Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria
© Kaczmarzyk et al. 2016
Received: 5 January 2016
Accepted: 8 January 2016
Published: 21 January 2016
Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty acid (C10–C14) substrates in vitro. Furthermore, we used AAE15 to complement a Synechocystis aas deletion mutant and showed that the new strain preferentially incorporates supplied medium chain fatty acids into internal lipid molecules. Based on this data we propose that AAE15 can be utilized in metabolic engineering strategies for cyanobacteria that aim to produce compounds based on medium chain fatty acids.
In recent years metabolic engineering has benefited from advances in gene synthesis and assembly that allow the implementation of complex biosynthetic pathways into a variety of microorganisms (Keasling 2012; Yadav et al. 2012; Seo et al. 2013). One focus of current research is the establishment of biosynthetic pathways for production a variety of oleo compounds such as fatty acids, alcohols, and alkanes in hosts such as yeast, Escherichia coli, and cyanobacteria (Steen et al. 2010; Lennen and Pfleger 2013; Pfleger et al. 2015; Savakis and Hellingwerf 2015). A cyanobacteria production host is particularly attractive, as their carbon and energy requirements are minimal. However, cyanobacteria-based production of fatty acids, fatty alcohols and alka(e)nes has been limited to several proof-of-principle studies (Liu et al. 2011; Tan et al. 2011; Ruffing and Jones 2012; Kaiser et al. 2013; Wang et al. 2013; Ruffing 2014; Yao et al. 2014). We strive for the utilization of cyanobacteria for the production of oleochemical compounds. For biosynthetic production of oleochemicals, intrinsically synthesized fatty acids should serve as substrates for the diverse downstream metabolic pathways. In cyanobacteria, fatty acids are chemically activated by acyl-ACP synthetase (AAS) (Kaczmarzyk and Fulda 2010). Acyl-ACP synthetases can therefore play a critical role in metabolic engineering strategies for oleochemicals. In this work we were interested in the closer characterization of an Arabidopsis enzyme capable of generating acyl-ACPs and to evaluate its potential for pathway engineering in cyanobacteria.
In Arabidopsis enzymes capable of activating fatty acids belong to a superfamily of acyl-activating enzymes (AAEs), which consists of 63 members, and is divided into seven clades based on sequence similarities (Shockey et al. 2003). Clade I contains eleven members and long chain acyl-CoA synthetase (LACS; C16–C20) activity has been confirmed for nine of these (Shockey et al. 2002). The conversion of very similar fatty acid substrates is reflected by characteristic features of the amino acid sequences of the proteins of clade I. In particular, clade I AAEs differ from all other AAEs by the presence of an amino acid stretch separating two highly conserved sequence motifs. Interestingly, this amino acid linker is remarkably longer in the two remaining proteins of clade I, for which initial tests were unable to proof LACS activity (Shockey et al. 2002). These proteins called AAE15 and AAE16 and encoded by At4g14070 and At3g23790, respectively, include an amino acid linker of approximately 70 amino acid residues, compared to about 40 amino acids found in eukaryotic LACSs (Shockey et al. 2002).
It was proposed previously that Arabidopsis AAE15 is a plastidial AAS (Koo et al. 2005). The conclusions were drawn from experiments in which plant extracts of Arabidopsis wild type and AAE15 and AAE16 knock-out lines were incubated in the presence of radioactive labeled medium chain fatty acids. We showed later that acyl activating enzymes characterized by the presence of a linker motif of 68–74 amino acid residues indeed have AAS activity (Kaczmarzyk and Fulda 2010). Sequences of this type could be found in sequenced genomes of almost all organisms performing oxygenic photosynthesis.
In a recent report, Beld et al. (2014) analyzed the activity of Arabidopsis AAE15 using a more direct approach. The enzyme was expressed in E. coli, and tested in acyl-CoA synthetase and AAS assays. It was concluded that AtAAE15 was a poor enzyme in both assays (Beld et al. 2014).
We were interested in further characterization of Arabidopsis AAE15, and its activity in Synechocystis sp. PCC6803. In this work, we expressed AAE15 heterologously in insect cells, purified it, and analyzed its enzymatic activity in vitro. We demonstrated AAS activity for AAE15 with some specificity for medium chain fatty acids (C10:0–C14:0). Moreover, we expressed AAE15 in the background of an AAS deletion mutant of Synechocystis sp. PCC6803. This mutant is unable to incorporate exogenously added fatty acids into lipids, and secrete free fatty acids to the culture media (Kaczmarzyk and Fulda 2010). Feeding experiments with radiolabeled fatty acids confirmed medium chain fatty acid specificity of AAE15.
Materials and methods
Heterologous expression of tagged AAE15 in insect cells
For heterologous expression the Bac to Bac Baculovirus Expression System (Thermo Fisher Scientific) was used. Two variants of AAE15 (At4g14070) were cloned in frame with the N-terminal 6xHis tag of the pFastBac™HT. The first clone corresponds to the complete open reading frame including the native start codon. For the second clone the predicted plastidial targeting signal was removed, leading to an N-terminal deletion of 195 bps. The vector pUNI51 carrying At4g14070 served as a PCR template, and full length and truncated versions of the gene were amplified using a forward primer introducing a NcoI restriction site, and a reverse primer including the stop codon, introducing a NotI restriction site. The primers sequences were 5′-AGATCCATGGAAATTCGTCTGAAACCT-3′ (forward 1), 5′-AGTACCATGGCTTGCGAGTCAAAGGAAAAAGAAG-3′ (forward 2), and 5′-AGTAGCGGCCGCTTAACTGTAGAGTTGATCAATC-3′ (reverse). PCR products were cloned into pGEMT-vector (Promega), verified by sequencing, and subsequently transferred into pFastBac™HT. The vectors were used to transform competent DH10Bac E. coli cells. Bacmid DNA was isolated and used to transfect Sf9 cells. A recombinant Baculovirus stock P1 was used to infect cells to produce a P2 Baculovirus stock, which was titered and used to infect insect cells for protein expression. Sf9 cells were infected at MOI 3 and grown at 27 °C as adherent cultures in T-75 culture flasks using Sf-900 II SFM media supplemented with penicillin at 50 U mL−1, and streptomycin at 50 μg mL−1.
Isolation and purification of recombinant protein from insect cells
Cells from two T-75 flasks were harvested 72 h after infection, washed once with PBS, and resuspended in 1 mL of extraction buffer (50 mM Tris–HCl pH 7.8, 150 mM NaCl). Cells were disrupted by sonication (2 × 30 s on ice) with Branson Sonifier Cell Disruptor B15, and cell debris was removed by centrifugation at 3500g at 4 °C for 15 min. Aliquots of the supernatant were saved for Western blot analysis and activity assays, and the remaining volume was centrifuged at 100,000g at 4 °C for 1 h to isolate the membrane fraction. The membranes pellet was resuspended in 300 μL of solubilization buffer (50 mM Tris–HCl, pH 7.8, 150 mM NaCl, 2 % Triton X-100), incubated at 4 °C overnight with agitation to release membrane-bound proteins, and clarified by centrifugation at 100,000g at 4 °C for 30 min. To purify His-tagged proteins the supernatant was applied to 800 μL of BD TALON resin (BD Biosciences) and agitated for 4 h at 4 °C to enable protein binding. The resin was transferred to a gravity-flow column and washed first with the solubilization buffer, and then with the same buffer supplemented with 20 mM imidazole to remove non-specifically bound proteins. The target protein was eluted with the solubilization buffer containing 100 mM EDTA. Fractions of 200 μL were collected and dialyzed overnight against 400 mL of the solubilization buffer at 4 °C. Protein concentration in cellular lysates and membrane suspensions was determined using Bradford assays. Protein concentration in the sample of the purified protein was not determined.
Protein samples were separated on standard 10 % SDS polyacrylamide gels and transferred to the Optiran BA-S 83 membrane (Schleicher and Schuell). Membranes were blocked with 3 % BSA in TBST buffer (10 mM Tris–HCl, 150 mM NaCl, 0.1 % Tween 20, pH 8.0), and probed with TetraHis-Antibody (Qiagen). As secondary antibody a peroxidase conjugated anti-mouse antibody was employed and the signals were detected by chemiluminescence using ECL Western Blotting Kit (Amersham).
The AAS activity was measured according to the protocol described before (Rock and Cronan 1981). The assay buffer contained 2.5 mM Tris–HCl (pH 8.0), 2 mM dithiothreitol, 0.25 mM MgCl2, 5 mM ATP, 10 mM LiCl, 2 % Triton X-100, 15 μM acyl-carrier-protein (ACP; from E. coli K12), and 30 μM [1-14C] fatty acid (specific activity 53.7–60 mCi mmol−1) in a total volume of 40 μL. The assays were initiated by adding defined amounts of protein sample (50 μg of total protein when crude cellular extracts were used as source of enzyme, and 10 μL of purified protein), and were conducted at 37 °C for 30 min. Transferring the assay volume to filter disks stopped the assays. The filter disks were dried and subsequently washed twice with 20 mL of chloroform: methanol: acetic acid (3:6:1, v/v/v) to remove unreacted free fatty acids. Control assays using only free fatty acids demonstrated quantitative removal of the labelled fatty acids by the two washing steps. The radioactivity was determined by liquid scintillation counting (Liquid Scintillation Analyser 1900 TR, Fa. Canberra Packard).
To make sure that all fatty acid substrates are accessible to the enzyme, positive control assays were performed, in which purified AAS from Synechococcus elongatus PCC 7942 was used. Synechococcus AAS was characterized before, and showed broad substrate specificity (C12–C18) (Kaczmarzyk and Fulda 2010).
Lipid analytical methods
Pre-cultures of Synechocystis wild type and mutant strains were diluted to OD730 0.2 in 15 mL BG11, and cultures were grown for 3 days. Cells of 10 mL culture were harvested, and washed twice in 0.1 M NaHCO3. Intracellular and extracellular lipid extractions were performed according to the protocol established before (Bligh and Dyer 1959). Fatty acids were converted to their methyl esters according to modified protocols described earlier (Christie 1982; Stumpe et al. 2001). The fatty acid methyl esters were analyzed by gas chromatography using a Shimadzu GC-2010 gas chromatograph equipped with a Stabilwax column (Restek).
Fatty acid uptake assay
Cyanobacterial cells were collected from 10 mL cultures at OD750 1 by centrifugation, resuspended in 2 mL of fresh BG11 medium, and transferred to a 2 mL microcentrifuge tubes. Radiolabeled [1-14C] fatty acids (lauric, specific activity 57 mCi mmol−1, myristic 55 mCi mmol−1, palmitic 60 mCi mmol−1, stearic 58 mCi mmol−1, oleic 56 mCi mmol−1, linolenic 53.7 mCi mmol−1; Amersham Biosciences) were individually added in amounts corresponding to 0.22 μCi, and the tubes were placed on a platform shaker under light and incubated for 15 h. Cells were pelleted and washed twice with 0.1 M NaHCO3. Total lipid extracts were prepared as follows: 1.5 mL chloroform: methanol (2:1, v/v) acidified with HCl were added to the cell pellets in 2 mL tubes, and lipids were extracted for 4 h under shaking. Afterwards 500 μL 0.45 % NaCl was added, the tubes were shaken briefly, and centrifuged at 2000g for 2 min for phase separation. The lower phase was transferred to a new tube, dried under a stream of nitrogen and resuspended in 20 μL of chloroform: methanol (1:1, v/v). Different lipid classes were separated by thin layer chromatography using acetone: toluene: water (91:30:8, v/v/v) as solvent and were visualized by fluorography. Signals were detected with an image analyzer (FLA-3000, Fujifilm).
Cyanobacteria strains and growth conditions
Liquid cultures of the glucose-tolerant Synechocystis sp. PCC 6803 and mutant strains were grown photoautotrophically in BG11 media buffered to pH 7.8 with 25 mM HEPES at 30 °C, with 45 μE s−1 m2 illumination in a climatic chamber (Percival Climatics SE-1100). For fatty acid profiles analysis, cultures were grown under 1 % (v/v) CO2 conditions. Mutant strains were cultivated in BG11 containing an appropriate antibiotic for the selection (kanamycin 25 μg mL−1, and/or chloramphenicol 20 μg mL−1). To prepare solid media 0.3 % (w/v) sodium thiosulfate pentahydrate and 1.5 % (w/v) agar were added to the buffered BG11 media. The plates were incubated under illumination with 25 μE s−1 m2.
A ∆aas deletion strain, in which a kanamycin resistance cartridge replaced part of the coding region of the gene slr1609, was created before (Kaczmarzyk and Fulda 2010). This strain was used as a host to overexpress homologous acyl activating enzyme from Arabidopsis thaliana: AAE15 (At4g14070).
List of primers
In the second strategy the ∆aas Synechocystis host was transformed with a replicative plasmid pJA2c, carrying AAE15, devoid of the plastidial targeting signal, under the control of psbA2 promoter. The pJA2c vector was constructed by Huang et al. (2010), and modified later (Anfelt et al. 2013), and contains chloramphenicol resistance gene as a selection marker. The primers used for amplification of AAE15 were as follows: forward (adding XbaI restriction site) 5′-GACCTCTAGAATGTGCGAGTCAAAGGAAAAAGAAG-3′, reverse (adding SpeI restriction site) 5′-CTACACTAGTTTAACTGTAGAGTTGATCAATC-3′.
Synechocystis cells were collected from 6 mL cultures at OD730 1, and total RNA was isolated with GeneJET RNA Purification Kit (Thermo Scientific) according to manufacturer’s instructions with the following modifications: lysozyme concentration in TE buffer was 40 mg L−1, and cells were disrupted by vortexing with glass beads for 15 min. DNA was removed with RapidOut DNA Removal Kit (Thermo Scientific).
RT-qPCR was performed in the CFX96 Real-Time PCR Detection System (Bio-Rad) with iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad). All reactions were performed in duplicate, and no-RT controls were included. As a reference gene rpoB (sll1787) encoding RNA polymerase beta subunit was used. The list of primers is provided in Table 1.
AAE15 has AAS activity in vitro, with specificity for medium chain fatty acids
Complementation of Synechocystis aas knockout with Arabidopsis AAE15
In order to examine the activity of Arabidopsis AAE15 in vivo, we performed feeding of radioactive fatty acids. A Synechocystis strain lacking its endogenous AAS (∆aas) was used as a host to express Arabidopsis AAE15. The AAE15 expression cassette was integrated into the chromosome of the cyanobacteria ∆aas strain by homologous recombination to create ∆aas:AAE15. Complete segregation of the newly generated strain was confirmed by PCR. Wild type Synechocystis and the ∆aas strain served as positive and negative control, respectively in fatty acid uptake assays.
Intensities of spots representing FFA and MGDG
Expression of Arabidopsis AAE15, resulted in changes in intracellular and extracellular free fatty acids pools in the cyanobacterial ∆aas strain
Gene expression level for acyl activating enzymes in different strains
In this work we characterized the Arabidopsis AAE15 enzyme in Synechocystis sp. PCC6803. We were particularly interested in evaluating the possibility to introduce modified substrate specificity into the cyanobacterial fatty acid metabolism. To obtain first insight into its enzymatic parameters we expressed Arabidopsis AAE15 in insect cells, and determined its AAS activity in vitro. In a recent report it was concluded upon heterologous expression in E. coli that the enzyme possesses poor activity in both acyl-CoA synthetase and AAS assays (Beld et al. 2014). In that study the full-length protein was expressed in the E. coli strain BL21. We propose that removing of the N-terminal transit peptide is essential to detect a robust AAS activity of AAE15 in expression hosts other than plants. A construct expressing such truncated protein resulted in significant AAS activity with decanoic, lauric and myristic acid (Fig. 2a, b).
It was hypothesized previously that AAE15 may be involved in acyl editing of membrane lipids in Arabidopsis cells (Koo et al. 2005). Thus, in addition to medium chain fatty acids, we tested fatty acid substrates typically found in Arabidopsis lipids: C16, and C18 fatty acids with 0–3 double bonds. Our in vitro activity assays showed that AAE15 is an AAS with strong preference for C10, C12, and C14 substrates, but can also activate other fatty acids with greatly reduced efficiency. The results indicated considerably different substrate specificity compared to the Synechocystis endogenous AAS (Kaczmarzyk and Fulda, 2010).
The in vitro data was confirmed by in vivo experiments. When we replaced the native aas gene of Synechocystis by AAE15 and fed the mutant strain with labeled fatty acids the results again indicated a very clear preference for medium chain fatty acids. In contrast, the endogenous AAS activity of the Synechocystis wild type strain mediated comparable incorporation of all offered fatty acids and showed no particular substrate specificity (Fig. 3).
On the other hand, our data showed also that a more robust expression of AAE15 is able to complement the inactivation of the endogenous AAS protein in Synechocystis. When the truncated version of AAE15 lacking the plastidial targeting signal was expressed under control of the strong psbA2 promoter the fatty acid secretion phenotype of the cyanobacterial aas knockout strain was revoked, indicating that all fatty acids that could be detected in the culture media of the ∆aas strain, were activated and recycled in the strain complemented, ∆aas:pJA2AAE15.
The AAE15 enzyme could be a useful tool for metabolic engineering projects aimed at the biosynthesis of medium chain fatty acid-derived products. There has been growing interest in engineering microorganisms for fatty acid-derived chemicals and fuels (Steen et al. 2010; Lennen and Pfleger 2013; Pfleger et al. 2015; Savakis and Hellingwerf 2015). One of the challenges is to tailor the carbon chain length in order to obtain the desired properties of the final fatty acid-derived products. For example, medium chain length fatty acids are extensively used for the production of soap and detergents (Dyer et al. 2008), and medium chain length alkanes are main components of jet fuel (Kallio et al. 2014). Reports addressing the chain length issue propose expression of acyl-ACP thioesterases with medium chain fatty acids specificity, as enzymes that can control the length of the end product (Zheng et al. 2012; Choi and Lee 2013; Howard et al. 2013; Liu et al. 2013; Torella et al. 2013; Youngquist et al. 2013) Enzymes involved in oleochemical biosynthesis pathways usually require a CoA- or ACP-activated derivative of the fatty acid substrate. In cyanobacteria, fatty acid metabolism relies on ACP-thioesters, which are the preferred substrates of acyl transferases (Weier et al. 2005) in lipid synthesis, and the acyl-ACP reductase of the alkane synthesis pathway (Schirmer et al. 2010). A strategy aimed at the production of medium chain length fatty alcohols in E. coli was published recently (Youngquist et al. 2013). An AAS such as AAE15 that can efficiently deliver activated medium chain fatty acids to downstream metabolic pathways is of significant biotechnological interest.
This work was supported by the Deutsche Forschungsgemeinschaft (German Research Foundation; grant no. FU 430/3–1) and by the Swedish Foundation for Strategic Research (SSF) grant number RBP14-0013.
The authors declare that they have no competing interests.
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