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Functional composite part for LCFA transport and metabolism

1.Introduction

To tackle the obesity problem, the iGEM team RDFZ_China 2022 resorted to engineered microbe. The team aimed to design a gut microbe with enhanced ability to transport and metabolize LCFA, in order to absorb and digest the LCFA in human intestine, and thus reduce the fat absorption of the host.

The team provides both the FadL and FadD within the composite part, which give an enhanced performance of LCFA transport and metabolism.

2.Protein and function overview

The LCFA transport and metabolism pathway mainly consists of 4 proteins, FadD, FadL, FadE and FadR. FadL is the LCFA transport protein, enabling facilitated transport of exogenous LCFA, while FadD is the main Fatty Acyl-CoA Synthetase in E.coli Nissle 1917. FadE is responsible for further dehydrogenation of LCFA-CoA to produce fatty enoyl-CoA, the first compound in the β oxidation pathway, while FadR is a transcription factor that regulates the n fatty acid biosynthesis and fatty acid import, activation, and oxidation.

Exogenous LCFA can be transported by FadL to enter the periplasm, and traverse into the cell after modifications. FadD which binds to the outer membrane of the cell abstracts the LCFA molecule and synthesize LCFA-CoA. This unidirectional transportation is given the name vectorial transport. After LCFA-CoA are sequestered inside the cell by vectorial thioesterification with FadD, they can bind to the repressor protein FadR. This binding to FadR causes it to dissociate from operator sites on the fatty acid degradative (Fad) regulon, relieving the repression on the regulon transcription such that Fad genes can be expressed. Afterwards, FadE catalyzes the formation of fatty enoyl-CoA, and LCFA can thus enter the be β oxidation pathway and be metabolized. (Yoo, 2001)

Figure1. Components of the LCFA transport pathway (Bae,2014)

3.Individual components

FadL

Introduction to FadL

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. 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). FadL is necessary for transmembrane movement of fatty acids, which cannot freely move through the E. coli cell membrane. (Black, P. N., 1988) Once embedded in the membrane, FadL binds to extracellular long-chain fatty acids with high affinity, and can transports these fatty acids across the outer membrane into the periplasm (Liu, H. 2012). 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 (Maloy et al., 1981). However, in the E. coli model system, fatty acid import not only is dependent on the membrane-bound transporter, FadL, but also requires the FACS, FadD (see below). This is because FadD catalyzes the esterification of LCFA with CoA, and thus trap the LCFA, rendering the whole process unidirectional. (Black, P. N., 1988)

Figure2. FadL facilitate the transportation of exogenous LCFA through the outer membrane.

Other features and applications of FadL

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.

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).

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.

FadD

Introduction to FadD

FadD is a soluble fatty acyl CoA synthetase(FACS) endogenous to Escherichia coli. It is classified as an AMP-forming fatty acid CoA ligase, meaning that it combines fatty acids with Coenzyme A molecules in a reaction that is powered by converting ATP to AMP.

Figure3.FadD catalyze the ligation of CoA to LCFA

Fatty Acyl-CoA Synthetase(FACS) is used in conjunction with thioesterase to greatly increase the concentration of Fatty Acyl-CoA present in the cell, to increase the amount of bioproducts produced. It is used to produce Fatty Alcohols and Wax Esters in the 2011 Utah_State iGEM project.

Figure4.Project of team 2011 Utah_State

FadD can be found within the cell by the plasma membrane, where it is non-integrally associated. Its activity is enhanced by the membrane lipids or detergents nearby. The protein has a molecular weight of 62,028 Daltons, though it forms a dimer with a molecular weight of about 120,000. It is contained within a 2.2 kilobase fragment of the E. coli genome, as part of a fatty acid degradative regulon along with fadBA, fadE, and fadL, all under control of repressor FadR. Transcription starts 60 base pairs upstream of the translation start. After the translational stop is a GC-rich inverted repeat and a 8T transcriptional terminator. Two FadR operator sites are found at -13 to -29 and -99 to -115. A rare UUG codon at translation initiation may downregulate expression. (Black, 1992)

FadD activates both medium and long chain fatty acids into fatty acyl CoA thioesters, which are substrates for beta oxidation, phospholipid biosynthesis, and cellular signalling. Beta oxidation is the pathway that degrades fatty acids, which can be regulated by fatty acyl CoA thioesters. (Yoo, 2001).

Chemical Reactions, Substrate Specificity, and Kinetics of FadD

Mechanistic equation:

FA + ATP = FA-AMP (needs Mg2+), FA-AMP + CoASH = FA-SCoA.

Figure5. The chemical reaction catalyzed by FACS FadD

FadD activates fatty acids by converting their carboxyl group into an acyl-CoA thioester, which is a stronger electrophile. Fatty acids enter the FadD active site from the membrane through a narrow channel that faces the inner membrane while ATP enters through a distinct large channel. Binding these molecules causes FadD to undergo ligand-induced conformational changes. The molecules then form an AMP-FA intermediate, which the flexible C-terminal clamps in order to position the intermediate and prevent its escape. CoA, the final substrate, binds to FadD after the fatty acid and ATP. CoA enters the FadD active site via a third channel and attacks the new bond, generating FA-CoA and AMP. Supposedly, when CoA bonds to a long chain fatty acid, AMP is pushed from the active site by the LCFA-CoA product, but this push is less pronounced with MCFA. (Ford, 2015)

FadD belongs to a class of adenylate forming enzymes, whose fatty acid tunnel length determines substrate specificity by accommodating a long hydrophobic tail. FadD has broad chain length specificity with a Vmax ranging from 2632 nmol/min/mg protein for C12 to 135 for C6. However, its maximal activity is reserved for fatty acids with carbon numbers ranging between 12 and 18, activating both mono- and poly-unsaturated fatty acids. The thioesters synthesized are destined for degradation or phospholipid incorporation. FadD has lower activity on medium chain fatty acids with 6 to 12 carbons. Downstream beta oxidation enzymes also have poor activity on MCFA. (Black, 1992)

Protein Function & Overview

FadD is a soluble fatty acyl CoA synthetase endogenous to Escherichia coli. It is classified as an AMP-forming fatty acid CoA ligase, meaning that it combines fatty acids with Coenzyme A molecules in a reaction that is powered by converting ATP to AMP. FadD activates both medium and long chain fatty acids into fatty acyl CoA thioesters, which are substrates for beta oxidation, phospholipid biosynthesis, and cellular signalling. Beta oxidation is the pathway that degrades fatty acids, which can be regulated by fatty acyl CoA thioesters. (Yoo, 2001)

FadD can be found within the cell by the plasma membrane, where it is non-integrally associated. Its activity is enhanced by the membrane lipids or detergents nearby. The protein has a molecular weight of 62,028 Daltons, though it forms a dimer with a molecular weight of about 120,000. It is contained within a 2.2 kilobase fragment of the E. coli genome, as part of a fatty acid degradative regulon along with fadBA, fadE, and fadL, all under control of repressor FadR. Transcription starts 60 base pairs upstream of the translation start. After the translational stop is a GC-rich inverted repeat and a 8T transcriptional terminator. Two FadR operator sites are found at -13 to -29 and -99 to -115. A rare UUG codon at translation initiation may downregulate expression. (Black, 1992)

After long chain fatty acyl-CoA are sequestered inside the cell by vectorial thioesterification with FadD, they can bind repressor protein FadR. This binding to FadR causes it to dissociate from operator sites on the fatty acid degradative (Fad) regulon, relieving the repression on the regulon transcription such that Fad genes can be expressed. (Yoo, 2001)

Chemical Reactions, Substrate Specificity, and Kinetics

Mechanistic equation: FA + ATP = FA-AMP (needs Mg2+), FA-AMP + CoASH = FA-SCoA. FadD activates fatty acids by converting their carboxyl group into an acyl-CoA thioester, which is a stronger electrophile. Fatty acids enter the FadD active site from the membrane through a narrow channel that faces the inner membrane while ATP enters through a distinct large channel. Binding these molecules causes FadD to undergo ligand-induced conformational changes. The molecules then form an AMP-FA intermediate, which the flexible C-terminal clamps in order to position the intermediate and prevent its escape. CoA, the final substrate, binds to FadD after the fatty acid and ATP. CoA enters the FadD active site via a third channel and attacks the new bond, generating FA-CoA and AMP. Supposedly, when CoA bonds to a long chain fatty acid, AMP is pushed from the active site by the LCFA-CoA product, but this push is less pronounced with MCFA. (Ford, 2015)

FadD belongs to a class of adenylate forming enzymes, whose fatty acid tunnel length determines substrate specificity by accommodating a long hydrophobic tail. FadD has broad chain length specificity with a Vmax ranging from 2632 nmol/min/mg protein for C12 to 135 for C6. However, its maximal activity is reserved for fatty acids with carbon numbers ranging between 12 and 18, activating both mono- and poly-unsaturated fatty acids. The thioesters synthesized are destined for degradation or phospholipid incorporation. FadD has lower activity on medium chain fatty acids with 6 to 12 carbons. Downstream beta oxidation enzymes also have poor activity on MCFA. (Black, 1992)

Interactions with other Proteins: Repression by FadR and Cleavage by Omp

The FadD gene contains two FadR binding sites. The first operator (located from -13 to -29) has 12/17 consensus and the second operator (-115 to -98) has 9/17 consensus. FadD’s operator sites have Keq equal to 10-9 M and 10-8 M, compared to Keq equal to 3 x 10-10 M for FadB’s operator site, the strongest binding site for FadR in E. coli. Long chain fatty acyl CoA prevents FadR from having affinity for operator 1, which would otherwise turn off fadD transcription. (Black, 1992) FadD is a substrate for OmpT in vitro, which cleaves the 62 kDa protein into a 42.97 kDa C-terminal fragment and a 19.39 kDa N-terminal fragment. The cleavage site between residues lysine 172 and arginine 173 is a linker domain that connects the ATP and LCFA binding C-terminal to the N-terminal. The outer membrane serine protease OmpT has dibasic residue specificity with its active site on the cell surface. (Yoo, 2001)

Cleavage did not affect binding ability and the fragments remained associated afterwards. OmpT cleaving FadD results in FadD’s new Km and Vmax values twice as high, but catalytic efficiency remains similar. Enzymatic activity is retained because the hydrocarbon chain of fatty acids interacts with the 43 kDa fragment and the AMP binding signature motif binds nucleotides in the same 43 kDa fragment. Adjacent to where the hydrocarbon binds, consensus 25 amino acid fatty acyl CoA signature motif is involved in substrate specificity and binding. While all the binding domains are within the 43 kDa C-terminal fragment, it is unstable alone. The N-terminal remains associated because it is required for structural stability. (Yoo, 2001)

When the substrate oleate was present, it inhibited the protease OmpT by changing FadD conformation to protect the cleavage site. Adding ATP allowed FadD to reset its conformation, so the site was exposed again. Sensitivity to proteolysis is correlated with increased mobility and flexibility, so changing the conformation of the flexible hinge by binding LCFA or ATP or interacting with detergent can alter the accessibility of the OmpT cleavage site. (Yoo, 2001)

Mutations

Mutations clustered on the face of FadD where AMP exits enhance activity by aiding product exit. Growth rate increased with a wider AMP exit channel from the active site, although some of the mutants had decreased activity on long chain fatty acids. FadD mutations that increase the rate of reaction do not enhance affinity for MCFA. ATP binding precedes and enhances fatty acid binding, so mutants that increase activity facilitate AMP exit and ATP entry from the active site. Removing amino acid side chains surrounding the ATP/AMP channel destabilizes the closed conformation. Mutation eases transition to open state by enhancing AMP exit. These mutations affect the structure of the AMP exit channel or interact with the C-terminal domain. (Ford, 2015)

Bioengineering

Synthetic biologists have engineered E. coli to be a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. When paired with fadE (acyl-CoA dehydrogenase) disruption and tesA (acetyl-CoA thioesterase) overexpression, overexpression of fadD has enhanced the synthesis of fatty alcohols, olefins, and free fatty acids. For all these biofuels, other steps were involved to maximize production: fatty alcohols required accABCD (acetyl-CoA carboxylase) and acrI (acyl-CoA reductase from A. baylyi) co-overexpression, olefins required oleABCD (beta-ketoacyl-ACP synthase from S. maltophilia) overexpression, and FFA required fatty acid synthase (fabH, fabD, fabG, fabF) and accABCD overexpression. (Clomburg, 2010) (Colin, 2011)

Related proteins

Examining fadD’s amino acid sequence alongside rat and yeast acyl CoA synthetases reveals a high degree of similarity in the carboxyl end (353-455), which might be an ATP binding domain because it is also shared with firefly luciferase. High similarity to the other acyl CoA synthetases at a central location (200-273) might indicate a fatty acid or Coenzyme A binding domain. (Black, 1992)

There is a second acyl-CoA synthetase in E. coli: FadK. FadK has a higher relative rate on medium chain fatty acids than long chain, but lower absolute activity in comparison to FadD. FadK is only expressed under anaerobic conditions. An unidentified H+/LCFA cotransporter might be present in the inner membrane to interact directly with acyl CoA synthetase. (Ford, 2015)

Uptake and degradation of fatty acids (FadL +FadD)

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. FadD Fatty Acyl-CoA Synthetase (Ligase) from E. coli.The FadD Fatty Acyl-CoA Synthetase (Ligase) converts fatty acids into fatty acyl-CoA using ATP.

Synthesized FadL and FadD function：

We examined the function of the bioengineered product FadL and FadD. We added Palmitic acid—a long chain fatty acid frequently contained in fast foods— into standard LB liquid medium with engineered bacteria. The bacteria culture was shake-nurtured for 24 hours under 220rmp and 37 degrees Celsius. Then, we performed Differential Centrifugation to the sample, applying 1000rpm for 1 minute to isolate and remove unreacted Palmitic acid in the supernatant, then 3000rpm for 10 minutes to collect bacteria. Supernatant was then diluted with distilled water. The concentration of Palmitic acid in the supernatant and in the bacteria are measured using photospectrometer.

The testing was repeated for 3 times to ensure credibility.

The result is as shown:

Table. Results for FadL and FadD function test

The total concentration of Palmitic acid the treatment group with both FadL and FadD genes is significantly lower than that of Negative control, which contain only the plasmid carrier but neither of the two genes, indicating that the LCFA intake and metabolization are achieved. Meanwhile, the treatment group with only FadL gene has a lower extracellular concentration and higher intracellular concentration of Palmitic acid than does negative control, indicating successful LCFA transportation.  The treatment group with only FadD gene, on the other hand, has a lower intracellular concentration of Palmitic acid than does negative control, indicating successful LCFA metabolism. Taken as a whole, the system can achieve the goal of Palmitic acid intake and metabolization in vitro, and both synthesized proteins involved in the process, FadL and FadD, are functional and effective.

Optimum pH and temperature:

  Secondly, we measured the optimum pH and temperature of our engineered bacteria to ensure their survival and appropriate functioning inside human intestine. We nurtured bacteria in pH 5~9 and temperature 25~50 degree Celsius and measured the Palmitic acid concentration in bacteria cultures using similar approach like in step one. Bacteria are treated under different pH and temperature and shake-nurtured in LB culture for 60 hours under 220rpm. Within each system, the initial Palmatic acid concentration is set at 20g/L. The consumption of Palmitic acid is then measured using the same approach in step one: centrifugating the bacteria liquid culture first at 1000rpm for 1 minute to separate Palmitic acid remaining in the culture and at 3000rpm for 10 minutes to collect bacteria, and then use photospectrometer to measure palmitic concentration in and out of the cells. The total amount of remaining Palmitic acid is deducted from the initial concentration to get total Palmitic acid consumption, which indicates the functioning of bacteria under different environment settings.

The results are represented in the figures:

Figure6.Results for the optimum pH

Figure7.Results for the optimum temperature

In the figures, Palmitic acid consumption peaks at pH close to 7 and temperature close to 40 degrees Celsius, positively indicating that the performance of bacteria is the best at pH close to 7 and temperature close to 40 degrees Celsius, a temperature and acidity environment like that of human intestine, demonstrating that the bacteria could survive and function under human intestines. 

Bacteria Function and Survival at Simulated Human Intestinal Environment:

  Finally, we tested the function engineered bacteria in a simulated intestinal environment. To simulate the environment of intestine, the pH is controlled at 7 and the temperature is controlled at 37 degrees Celsius. Moreover, the system is kept anaerobic to simulate the scarcity of oxygen inside human intestine. To achieve this, the Erlenmeyer bottle that contains the experimental system will be put inside an anaerobic bag. Bile salt, Pancreatic juice, and lipase (purchased as bottles of powders, not taken from any animal or human) will be added to the bottle to simulate real contents of human intestine. Considered the main source of lipids in human body, 24 degrees Celsius Palm oil is dissolved into LB medium at temperature above 24 degrees Celsius. The experimental system that contains LB medium, Palm oil, Bile salt, Pancreatic juice, and lipase was shake-nurtured for 60 hours under 300 rpm. Palm oil consumption is measured after cooling the sample and weighing the gained solid. Palmitic acid consumption is measured using the same centrifugation approach as in the step of measuring the optimum pH and temperature.  After centrifuging and diluting, OD value at 600 nm can be measured to determine the relative concentration of bacteria bodies in the system.

The result of Palmitic acid consumption is represented in the figure:

Figure8. results for simulated intestinal condition test

Both the test and the controlled bacteria can survive under simulated intestinal environment. However, Palmitic acid consumption of engineered bacteria that carries both genes, as indicated by the blue line, is almost twice of that of controlled bacteria that contains only the plasmid, as indicated by the red line. However, the engineered bacteria performs slightly less effective than it would have at optimum environment. 

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

Black, P. N., DiRusso, C. C., Metzger, A. K., & Heimert, T. L. (1992). Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. The Journal of biological chemistry, 267(35), 25513–25520.

Yoo, J. H., Cheng, O. H., & Gerber, G. E. (2001). Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT. The Biochemical journal, 360(Pt 3), 699–706. https://doi.org/10.1042/0264-6021:3600699

Ford, T. J., & Way, J. C. (2015). Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids. PeerJ, 3, e1040. https://doi.org/10.7717/peerj.1040

Clomburg, J. M., & Gonzalez, R. (2010). Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology. Applied microbiology and biotechnology, 86(2), 419–434. https://doi.org/10.1007/s00253-010-2446-1

Colin, V. L., Rodríguez, A., & Cristóbal, H. A. (2011). The role of synthetic biology in the design of microbial cell factories for biofuel production. Journal of biomedicine & biotechnology, 2011, 601834. https://doi.org/10.1155/2011/601834

Uptake and degradation of fatty acids(FadL +FadD)

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. FadD Fatty Acyl-CoA Synthetase (Ligase) from E. coli.The FadD Fatty Acyl-CoA Synthetase (Ligase) converts fatty acids into fatty acyl-CoA using ATP.

Sequence and Features