Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis
- Lei Zhu†1,
- Juanli Cheng†1,
- Biao Luo1,
- Saixiang Feng1,
- Jinshui Lin1,
- Shengbin Wang1,
- John E Cronan2, 3 and
- Haihong Wang1Email author
© Zhu et al; licensee BioMed Central Ltd. 2009
Received: 29 January 2009
Accepted: 04 June 2009
Published: 04 June 2009
The original anaerobic unsaturated fatty acid biosynthesis pathway proposed by Goldfine and Bloch was based on in vivo labeling studies in Clostridium butyricum ATCC 6015 (now C. beijerinckii) but to date no dedicated unsaturated fatty acid biosynthetic enzyme has been identified in Clostridia. C. acetobutylicium synthesizes the same species of unsaturated fatty acids as E. coli, but lacks all of the known unsaturated fatty acid synthetic genes identified in E. coli and other bacteria. A possible explanation was that two enzymes of saturated fatty acid synthesis of C. acetobutylicium, FabZ and FabF might also function in the unsaturated arm of the pathway (a FabZ homologue is known to be an unsaturated fatty acid synthetic enzyme in enterococci).
We report that the FabF homologue located within the fatty acid biosynthetic gene cluster of C. acetobutylicium functions in synthesis of both unsaturated fatty acids and saturated fatty acids. Expression of this protein in E. coli functionally replaced both the FabB and FabF proteins of the host in vivo and replaced E. coli FabB in a defined in vitro fatty acid synthesis system. In contrast the single C. acetobutylicium FabZ homologue, although able to functionally replace E. coli FabZ in vivo and in vitro, was unable to replace FabA, the key dehydratase-isomerase of E. coli unsaturated fatty acid biosynthesis in vivo and lacked isomerase activity in vitro.
Thus, C. acetobutylicium introduces the double of unsaturated fatty acids by use of a novel and unknown enzyme.
Only one of the three C. acetobutylicium fabF homologues can functionally replace E. coli FabF in vivo
Effects of growth temperature on fatty acid compositions (% by weight)of fabF strain MR52 carrying plasmids encoding C. acetobutylicium fabF1.
The C. acetobutylicium fabF1 gene can functionally replace E. coli FabB
Fatty acid compositions (% by weight)of fabB strain K1060 transformed with plasmids encoding either C. acetobutylicium fabF1 or E. coli fabB.
Functional analysis of C. acetobutylicium FabZ in vivo
Composition of fatty acids of strain HW7
Fatty acid composition (% by weight)
In vitro assay of C. acetobutylicium FabZ and FabF1 activities
Although C. acetobutylicium, C. beijerinckii and E. coli synthesize the same species of unsaturated fatty acids  and Clostridia are thought to follow the same synthetic mechanism as E. coli , the enzyme that introduces the cis double bond of the unsaturated fatty acids remains unknown. Like other Clostridia the C.acetobutylicium genome encodes none of the three known anaerobic unsaturated fatty acid synthesis pathways denoted by the presence of genes encoding FabM, FabA or FabN proteins. One possibility was that the single FabZ of this bacterium could somehow partition acyl chains between the saturated and unsaturated branches of the pathway. However, our in vivo and in vitro data show that C. acetobutylicium FabZ cannot synthesize the first intermediate in unsaturated fatty acid synthesis. Hence, Clostridia must contain a novel enzyme that introduces the cis double bond. Note that the proposed isomerase activity of the C. acetobutylicium FabZ was not unreasonable. C. acetobutylicium FabZ shares 51.4 and 59.3% identical residues with E. faecalis FabN and FabZ, respectively, and there is no sequence signature that denotes isomerase ability [9, 23, 24]. This is because the isomerase potential of 3-hydroxyacyl-ACP dehydratases is not determined by the catalytic machinery at the active site but rather by the β-sheets that dictate the orientation of the central α-helix and thus the shape of the substrate binding tunnel [23, 24]. We are currently seeking the gene(s) that encode the enzyme responsible for cis double bond introduction in C. acetobutylicium.
In contrast to FabZ, the single 3-ketoacyl-ACP synthase (FabF) of this bacterium performs the elongation functions required in both branches of the fatty acid synthetic pathway. This protein can both elongate palmitoleoyl-ACP to cis- vaccenoyl-ACP as does FabF in E. coli and also elongates the cis double bond containing product of FabA as does E. coli FabB. However, C. acetobutylicium FabF, was unable to perform the two tasks simultaneously and thus differs from Enterococcus faecalis FabO . Although the C. acetobutylicium FabF and E. faecalis FabO proteins are 45–46% identical to E. coli FabF, they are only 55% identical to one another. Hence, each of the three proteins is distinct from the other two. The finding that C. acetobutylicium FabF was unable to perform the two tasks simultaneously could be due to the intrinsic temperature sensitivity of FabF1 and to the enzyme undergoing a type of kinetic confusion in this unnatural setting. Perhaps the intermediates of one branch of the pathway act (in effect) as inhibitors of the other branch. In this scenario the presence of the E. coli enzyme (either FabB or FabF) would result in the inhibitory intermediates being converted to long chain acyl chains, thereby freeing the C. acetobutylicium FabF to operate in the other branch. The complex task faced by FabF1 upon expression in an E. coli strain lacking both FabB and FabF is illustrated by the effects of overproduction of FabA and FabB in E. coli . Overproduction of FabA results in increased production of saturated fatty acids rather than the increase in unsaturated fatty acid levels that might have been expected . In contrast overproduction of FabB has the opposite result; unsaturated fatty acid levels are increased . However, if the two enzymes are simultaneously overproduced, the fatty acid composition returns to normal . These counter-intuitive results are due to the fact that FabA catalyzes reversible reactions whereas the FabB reaction is irreversible. Hence, when FabB activity is limiting, any excess cis-3-decenoyl-ACP produced by FabA can be isomerized back to trans-2-decenoyl-ACP and upon FabI action, this acyl chain can enter the saturated arm of the pathway. However, when FabB is in excess, it catalyzes the irreversible elongation of cis-3-decenoyl-ACP and thereby pulls the flow of carbon toward the unsaturated branch of the pathway. Thus, it would seem a surprising finding if the C. acetobutylicium FabF was able to accurately partition acyl chains between the two branches of the fatty acid synthetic pathway of a foreign organism.
It should be noted that it was not unexpected that the FabF homologue encoded within the fab gene cluster was the only FabF homologue that functioned in fatty acid synthesis. There are good arguments against the other two homologues having this function. The CAC2008 ORF in located within a cluster of genes that appear involved in synthesis of a glycosylated product of a hybrid polyketide-nonribosomal polypeptide pathway. If so, the CAC2008 ORF would be involved in synthesis of the polyketide moiety. The CAA0088 ORF is encoded on the C. acetobutylicium megaplasmid required for the late steps of solvent production by this organism. C. acetobutylicium survives loss of the megaplasmid  and therefore the CAA0088 ORF cannot encode an enzyme essential for fatty acid synthesis (although it could still provide FabF function). Note that it has been recently reported that the single FabF protein of the distantly related gram positive bacterium Lactococcus lactis can also perform the FabB reaction as well as that of FabF.
Unsaturated fatty acid synthesis in Clostridia cannot be explained by a plenipotent FabZ indicating that these bacteria encode a novel enzyme that introduces the cis double bond. In contrast the Clostridia FabF protein has the functions of both of the long chain 3-ketroacyl-ACP syntheases of E. coli. The diversity of bacterial enzymes used for synthesis of the cis double bond of unsaturated fatty acids is unexpected because the remainder of the fatty acid synthetic enzymes is well conserved among very diverse bacteria.
Bacterial strains, plasmids and growth conditions
The E. coli strains and plasmids used in this study are listed in Additional file 1. Luria-Bertani medium was used as the rich medium for E. coli. The phenotypes of fab strains were assessed on rich broth (RB) medium . Oleate neutralized with KOH was added to RB medium at final concentration of 0.1% and solubilized by addition of Brij 58 detergent to final concentration of 0.1 to 0.2%. Antibiotics were used at the following concentrations (in mg/L) sodium ampicillin, 100; chloramphenicol, 30; kanamycin sulfate and rifampicin, 200. L-Arabinose and D-fucose were used at concentrations of 0.01%. Isopropyl-β-D-thiogalactoside (IPTG) was used at final concentration of 1 mM.
Recombinant DNA techniques and construction of plasmids
Restriction enzymes, T4 DNA ligase and Taq DNA polymerase were from Invitrogen or New England Biolabs unless indicated otherwise. All enzymatic reactions were carried out according to the manufacturer's specifications. Qiagen products were used to isolate plasmids, purify DNA fragments from agarose gels and purify PCR products. Plasmids were introduced into E. coli strains by CaCl2-mediated transformation. C. acetobutylicium ATCC824 genomic DNA was extracted using the GNOME DNA kit (Bio 101). DNA sequencing and the synthesis of oligonucleotides were done at the University of Illinois Keck Genomics Center.
The C. acetobutylicium fabF homologues were amplified from genomic DNA using the primers fabF1, fabF2 and fabF3 (Additional file 1). The PCR products were cloned into vector pCR2.1TOPO to give plasmids pHW40 (fabF1), pHW41 (fabF2) and pHW42 (fabF3). Plasmids pHW40 and pHW42 were then digested with EcoRI, the appropriate fragments were isolated and these were ligated into pHSG576  digested with the same enzyme to give plasmids pHW33 and pHW35, respectively. The orientation of the C. acetobutylicium ORFs in these plasmids were such that the genes would be transcribed by the vector lac promoter. The HindIII-XhoI fragment of pHW41 was ligated into vector pSU20  digested with the same enzymes to give pHW43 which was then digested with HindIII plus SalI and the fabF2-containing fragment was inserted into the same sites of vector pHSG576 to give pHW34. Plasmids pHW16, pHW31 and pHW32 were constructed as follows. The upstream primers were primers12, 34 and 56 (Additional file 1) and the downstream primer was the M13 (-) forward primer. Plasmids pHW33, pHW34 and pHW35 were used as templates for PCR amplification. The products were cloned into vector pCR2.1 TOPO to yield pHW16, pHW31 and pHW32, respectively. The BspHI-PstI fragments of pHW16 and pHW32 were then ligated into NcoI and PstI sites of pBAD24  to give plasmids pHW36 and pHW38, respectively. Likewise, the BspHI-HindIII fragment of pHW31 was inserted into the NcoI and HindIII sites of pBAD24 to yield pHW37.
The fabZ homologue was amplified by PCR using C. acetobutylicium genomic DNA as template with primers Zprimer1 and Zprimer2 (Additional file 1). The PCR product was inserted into pCR2.1 TOPO vector to give pHW15. The BspLU11I-HindIII fragment of pHW15 was inserted into the sites of pBAD24 digested with NcoI and HindIII to give pHW22. The BspHI-EcoRI fragments of pHW15 and pHW16 was inserted into the NcoI and EcoRI sites of pET28b to give pHW39 and pHW28, respectively. By site-directed mutagenesis using the primers listed in Additional file 1, an NdeI site was introduced into pHW39 and pHW28. The NdeI-EcoRI fragment of this two new plasmids were inserted into the NdeI and EcoRI sites of pET28b to give pHW74 and pHW76. To increase FabZ expression, 24 codons that correspond to rare E. coli tRNA species were substituted with codons favored in E. coli by site-directed mutagenesis using the primers listed in Additional file 1 to give pHW74m. The NcoI-HindIII fragment of pHW74m was inserted into the NcoI and HindIII sites of pBAD24 to give pHW22m.
Construction of an E. coli fabZ Deletion Strain
A linear DNA fragments carrying a kan cassette was amplified from pKD13 by PCR [9, 31] using primers, HZ1 and HZ2 listed in Additional file 1. These primers were homologous at the 3' end for priming sequences in pKD13 and contained 45-nucleotide extensions at the 5' end homologous to the E. coli fabZ sequence. The 1.4 kb PCR product was purified, treated with DpnI, and then introduced into a pHW22-containing derivative of DY330 a strain lysogenic for a defective prophage that contains the recombination genes under control of temperature-sensitive c I-repressor . The transformed cells were spread on LB plates containing ampicillin, kanamycin and arabinose. The E. coli fabZ deletion strain, HW7, was verified by PCR using primers P1, P2 plus HZ1, and HZ2.
Analysis of phospholipid fatty acid compositions
Cultures (5 ml) were grown aerobically at different temperatures in RB medium overnight. The cells were then harvested and the phospholipids extracted as described previously . The fatty acid compositions were analyzed by mass spectroscopy as described previously [9, 14]. For analysis of radioactive fatty acids, 100 μl of a culture grown overnight in LB medium was transferred into 5 ml of RB medium supplemented with 0.1% cis-9, 10-methylenehexadecanoic acid (a cyclopropane fatty acid) plus 0.01% L-arabinose. After incubation of these cultures for 1 h, 5 μCi of sodium [1-14C] acetate was added and the culture allowed continuing growth for 4 h. The phospholipids were then extracted as described above. The phospholipid acyl chains were converted to their methyl esters, which were separated by argentation thin-layer chromatography, and analyzed with autoradiography 
Expression of plasmid-encoded proteins
To assay expression of the products of C. acetobutylicium fabF1 and fabZ, pHW28 and pHW39 were introduced into E. coli strain BL21 (DE3), which encodes T7 RNA polymerase under the control of the IPTG-inducible lacUV5 promoter. The products of the cloned gene were selectively labeled with [35S]methionine as described . The proteins were separated on a sodium dodecyl sulfate-12% polyacrylamide gel (pH 8.8). The destained gels were dried, and the labeled proteins were visualized by autoradiography . The molecular mass markers (Bio-Rad, Richmond, Calif) were rabbit phosphorylase, bovine serum albumin, rabbit actin, bovine carbonic anhydrase, trypsin inhibitor and hen egg white lysozyme.
Purification of FabF1 and FabZ
Plasmid pHW76 and pHW74m were introduced into strain BL21 (DE3), respectively, and the proteins were overexpressed and purified as described previously. The enzymes were homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The E. coli FabD, FabH, FabG, FabA, FabZ, FabB, FabI and Vibrio harveyi AasS proteins were purified by their hexahisitidine tags described previously [18, 20].
Assay of FabF1 and FabZ activity in vitro
Fatty acid synthesis was reconstituted in vitro to assay FabF1 and FabZ activity using the purified enzymes that catalyze the fatty acid biosynthesis essentially. The assay utilized the AasS acyl-ACP synthetase from Vibrio harveyi  to generate 3-hydroxydecanoyl-ACP. The reaction mixtures to synthesize 3-hydroxydecanoyl-ACP contained 20 μM ACP, 10 mM ATP, 10 mM MgSO4, 5 mM DTT, 0.1 M sodium phosphate buffer (pH 7.0), 100 μM 3-hydroxydecanoic acid (Sigma) and AasS (0.2 μg) in a final volume of 50 μl. The mixtures were incubated at 37°C for 1 h. To assay C. acetobutylicium FabF1, the following incubation 1 μg each of E. coli FabD, FabG and FabA, 100 μM NADPH, 100 μM NADH, 100 μM malonyl-CoA, and 1 μg of either E. coli FabB or C. acetobutylicium FabF1 was added. To assay C. acetobutylicium FabZ, the following incubation contained 1 μg each of E. coli FabD, FabG and FabB, 100 μM NADPH, 100 μM NADH, 100 μM malonyl-CoA, and 1 μg of E. coli FabA or C. acetobutylicium FabZ was added. The resulting mixture was incubated for an additional 1 h and the reaction products were analyzed by conformationally sensitive gel electrophoresis on 20% polyacrylamide gels containing 2.5 M urea [20, 24]. The gel was stained with Coomassie Brilliant Blue R250.
This work was supported by the President Foundation of South China Agricultural University and NIH grant AI15650. We are grateful to Professor Hiroshi Kobayashi (Graduate School of Pharmaceutical Sciences, Chiba University Japan) for critical reading.
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