Engineering acyl carrier protein to enhance production of shortened fatty acids
- Xueliang Liu†1, 2,
- Wade M. Hicks†1, 3Email author,
- Pamela A. Silver1, 3 and
- Jeffrey C. Way1Email author
© Liu et al. 2016
Received: 29 October 2015
Accepted: 7 January 2016
Published: 2 February 2016
The acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids.
We constructed a homology model of the Synechococcus elongatus ACP, showing a hydrophobic pocket harboring the growing acyl chain. Amino acids within the pocket were mutated to increase steric hindrance to the acyl chain. Certain mutant ACPs, when over-expressed in Escherichia coli, increased the proportion of shorter chain lipids; I75 W and I75Y showed the strongest effects. Expression of I75 W and I75Y mutant ACPs also increased production of lauric acid in E. coli that expressed the C12-specific acyl-ACP thioesterase from Cuphea palustris.
We engineered the specificity of the ACP, an essential protein of fatty acid metabolism, to alter the E. coli lipid pool and enhance production of medium-chain fatty acids as biofuel precursors. These results indicate that modification of ACP itself could be combined with enzymes affecting length specificity in fatty acid synthesis to enhance production of commodity chemicals based on fatty acids.
KeywordsAcyl carrier protein Protein engineering Thioesterase Free fatty acid Lauric acid
With the continuous rise in global energy needs and adverse climate changes, development of cleaner and renewable alternatives to fossil fuels has become paramount. Microbial synthesis of biofuels is an attractive, renewable alternative to fossil fuels [1–3]. Organisms naturally synthesize large quantities of fuel-like hydrocarbons in the form of lipids, which are used in cell membranes and other molecules. In microbes, the end products of fatty acid metabolism are long acyl chains consisting mostly of 16–18 carbons. When extracted for fuels, these long-chain carbon molecules remain solid at room temperature and lack favorable physical properties such as higher volatility and lower viscosity. Such properties are characteristic of medium-length (8–12) carbon chains used ubiquitously in fuels for vehicles and jets.
We found that over-expressing certain mutant ACPs altered the composition of the cellular lipid pool and increased production of certain medium-chain fatty acids. Our findings could be useful for microbial production of transportation biofuels based on metabolically engineered pathways.
Results and discussion
To enhance production of medium-chain fatty acids, we constructed mutants of ACP designed to decrease the acyl chain pocket size (Fig. 2). Variants of the cyanobacterial (S. elongatus) ACP were expressed in an E. coli host. We chose S. elongatus ACP due to its potential compatibility with recently discovered enzymes of the cyanobacterial alkane biosynthesis pathway , which could enable microbial synthesis of fatty alcohol or alkanes. The native E. coli ACP gene was left intact, as we found that its knockout could not be rescued by complementation from expression of wild-type E.coli ACP encoded on a plasmid (data not shown). To determine which hydrophobic residues of S. elongatus ACP lined the inner, acyl chain pocket, we constructed a structural homology model using the published crystal structure of E. coli ACP bound to a C10 fatty acyl chain (2FAE) as a template (Fig. 2). We constructed a number of single amino acid mutants by exchanging small hydrophobic side-chain residues, such as isoleucine or leucine, with bulkier hydrophobic side-chains such as phenylalanine, methionine, tyrosine, or tryptophan. ACPs initially fold into an inactive apo state. Conversion to the active holo state is achieved through post-translational modification whereby 4′-phosphopantetheine is transferred from co-enzyme A (CoA) to a specific serine residue on the apo-ACP (Ser39 on S. elongatus ACP) [8, 16]. Acyl carrier protein over-expression may reduce the CoA pool and lead to toxic accumulation of apo-ACP, which inhibits sn-glycerol-3-phosphate acyltransferase [16, 17], so as a quick check for functional expression of recombinant ACPs, we measured culture growth kinetics over 15 h. Compared to controls, cells over-expressing wild-type (‘WT’) E. coli ACP (Ec-ACP), WT S. elongatus ACP (Se-ACP), or mutant Se-ACPs all showed suppressed growth at low levels of induction and worsened at higher induction levels (Additional file 1: Figure S1; Additional file 2: Figure S2), suggesting that these recombinant cyanobacterial ACPs were expressed and properly folded.
To explore the potential to further skew cellular lipids toward short-chain lengths, particularly those shorter than 14 carbons long, we introduced secondary point-mutations in addition to the Se-ACP I75 W or I75Y mutations. Amino acids with small hydrophobic side-chains such as isoleucine, valine, or alanine were exchanged for a bulkier methionine, a polar glutamine, or a hydrophilic arginine. Double mutant Se-ACPs did not significantly increase the C14:C16 ratio beyond either single I75 W or I75Y mutation alone (Additional file 3: Figure S3), and did not cause observable production of chains shorter than C14.
As an additional control, the Se-ACP serine 39 residue, which is post-translationally modified with 4-phosphopantetheine, was mutated to alanine (S39A), thereby generating an inactive, obligate apo-ACP. Over-expressing this inactive ACP resulted in similarly low C14:C16 ratio compared to WT (Fig. 3). Growth was suppressed by over-expressing this mutant protein, suggesting that the protein was correctly folded [16, 17].
In sum, we have shown that ACP, an essential protein in fatty acid metabolism, can be modified by site-directed mutagenesis to skew cellular lipid pools toward smaller acyl chain lengths. Specifically, expressing certain mutant ACPs enhanced the level of C14 fatty acids in membrane lipids, and by co-expressing mutant ACPs with a chain-length specific thioesterase production of a medium-chain free fatty acid (lauric acid) was enhanced. These results are consistent with a hypothesis that bacterial ACPs influence lipid chain-length during fatty acid synthesis. Other enzymes involved in fatty acid synthesis also likely affect chain-length, and engineering modified acyl chain specificity has been similarly achieved. For example, FabB and FabF catalyze elongation of fatty acid chains (Fig. 1), and have a clearly defined pocket that should accommodate carbon chains up to about 18 . Val et al. engineered the FabF pocket to accommodate a maximum of six carbons . Similarly, the cyanobacterial aldehyde decarbonylase solved structure [21, 22] contains electron density corresponding to a C18 fatty acid or aldehyde; Khara et al. modified this enzyme to have specificity for medium-chain substrates . The C8-, C12-, and C14-specific plant-derived acyl-ACP thioesterases apparently also control length of fatty acid products, although the underlying structural mechanisms have not been identified. Since FFAs contain the hydrophilic carboxylic acid functional group, they are not ideal fuel molecules. Instead, FFAs can act as precursors to further enzymatic modification for transformation into highly desired fuel molecules such as fatty alcohols and alkanes. Engineering such enzymes (e.g., aldehyde decarbonylases, acyl-ACP reductases, and carboxylic acid reductases) toward shorter carbon chain substrate recognition will likely be key to tailoring biofuel formulations. To achieve the ultimate goal of efficient biofuel synthesis, it may be necessary to engineer the length specificity of several enzymes—most such enzymes have evolved to handle chains of 16–18 carbons, but shorter chains are desired in fuels. This technology could help to optimize biofuel yield and molecular makeup, which would benefit the goal of developing energy sources alternative to fossil fuels.
The structural model of Se-ACP harboring a decanoyl-chain was obtained by homology to the published x-ray crystal structure of the E. coli decanoyl-ACP (2FAE) using SWISS-MODEL .
Double-stranded DNA encoding E. coli and S. elongatus ACP genes were synthesized as gBlocks (Integrated DNA Technologies) and cloned into the pCDF-Duet vector by Gibson Assembly . Single- and double-amino acid mutations of the Se-ACP gene were incorporated during DNA synthesis. An empty pCDF-Duet-1 vector (Millipore) without the ACP gene was included as control. Plasmids were sequence-verified and transformed into E. coli BL21(DE3). For FFA production, the C12 thioesterase gene (UcFatB2 from C. palustris) was cloned into pET-Duet-1 vector (Millipore) and transformed into strains harboring the plasmids carrying the ACP variants.
Growth kinetics assay
ACP expressing strains in triplicates were inoculated from single colonies representing independent transformants into LB medium, grown overnight to saturation, and back-diluted into M9 minimal media containing 0.4 % glucose. The cultures were grown to mid-exponential phase (OD ~0.4), dispersed into 96-well plates, induced with various concentrations of IPTG, and left to grow shaking at 37 °C in a plate reader (BioTek NEO). The optical densities (OD) of the cultures were recorded every 5 min over 15 h by the plate reader. The growth curves, as well as the final OD after 15 h were compared among the strains to quantify growth suppression by ACP over-expression.
Analysis of cellular lipid composition
ACP expressing strains in triplicates were inoculated in LB, grown overnight, and back-diluted into M9 minimal media containing 3 % glucose. The cultures were grown to an optical density of 0.4, induced with 1 mM IPTG, and grown for six more hours at 37 °C. For the time course experiment (Fig. 4), the cultures were left to grow for up to 24 h. After growth, 10 ml of cell culture was used for extraction and analysis, corresponding to wet biomass weights (pellet) of around 5 mg (ACP over-expressing, growth defect) to 10 mg (not inducing ACP). The cells were pelleted and resuspended in 1:1 methanol:chloroform with 2 % glacial acetic acid for lysis, hydrolysis of membrane lipids, and solubilization of fatty acids into the organic phase. Octanoate (C8 fatty acid) was added into the mixture as an internal standard. After vigorous mixing by vortexing, the organic phase was transferred by glass pipettes into glass vials, and the chloroform solvent was evaporated by nitrogen. The vials were then treated with methanol containing 1.25 M HCl at 50 °C for 15 h to catalyze methylation of the fatty acids. The reaction was quenched by adding 5 ml of 100 mg/ml sodium bicarbonate. 0.5 ml hexane was added and the mixture was vortexed vigorously before the hexane phase containing the FAME was extracted and subsequently analyzed on a GC–MS (Agilent 6890/5975) . First a standard set of FAMEs with varying chain lengths was run on the GC–MS in scan mode to determine the identity of each fatty acid peak based on the elution time for each fatty acid and comparison of its fragment profile to those in the NIST database (via Agilent ChemStation software). Fatty acid peaks from the extracted cell samples were also identified using scan mode. To quantify peak areas, the background was minimized using Selective Ion Mode (SIM) whereby the elution times were used to determine fatty acid identity and only the most dominant mass peaks pertaining to each fatty acid methyl ester were counted. For calibration of concentrations, standard curves for C14 and C16 FAMEs dissolved in hexane were taken in the range of 0.1–400 mg/L. A linear fit of hexane background-subtracted peak area to known concentration was extracted in the 0.1–6.215 mg/L range to cover the range of concentrations seen in the cell samples. Molar concentration was determined by dividing mass concentration (mg/L) by the molecular weight of C14 FAME (242 g/mol) or C16 FAME (270.4 g/mol). To compare the proportions of different chain lengths in each sample, the molar concentration ratio of C14 to C16 FAME was taken.
Analysis of free fatty acid (FFA)
ACP and C12 thioesterase-expressing strains in triplicates were grown in M9 minimal media containing 3 % glucose and induced with IPTG as described above. After 6 or 24 h of growth, five microliters of each culture (cells and media, as medium chain FFA may be secreted) were transferred to wells of a new 96-well plate for high-throughput spectrometric determination of FFA concentration using the Roche Free Fatty Acid Kit (Product Number 11383175001). The FFA is first converted via acyl-CoA synthetase into acyl-CoA, which is then oxidized in the presence of acyl-CoA oxidase to enoyl-CoA, releasing H2O2 in the process that converts 2,4,6-tribromo-3-hydroxy-benzoic acid (TBHB) and 4-aminoantipyrine (4-AA) to a red dye detectable by spectrometer at 546 nm. To specifically detect lauric acid, cultures of ACP plus thioesterase-expressing cells were lysed and extracted with chloroform. The FFA was ethylated and run on the GC–MS to determine the spectrum of chain lengths.
acyl carrier protein
free fatty acid
XL and WMH contributed equally to this work. XL designed and performed the experiments, analyzed the data, and drafted the manuscript. WMH, PAS, and JCW designed the experiments, analyzed the data, and revised the manuscript. All authors read and approved the manuscript.
The authors would like to thank Dr. Joseph Torella and Dr. Tyler Ford for providing the medium-chain thioesterase and helpful discussions in general.
The authors declare that they have no competing interests.
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