFA production via induction of AcrAB expression. Overexpression of AcrAB might in turn promote interaction with TolC, a key component of the multidrug efflux transporter, to form the AcrAB-TolC complex, promoting secretion of internally produced FFAs.
The outer membrane porin GusC is known to be responsible for uptake of β- glucuronides[122]. Expression of GusC is induced by two kinds of effector molecules, glucuronate (a sugar acid derived from glucose) and β-glucuronides (compounds synthesized by linking glucuronate to another substance via a glycosidic bond)[123]. GusC expression has been shown to decrease during the transition of cells from exponential phase to stationary phase[124]. GusC has homology to the Occ (outer membrane carboxylate channels) family, which requires substrates with carboxyl groups for efficient import[125, 126]. Occ family members have specificity toward a wide range of substrates, including basic amino acids, glucuronate, lactate, and vanillate[125, 127]. Occ family members also recognize and import carbapenem, an antibiotic with high molecular weight and a carboxyl group[128, 129]. However, the mechanism for the increase in FFA production observed upon deletion of gusC is not readily explained by these previously identified transport activities.
MdlA is a putative multidrug resistance-like ABC exporter[130], and its inactivation resulted in a 15% increase in FFA production (Fig. 4.1). However, the mdlA deletion mutant did not exhibit increased extracellular FFA levels compared with the control strain, indicating that MdlA is not involved in fatty acid transport (Fig. 4.1). Previously, the expression level of MdlA was shown to decrease 14-fold when E. coli cells enter stationary phase in glucose minimal medium[131]. Thus, MdlA has been predicted to be involved in steady-state growth in E. coli. Although it is difficult to explain the relationship between inactivation of MdlA and improved FFA production, our results nonetheless suggest that MdlA plays at least an indirect role in FFA production.
extracellular FFA, but a similar proportion of extracellular FFAs (41% of total FFA content), compared with the envR deletion strain (Fig. 4.5). Deletion of fadL, encoding an outer membrane protein responsible for uptake of LCFAs, has been shown previously to increase extracellular FFA production (30-40% of total FFA content)[39]. Deletion of fadL in the triple knockout strain resulted in an 8% decrease in total FFA production, but an increase in the percent of extracellular FFAs to total FFAs (Fig. 4.5 and Table S2). This result indicates that deletion of envR and fadL has a synergistic effect on extracellular FFA levels. A further increase in FFA production was observed when deletion of fadL was accompanied by deletion of ompF, encoding an outer membrane porin responsible for uptake of SCFAs and MCFAs (Fig. 4.5 and Table 4.3). This result implies that inactivation of OmpF and FadL in the triple knockout strain blocked uptake of SCFAs, MCFAs, and LCFAs produced by the action of ʹTesA, while permitting FFA export. This mutant exhibited a three-fold increase in extracellular FFA production compared with the triple knockout strain, accounting for approximately 50% of total FFA production 72 h after induction (Fig. 4.6).
Table 4.3. Results of batch fermentations with engineered strains Strain Genotype description Intracellula
r FFAa
Extracellul ar FFAa
Ratio of extra
FFA
SBF06 ‘TesA 62.27 27.87 30.94
SBF22 ΔenvR, ‘TesA 80.12 52.74 39.67
SBF23 ΔgusC, ‘TesA 107.16 46.16 30.08
SBF24 ΔmdlA, ‘TesA 77.57 32.11 29.17
SBF25 ΔenvR, ΔgusC, ΔmdlA, ‘TesA 93.39 65.15 41.11
SBF26 ‘TesA, GusC 44.52 21.93 33.03
SBF27 ‘TesA, MdlA 65.28 25.52 28.22
SBF28 ‘TesA, AcrAB 87.95 57.49 39.44
SBF29 ΔenvR, ΔacrAB, ‘TesA 68.71 23.65 25.53
SBF30 ΔompF, ‘TesA 70.05 31.47 30.97
SBF31 Δaas, ‘TesA 60.91 27.10 31.54
SBF32 ΔfadL, ‘TesA 65.67 34.40 34.33
SBF33 SBF25 with ΔompF 101.31 68.35 40.29
SBF34 SBF25 with Δaas 88.34 57.50 37.38
SBF35 SBF25 with ΔfadL 79.10 67.37 46.00
SBF36 SBF25 with ΔompF 85.40 60.40 41.35
SBF37 SBF25 with ΔompF, ΔfadL 96.82 85.92 47.02
SBF38 SBF25 with Δaas, ΔfadL 78.45 45.35 36.78
SBF39 SBF25 with ΔompF, Δaas, ΔfadL 100.61 83.99 45.43
Note: All data were obtained or calculated after 48h of post-induction.
a. The concentration of FFA was normalized by value of OD600 (mg/L/OD).