Part:BBa_K861500

FadL, Long-chain fatty acids transporter from E.coli str. K12

Long-chain fatty acids are transported across the cell membrane via a transportation/acyl-activation mechanism involving an outer membrane protein, FadL. The FadL gene encodes a 43,000-dalton membrane protein (FLP) which has been implicated as being essential for LCFA transportation.

William_and_Mary 2020 Contribution: FadL Literature Review

Endogenous to Escherichia coli, the FadL gene encodes a 33 kDalton outer membrane protein associated with the import of fatty acids. FadL works as one of multiple proteins necessary for the import and export of fatty acids. For example, FadD activates fatty acids by attaching Coenzyme A shortly after import by FadL. The fatty acid imported by FadL can be metabolized by the cell for carbon.

The FadL protein is inserted into the cell membrane due to a signal peptide 27 amino acids in length( Black, P. N. 1991) One embedded in the membrane, FadL binds to extracellular long-chain fatty acids with high affinity.(Liu, H. 2012). FadL transports these fatty acids across the outer membrane into the periplasm (Liu, H. 2012). FadL is necessary for transmembrane movement of fatty acids, which cannot freely move through the E. coli cell membrane; FadL acts in the facilitated transport of free fatty acids into the cell. The spread of this process has been defined in terms of Vmax= 800 pmol/min/mg enzyme and Km= 87.3 µM for 18:1 fatty acids (Black, P. N., 1988, Maloy et al., 1981).

FadL deletion may be combined with the manipulation of other genes for applications related to fatty acid production and export. If FadL is deleted, fatty acid accumulates extracellularly, which may be desirable when engineering bacteria for fatty acid production. For example, in the paper (Liu, H. 2012), the authors found the amplification of TesA and the deletion of fadL in E. coli BL21 (DE3) improved extracellular fatty acid production. They found that, after promoting TesA and deleting FadL, E.coli produced 4.8 g L−1extracellular fatty acid with a production rate of 0.004 gh−1 g−1 dry cell. In another paper, (Shin, K. S et. al 2017), researchers found that the deletion of FadL increased fatty acid production in most cases. They noted that deletion of ompF or FadL individually, without any additional genetic manipulation, resulted in marginal improvements in FFA production (Shin, K. S et. al 2017). The authors did note that with the marginal increase in total free fatty acid increase was accompanied by a substantial increase in the percentage of extracellular free fatty acid. FadL deletion increased the percentage of extracellular fatty acid to 34% of total fatty acid (Shin, K. S et. al 2017). Interestingly, the researchers found that deletion of FadL did not always increase the production of FFA when combined with other gene deletion. For example, when envR, gusC, and mdlA were deleted in addition to FadL total FFA production was reduced by 10% (Shin, K. S et. al 2017). The graph below shows genes knocked out and how they contributed to FFA production.

Additionally, FadL has been shown to have a significant effect on the integrity of the bacterial outer membrane (Tan Z. et al. 2017). In cases where FadL expression was decreased, researchers attributed a subsequent decrease in membrane integrity to a disruption of the lipid biosynthesis pathway, as fatty acid import is the main carbon source for E.coli. Since lipids are the main component of the cell membrane, even slight changes in lipid concentration can greatly decrease membrane integrity. (Tan Z. et al. 2017). In one study, researchers observed that a decrease in FadL expression actually led to a decrease in fatty acid production after a prolonged period of time, due to the disruption of the lipid biosynthesis. The researchers tested using 6 different promoters which expressed varying levels of FadL mRNA to see if there was a correlation between FadL expression and fatty acid titer. After 72 hours, the researchers found a positive relationship between the abundance of FadL mRNA and the overall fatty acid titer(Tan Z. et al. 2017) . Although FadL has been shown to increase extracellular fatty acid, it may decrease the overall production of fatty acid after a long period of time. This concern should be kept in mind when using FadL to increase extracellular fatty acid.

References

Black, P. N. (1991). Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport. Journal of Bacteriology,173(2), 435-442. doi:10.1128/jb.173.2.435-442.1991 Black P. N. (1988). The fadL gene product of Escherichia coli is an outer membrane protein required for uptake of long-chain fatty acids and involved in sensitivity to bacteriophage T2. Journal of bacteriology, 170(6), 2850–2854. https://doi.org/10.1128/jb.170.6.2850-2854.1988 Higashitani, A., Nishimura, Y., Hara, H., Aiba, H., Mizuno, T., & Horiuchi, K. (1993). Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli. Molecular and General Genetics MGG,240(3), 339-347. doi:10.1007/bf00280384 Liu, H., Yu, C., Feng, D., Cheng, T., Meng, X., Liu, W., . . . Xian, M. (2012). Production of extracellular fatty acid using engineered Escherichia coli. Microbial Cell Factories,11(1), 41. doi:10.1186/1475-2859-11-41 Maloy, S. R., Ginsburgh, C. L., Simons, R. W., & Nunn, W. D. (1981). Transport of long and medium chain fatty acids by Escherichia coli K12. The Journal of biological chemistry, 256(8), 3735–3742. Shin, K. S., & Lee, S. K. (2017). Increasing Extracellular Free Fatty Acid Production in Escherichia coli by Disrupting Membrane Transport Systems. Journal of Agricultural and Food Chemistry,65(51), 11243-11250. doi:10.1021/acs.jafc.7b04521.s001 Tan, Z., Black, W., Yoon, J.M. et al. Improving Escherichia coli membrane integrity and fatty acid production by expression tuning of FadL and OmpF. Microb Cell Fact 16, 38 (2017). https://doi.org/10.1186/s12934-017-0650-8

Sequence and Features

Assembly Compatibility:

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  Illegal AgeI site found at 1102
  
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