Photosynthetic CO2 Conversion to Fatty Acid Ethyl Esters (FAEEs

Jan 27, 2017 - The atfA gene (A. baylyi),(2) the xpkA gene (Aspergillus nidulans),(17, 19) the pta gene (Bacillus subtilis),(17, 19) and the pdc and a...
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Photosynthetic CO2 Conversion to Fatty Acid Ethyl Esters (FAEEs) Using Engineered Cyanobacteria ABSTRACT: Metabolic engineering of cyanobacteria has received attention as a sustainable strategy to convert carbon dioxide to fatty acid-derived chemicals that are widely used in the food and chemical industries. Herein, Synechococcus elongatus PCC 7942, a model cyanobacterium, was engineered for the first time to produce fatty acid ethyl esters (FAEEs) from CO2. Due to the lack of an endogenous ethanol production pathway and wax ester synthase (AftA) activity in the wild-type cyanobacterium, we metabolically engineered S. elongatus PCC 7942 by expressing heterologous AftA and introducing the ethanol pathway, resulting in detectable peaks of FAEEs. To enhance FAEE production, a heterologous phosphoketolase pathway was introduced in the FAEE-producing strain to supply acetyl-CoA. Subsequent optimization of the cyanobacterial culture with a hexadecane overlay resulted in engineered S. elongatus PCC 7942 that produced photosynthetic FAEEs (10.0 ± 0.7 mg/L/OD730) from CO2. This paper is the first report of photosynthetic production of FAEEs from CO2 in cyanobacteria. KEYWORDS: cyanobacteria, metabolic engineering, fatty acid ethyl ester, CO2 conversion

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INTRODUCTION

Free fatty acids (FFAs) have been produced from CO2 in genetically modified Synechocystis sp. PCC 6803 by deleting acyl-ACP synthetase (slr1609), introducing a thioesterase (‘tesA), and weakening the cell wall layers.6 The engineered cyanobacterium secreted 197 mg/L FFAs. Also, Synechococcus sp. PCC 7002, an FFA-tolerant cyanobacterium, has been engineered for FFA production (130 mg/L) by knocking out the acyl-ACP synthetase/long-chain-fatty-acid CoA ligase ( fadD) and expressing Escherihcia coli thioesterase (‘tesA) and additional RuBisCO (rbcLS).7 In addition to FFAs, fatty alcohols8,9 (3 mg/g DCW) and fatty hydrocarbons9,10 (2.3 mg/ L/OD730) have been produced in engineered cyanobacteria by expressing fatty acyl-CoA reductase from Marinobacter aquaeolei VT8 and by expressing both acyl−acyl carrier protein reductase and aldehyde-deformylating oxygenase. However, metabolic engineering of cyanobacteria to produce fatty acid ethyl esters (FAEEs) from CO2 has not been reported yet. Biodiesel is composed of fatty acid methyl esters (FAMEs) and FAEEs that have similar characteristics with respect to chemical and physical fuel properties.11 FAEEs suitable for use in biodiesel were first produced from glucose and sodium oleate in engineered E. coli by expressing wax ester synthase (atfA) from Acinetobacter baylyi.12 Moreover, the combined expression of thioesterase ‘TesA and acyl-CoA ligase, the deletion of the β-oxidation pathway, and the introduction of a heterologous ethanol pathway (pdc and adh) from Zymomonas mobilis have resulted in FAEE production of 674 mg/L from glucose (20 g/L) alone. In Saccharomyces cerevisiae, FAEEs (520 mg/L) and ethanol (1.4 g/L) have been coproduced from glycerol (17 g/L) by expressing a bacterial acyltransferase.13 Moreover, overexpression of native acetyl-CoA carboxylase and fatty acid synthases (FAS1 and FAS2) with the deletion of the β-oxidation pathway and expression of AtfA from A. baylyi resulted in the production of FAEE (5.44 mg/L) and ethanol (4.1 g/L) from glucose (20 g/L).14 Further medium optimization

Fatty acid-derived chemicals used in the food, chemical, and energy industries (e.g., fragrances, surfactants, solvents, lubricants, and biofuel) are typically produced from plant and animal oils. Recently, microbial cell factories have been developed to produce fatty acid-derived chemicals (free fatty acids, fatty alcohols, fatty hydrocarbons, and fatty acid ethyl esters) from various carbon sources such as glucose, glycerol, and lignocellulose (a renewable biomass).1−3 In addition, the direct conversion of carbon dioxide to fatty acid-derived chemicals has received attention as a sustainable solution to respond to greenhouse gas emissions and energy supply problems.4 Metabolic engineering of cyanobacteria has been used to produce fatty acid-derived chemicals from CO2.5 Table 1. Bacterial Strains and Plasmids Used in This Study strain or plasmid strains E. coli HIT-DH5α S. elongatus PCC 7942 Se2Et Se1A-2Et Se1AXP-2Et

plasmids pSe1Bb1s-GFP pSe2Bb1k-GFP pSe1Bb1s-atfA pSe1Bb1s-atfAxpkA-pta pSe2Bb1k-pdcadh

relevant characteristics

references

F−(80d lacZ M15) (lacZYAargF) U169 hsdR17(r− m+) recA1 endA1 relA1 deoR96 wild-type (ATCC 33912)

RBC Bioscience

S. elongatus PCC 7942 NSII::Bb1k-pdc-adh S. elongatus PCC 7942 NSI::Bb1s-atfA NSII::Bb1kpdc-adh S. elongatus PCC 7942 NSI::Bb1s-atfA-xpkA-pta NSII::Bb1k-pdc-adh

this study

pUC, Spcr, LacI, Ptrc, NSI target sites, gfp gene pUC, Kmr, LacI, Ptrc, NSII target sites gfp genes pUC, Spcr, LacI, Ptrc, NSI target sites, atfA gene pUC, Spcr, LacI, Ptrc, NSI target sites, atfA, xpkA, pta genes pUC, Kmr, LacI, Ptrc, NSII target sites, pdc, adh genes

ATCC

this study this study

17 17 this study

Received: Revised: Accepted: Published:

this study this study

© 2017 American Chemical Society

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1, 2017 27, 2017 27, 2017 27, 2017 DOI: 10.1021/acs.jafc.7b00002 J. Agric. Food Chem. 2017, 65, 1087−1092

Letter

Journal of Agricultural and Food Chemistry

Figure 1. Schematic diagram of photosynthetic FAEE production from CO2 in engineered S. elongatus PCC 7942. (A) Schematic pathway for FAEE production in recombinant S. elongatus PCC 7942 strain by introducing a heterologous ethanol-producing pathway and coexpressing the atf gene encoding for a wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase from A. baylyi.12 The ethanol-producing pathway requires expression of the pdc gene encoding for heterologous Z. mobilis pyruvate decarboxylase and the adh gene encoding for Z. mobilis alcohol dehydrogenase. To supply the acetyl-coA pools, a heterologous phosphoketolase pathway coexpressing the xpkA gene encoding a phosphoketolase from A. nidulans and the pta gene encoding for a phosphotransacetylase from B. subtilis were required. (B) Schematic diagrams of the construction of S. elongatus PCC 7942 strains that produce FAEEs. The pdc-adh genes were introduced into neutral site II (NSII) for endogenous ethanol production. Subsequently, the heterologous atfA and atfA-xpkA-pta genes were introduced to neutral site I (NSI) of S. elongatus genomic DNA. A gel image showed colony-PCR results verifying recombinant S. elongatus strains using a pair of Se1-fw/rv and Se2-fw/rv for the NSI and NSII integrations, respectively. The DNA sequences were also verified. Target size of each PCR product for wild-type or mutant cyanobacteria: wild-type (1.6 kb), Se1A-2Et (5.9 kb), Se1AXP-2Et (9.3 kb) at NSI and wild-type (2.8 kb), Se2Et (7.8 kb), Se1A-2Et (7.8 kb), Se1AXP-2Et (7.8 kb) at NSII. The genotypes of the recombinant strains Se2Et, Se1A-2Et, and Se1AXP-2Et are described in Table 1. Designer 2.0 software (DNA2.0; Menlo Park, CA, USA) (see details in the Supporting Information). They were then synthesized (Genscript Inc., Piscataway, NJ, USA) for efficient heterologous expression in S. elongatus PCC 7942. The pdc and adh genes were also cloned into pSe2Bb1k-GFP to construct pSe2Bb1k-pdc-adh for endogenous production of ethanol. The atfA gene for FAEE production was cloned into pSe1Bb1s-GFP to construct pSe1Bb1s-atfA. To enhance the acetyl-CoA availability, additional xpkA and pta genes were cloned with pSe1Bb1s-atfA, yielding pSe1Bb1s-atfA-xpkA-pta. Transformation of S. elongatus PCC 7942. Transformation of S. elongatus PCC 7942 was performed as described previously.20 The SyneBrick vectors constructed in this study were transferred for chromosomal integration. Recombinant strains Se2Et, Se1A-2Et, and Se1AXP-2Et were obtained after transferring colonies to fresh selective plates to obtain a completely segregated mutant due to the oligoploid in cyanobacteria.21 The strains were confirmed by PCR to verify chromosomal integration of targets into either NSI or NSII (Figure 1). DNA sequences were also verified using Se1-fw (5′-AAG CGC TCC GCA TGG ATC TG-3′) and Se1-rv (5′-CAA GGC AGC TTG GAA GGG CG-3′) for NSI and Se2-fw (5′-GGC TAC GGT TCG TAA TGC CA-3′) and Se2-rv (5′-GAG ATC AGG GCT GTA CTT AC-3′) for NSII. Growth Conditions for FAEE Production from Engineered Cyanobacteria. Recombinant S. elongatus PCC 7942 for the production of FAEEs was cultivated at 30 °C under continuous fluorescent light (100 μmol photons/m2/s) measured using a LightScout Quantum meter (3415FXSE; Spectrum, Aurora, IL, USA). The cultivation was conducted using a sample volume of 100 mL (Duran bottle with a

and metabolic engineering of S. cerevisiae have enhanced FAEE production to 34 mg/L from glucose (20 g/L).13,15,16 Here, we report the first metabolic engineering of Synechococcus elongatus PCC 7942 to produce photosynthetic FAEEs from CO2. In this study, the toxicity of FAEE was alleviated in S. elongatus PCC 7942 using an in situ two-phase extraction. FAEE production was enhanced by replenishing intracellular acetyl-CoA via a heterologous phosphoketolase (PHK) pathway. Our strain will be further optimized to accelerate the development of biosolar cell factories to convert CO2 to FAEEs as a potential biofuel.

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MATERIALS AND METHODS

Strains and Plasmids. All bacterial strains and plasmids used in this study are listed in Table 1. E. coli strain DH5α was used for gene cloning and was grown in Luria−Bertani medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) at 37 °C on a rotary shaker at 200 rpm. When appropriate, the medium was supplemented with 50 μg/mL kanamycin and 100 μg/mL spectinomycin. All plasmids were derived from SyneBrick expression plasmids (standard vectors for chromosomal integration at neutral site I (NSI) or neutral site II (NSII); pSe1Bb1s-GFP, and pSe2Bb1k-GFP)17 using the BglBrick standard cloning method18 as a synthetic platform for gene expression in S. elongatus PCC 7942. The atfA gene (A. baylyi),2 the xpkA gene (Aspergillus nidulans),17,19 the pta gene (Bacillus subtilis),17,19 and the pdc and adh genes (Z. mobilis)2 were codon-optimized using Gene 1088

DOI: 10.1021/acs.jafc.7b00002 J. Agric. Food Chem. 2017, 65, 1087−1092

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The mass spectrometer was operated at 70 eV with selected ion monitoring (SIM), and the mass range was m/z 50−650. The ion source temperature was maintained at 220 °C. FAEE (C4−C24 even carbon numbers, no. 49454-U, Sigma-Aldrich) was used as an authentic standard for quantitative analysis. For the quantification of ethanol, a 1 mL cyanobacterial cell suspension collected from the collection bottle was centrifuged at 10000g for 10 min and filtered with a syringe filter (pore size of 0.2 μm). The filtered samples (1 μL) were analyzed by GC (model 6890; Agilent Technologies) equipped with an HP-INNOWAX polyethylene glycol column (30 m × 0.25 mm × 0.25 μm) and flame ionization detector (FID) under the following conditions: oven temperature from 50 to 240 °C heated at a rate of 10 °C/min, injector temperature of 250 °C, detector temperature of 250 °C with He carrier gas at a flow rate of 25 mL/min, and a split ratio of 10:1.

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RESULTS AND DISCUSSION S. elongatus PCC 7942 does not produce FAEEs by its natural metabolism due to lack of capability of producing ethanol and having activity of a wax ester synthase that esterifies acyl-CoA with ethanol. Thus, pathway engineering in S. elongatus PCC 7942 must be needed for heterologous FAEE production (Figure 1). Before FAEE production by the recombinant strains, the cellular toxicity of FAEEs to the cyanobacterial host was investigated by adding FAEEs into the medium (final concentration of 10 mg/L FAEE standard in the medium) 24 h after inoculation. The wild-type S. elongatus PCC 7942 showed severe growth inhibition with FAEE (Figure 2), although E. coli is tolerant up to 100 g/L.2 A previous work showed that the addition of free fatty acid (linolenic acid) inhibited growth of the wild-type cyanobacterium due to reduced photosynthesis, cell stress, and altered membrane permeability.23 To overcome the toxicity, a two-phase fermentation was used to extract FAEEs by adding a hexadecane overlay.22 The cyanobacterial growth in the presence of the hexadecane overlay with the addition of FAEE was restored to the wild-type level. Thus, we used a hexadecane overlay for in situ FAEE extraction. As the first step toward FAEE production, strain Se2Et coexpressing Pdc and Adh was constructed and cultivated with 5% (v/v) CO2 bubbling. This resulted in 232 ± 1.4 mg/L ethanol after 7 days (Figure 3, left panel). No FAEEs were detected. Subsequently, the wax ester synthase af tA gene was inserted into the ethanol-producing strain (Se2Et), resulting in strain Se1A-2Et. Strain Se1A-2Et showed 40% ethanol reduction compared to the Se2Et strain (Figure 3, middle panel), secreting 139 ± 5.6 mg/L ethanol into the medium. The formation of FAEEs from Se1A-2Et was determined using GC-MS analysis based on a hexadecane overlay extract. A chromatographic peak was detected at a retention time of 10.5, and its ion fragmentation pattern was matched to the palmitic acid ethyl ester (C18H36O2) peak of the authentic FAEE standard (Figure S1). Although the levels of FAEEs were too low (lower than the limit of detection, 42 μg/L) to determine the concentration, this was the first detection of FAEE formation from CO2 in cyanobacteria. The reason for the reduction in ethanol production in Se1A-2Et was not clear because corresponding endogenous ethanol was not esterified with acylCoA to produce FAEEs. Thus, metabolic engineering of S. elongatus PCC 7942 was applied to increase the production of FAEEs from CO2. In S. cerevisiae, production levels of FAEEs were successfully enhanced by overexpressing the phosphoketolase gene to increase the acetyl-CoA pool, a precursor of acyl-CoA.19 Previous studies

Figure 2. Cellular toxicity test of FAEEs to S. elongatus PCC 7942. (A) Wild-type cultivated and measured at OD730 under different conditions: (1) 5% (v/v) CO2 bubbling as a control, black bar; (2) 20% (v/v) hexadecane added to the control, blue bar; (3) 20% (v/v) hexadecane added to the control and addition of FAEEs (10 mg/L, final concentration in the medium), red bar; (4) control with added FAEEs, green bar. (B) Photographic image of the cyanobacterial cell culture bottle for the test. FAEEs (10 mg/L, final concentration in the medium) were added to the media of experiments 3 and 4, indicated by the red arrow at day 1 of (A). three-port cap) in BG-11 medium (UTEX) supplemented with 10 mM MOPS at pH 7.5, with 5% (v/v) CO2 gas (monitored by online gas analyzer) and 95% (v/v) filtered air supplied at a constant flow rate of 10 cm3/min into the medium.17,22 In addition, 10 μg/mL spectinomycin and 10 μg/mL kanamycin were supplemented for selection pressure. Then, 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added into the culture medium 24 h after inoculation for induction. Samples were collected in 20% (v/v) hexadecane for in situ FAEE extraction. Quantification of Photosynthetic FAEE and Ethanol. To quantify the secreted FAEEs, samples (200 μL) from the hexadecane layer in the culture were collected and diluted with ethyl acetate (800 μL) containing 10 mg/L methyl nonadecanoate as an internal standard. To quantify intracellular FAEEs, the cell suspension samples (600 μL) were added to 600 μL of ethyl acetate containing an internal standard. The samples were mixed with 150 mg of 0.1 mm zirconia/ silica beads. The cell samples were then homogenized with a beadbeater [90 s; 0.15 g glass beads (0.1 mm diameter)] and centrifuged at 13000g for 5 min. Samples from hexadecane/ethyl acetate and cell extract/ethyl acetate were analyzed using gas chromatography− mass spectrometry [GC-MS; Agilent 6890N GC series coupled with TOF-MS (LECO)]. The sample solution (2 μL) was injected in split mode (5:1) at an injector temperature of 250 °C and was separated in an Ultra-2 capillary column (17 m × 0.2 mm i.d., 0.11 μm film thickness; Agilent Technologies, Santa Clara, CA, USA). The oven temperature was 50 °C for 1.50 min, was ramped to 200 °C at 20 °C/min and then to 250 °C at 10 °C/min, and held for 3 min for a total run time of 17.00 min. Helium (99.9999%) was used as the carrier gas (1.0 mL/min constant flow at an oven temperature of 150 °C). 1089

DOI: 10.1021/acs.jafc.7b00002 J. Agric. Food Chem. 2017, 65, 1087−1092

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Figure 3. Photosynthetic FAEEs produced from CO2 in engineered S. elongatus PCC 7942. Engineered S. elongatus PCC 7942 strains were cultivated under 5% (v/v) CO2 bubbling in the presence of a 20% (v/v) hexadecane overlay. Measurements of cell growth, ethanol production (mg/L), and specific production of FAEEs (mg/L/OD730) were performed for the recombinant strains. In situ extracted samples from the hexadecane overlay using engineered S. elongatus strains were analyzed using GC-MS. The FAEE production in cells and the hexadecane overlay are represented as gray bars and green bars, respectively. Retention times of FAEEs and methyl nonadecanoate used as an internal standard (IS) are shown. The genotypes of the recombinant strains Se2Et, Se1A-2Et, and Se1AXP-2Et are described in Table 1. All data are the mean ± standard deviation from cultures run in triplicate.

an OD730 of 0.5, 1.5 ± 0.2 mg/L/OD730). Also, secreted FAEEs from Se1AXP-2Et (starting at an OD730 of 1, 4.9 ± 0.5 mg/L/OD730) increased 1.93-fold compared to Se1AXP-2Et (starting at an OD730 of 0.5, 2.5 ± 0.2 mg/L/OD730). Finally, Se1AXP-2Et produced FAEEs (10.0 ± 0.7 mg/L/OD730) from CO2. Interestingly, the strain Se1AXP-2Et (starting at an OD730 of 1) showed a 68% ethanol reduction compared to the Se2Et strain (Figure 3, right panel). The decreased patterns of both ethanol production and cyanobacterial cell growth were caused by increased production of FAEEs in cyanobacteria. Thus, further optimization of IPTG induction time and dosage could enhance the levels of FAEE production in Se1AXP-2Et. In addition, balancing the FAEE production and secretion and lowering the acyl-CoA dehydrogenase activity could also be crucial for sustainable cyanobacterial growth and FAEE production.24 Our FAEE production platform could be extended to test the use of various bacterial acyltransferases and to produce shortened FAEEs by engineering an acyl carrier protein with medium-chain fatty acid specificity.25,26 We demonstrated photosynthetic production of FAEEs from CO2 using metabolically engineered cyanobacteria for the first time. Redirecting carbon flux via the heterologous phosphoketolase pathway in recombinant cyanobacteria resulted in FAEE production from CO2. Culture optimization led to photosynthetic production of FAEEs (10.0 ± 0.7 mg/L/OD730) from CO2. For enhanced production, a systems biology approach27,28 (i.e., transcriptomics or metabolomics) could be useful for understanding the bottleneck of cellular metabolisms in cyanobacteria for production of FAEE and its cellular toxicity. Further optimization of our cyanobacterial strains is necessary to allow production of FAEEs from CO2. Recent programmable RNA-guided genome engineering and metabolic engineering29,30

have also shown that the bottleneck for acetyl-CoA-derived chemical production was due to the low levels of acetyl-CoA pools in cyanobacteria.17 This limitation has been overcome by increasing the acetyl-CoA pool from the pentose phosphate pathway through a heterologous phosphoketolase pathway. To increase FAEE production in S. elongatus PCC 7942, a heterologous phosphoketolase pathway was introduced, yielding an Se1AXP-2Et strain coexpressing phosphoketolase and phosphotransacetylase (Figure 1B). Strain Se1AXP-2Et showed 63% ethanol reduction compared to strain Se2Et (Figure 3, right panel), secreting 85.7 ± 7.6 mg/L ethanol into the medium. Intracellular FAEEs and secreted FAEEs were observed in the Se1AXP-2Et strain. Palmitic acid ethyl ester (C18H36O2, C16:0-ethyl ester) was quantified for both the hexadecane overlay and intracellular extract. Also, stearic acid ethyl ester (C20H40O2, C18:0-ethyl ester) was detected only in the intracellular extract. Specific production of the intracellular FAEEs gradually decreased from 1.4 ± 0.1 to 0.1 ± 0.02 mg/L/OD730 for the cultivations (10 days). On the other hand, secreted FAEEs in the overlay gradually increased from 0.7 ± 0.1 to 3.9 ± 0.6 mg/L for 10 days. However, 3 days after the inoculation, Se1AXP-2Et showed 45% growth reduction compared to its parental strain (Se1A-2Et). Production of intracellular FAEEs inhibited cyanobacterial cell growth. To reduce the possible intracellular FAEE inhibition, Se1AXP2Et was cultivated with higher density inoculum (starting at an OD730 of 1) than the previous culture (starting at an OD730 of 0.5) (Figure 3, right panel). Then, IPTG induction for Se1AXP-2Et was performed at an OD730 of 2. The amount of intracellular FAEEs produced from the Se1AXP-2Et (starting at an OD730 of 1, 4.6 ± 0.8 mg/L/OD730) increased 3-fold compared to the intracellular FAEEs produced from the Se1AXP-2Et (starting at 1090

DOI: 10.1021/acs.jafc.7b00002 J. Agric. Food Chem. 2017, 65, 1087−1092

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(3) Kim, S.; Cheong, S.; Chou, A.; Gonzalez, R. Engineered fatty acid catabolism for fuel and chemical production. Curr. Opin. Biotechnol. 2016, 42, 206−215. (4) Woo, H. M. Solar-to-chemical and solar-to-fuel production from CO2 by metabolically engineered microorganisms. Curr. Opin. Biotechnol. 2017, 45, 1−7. (5) Oliver, N. J.; Rabinovitch-Deere, C. A.; Carroll, A. L.; Nozzi, N. E.; Case, A. E.; Atsumi, S. Cyanobacterial metabolic engineering for biofuel and chemical production. Curr. Opin. Chem. Biol. 2016, 35, 43−50. (6) Liu, X.; Sheng, J.; Curtiss, R., 3rd Fatty acid production in genetically modified cyanobacteria. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 6899−6904. (7) Ruffing, A. M. Improved free fatty acid production in cyanobacteria with Synechococcus sp. PCC 7002 as host. Front. Bioeng. Biotechnol. 2014, 2, 17. (8) Yao, L.; Qi, F. X.; Tan, X. M.; Lu, X. F. Improved production of fatty alcohols in cyanobacteria by metabolic engineering. Biotechnol. Biofuels 2014, 7, 94. (9) Tan, X. M.; Yao, L.; Gao, Q. Q.; Wang, W. H.; Qi, F. X.; Lu, X. F. Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria. Metab. Eng. 2011, 13, 169−176. (10) Wang, W.; Liu, X.; Lu, X. Engineering cyanobacteria to improve photosynthetic production of alka(e)nes. Biotechnol. Biofuels 2013, 6, 69. (11) Rottig, A.; Wenning, L.; Broker, D.; Steinbuchel, A. Fatty acid alkyl esters: perspectives for production of alternative biofuels. Appl. Microbiol. Biotechnol. 2010, 85, 1713−1733. (12) Kalscheuer, R.; Stölting, T.; Steinbüchel, A. Microdiesel: Escherichia coli engineered for fuel production. Microbiology 2006, 152, 2529−2536. (13) Yu, K. O.; Jung, J.; Kim, S. W.; Park, C. H.; Han, S. O. Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase. Biotechnol. Bioeng. 2012, 109, 110− 115. (14) Runguphan, W.; Keasling, J. D. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metab. Eng. 2014, 21, 103−113. (15) Shi, S. B.; Valle-Rodriguez, J. O.; Siewers, V.; Nielsen, J. Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters. Biotechnol. Bioeng. 2014, 111, 1740−1747. (16) Thompson, R. A.; Trinh, C. T. Enhancing fatty acid ethyl ester production in Saccharomyces cerevisiae through metabolic engineering and medium optimization. Biotechnol. Bioeng. 2014, 111, 2200−2208. (17) Chwa, J. W.; Kim, W. J.; Sim, S. J.; Um, Y.; Woo, H. M. Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition. Plant Biotechnol. J. 2016, 14, 1768−1776. (18) Lee, T. S.; Krupa, R. A.; Zhang, F.; Hajimorad, M.; Holtz, W. J.; Prasad, N.; Lee, S.; Keasling, J. D. BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J. Biol. Eng. 2011, 5, 12. (19) de Jong, B. W.; Shi, S.; Siewers, V.; Nielsen, J. Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway. Microb. Cell Fact. 2014, 13, 39. (20) Golden, S. S.; Brusslan, J.; Haselkorn, R. Genetic engineering of the cyanobacterial chromosome. Methods Enzymol. 1987, 153, 215− 231. (21) Griese, M.; Lange, C.; Soppa, J. Ploidy in cyanobacteria. FEMS Microbiol. Lett. 2011, 323, 124−131. (22) Choi, S. Y.; Lee, H. J.; Choi, J.; Kim, J.; Sim, S. J.; Um, Y.; Kim, Y.; Lee, T. S.; Keasling, J. D.; Woo, H. M. Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha4,11-diene and squalene) by engineered cyanobacteria. Biotechnol. Biofuels 2016, 9, 202.

could be applied to improve the production of FAEEs from CO2 as a potential candidate for biodiesel.

Hyun Jeong Lee† Jaeyeon Choi† Sun-Mi Lee† Youngsoon Um† Sang Jun Sim§ Yunje Kim† Han Min Woo*,# †

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Clean Energy Research Center, Korea Institute of Science and Technology, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea § Department of Chemical and Biological Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea # Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00002. Figure S1: Mass spectra and retention times of an authentic FAEE standard (palmitic acid ethyl ester) and photosynthetic FAEE product from engineered S. elongatus PCC 7942. Codon-optimized DNA sequence for each target gene and its original genbank accession number (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(H.M.W.) E-mail: [email protected]. Phone: +82 31 290 7808. ORCID

Han Min Woo: 0000-0002-8797-0477

Funding

This work was financially supported by the Korea CCS R&D Center (KCRC) (Grant 2014M1A8A1049277) funded by the Korean government (Ministry of Science, Information, and Communications Technology (ICT) & Future Planning). Also, this work was partially supported by a Golden Seed Project (213008-05-1-WT911) grant funded by the Ministry of Agriculture, Ministry of Oceans and Fisheries. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank M. S. Jiwon Lee at the Korea Institute of Science and Technology for technical support. We also thank Prof. EonSeon Jin at Hanyang University for valuable comments.

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REFERENCES

(1) Peralta-Yahya, P. P.; Zhang, F. Z.; del Cardayre, S. B.; Keasling, J. D. Microbial engineering for the production of advanced biofuels. Nature 2012, 488, 320−328. (2) Steen, E. J.; Kang, Y.; Bokinsky, G.; Hu, Z.; Schirmer, A.; McClure, A.; Del Cardayre, S. B.; Keasling, J. D. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 2010, 463, 559−562. 1091

DOI: 10.1021/acs.jafc.7b00002 J. Agric. Food Chem. 2017, 65, 1087−1092

Letter

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DOI: 10.1021/acs.jafc.7b00002 J. Agric. Food Chem. 2017, 65, 1087−1092