Letter pubs.acs.org/JAFC
Direct Conversion of CO2 to α‑Farnesene Using Metabolically Engineered Synechococcus elongatus PCC 7942 ABSTRACT: Direct conversion of carbon dioxide (CO2) to value-added chemicals by engineering of cyanobacteria has received attention as a sustainable strategy in food and chemical industries. Herein, Synechococcus elongatus PCC 7942, a model cyanobacterium, was engineered to produce α-farnesene from CO2. As a result of the lack of farnesene synthase (FS) activity in the wild-type cyanobacterium, we metabolically engineered S. elongatus PCC 7942 to express heterologous FS from either Norway spruce or apple fruit, resulting in detectable peaks of α-farnesene. To enhance α-farnesene production, an optimized methylerythritol phosphate (MEP) pathway was introduced in the farnesene-producing strain to supply farnesyl diphosphate. Subsequent cyanobacterial culture with a dodecane overlay resulted in photosynthetic production of α-farnesene (4.6 ± 0.4 mg/L in 7 days) from CO2. To the best of our knowledge, this is the first report of the photosynthetic production of α-farnesene from CO2 in the unicellular cyanobacterium S. elongatus PCC 7942. KEYWORDS: cyanobacteria, metabolic engineering, farnesene, CO2 conversion
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INTRODUCTION Solar-to-chemical and solar-to-fuel production from CO2 have been developed using engineered microorganisms that assimilate CO2 and convert target chemicals and fuels using solar energy.1 Besides integrated bioelectrochemical systems, photosynthetic cyanobacteria have been metabolically engineered to redirect CO2 to value-added chemicals as biosolar cell factories. In cyanobacteria, approximately 5% of photosynthetic carbon flux is distributed to the terpenoid biosynthetic pathway.2 However, the corresponding free terpenes are not accumulated. Thus, to redirect the terpenoid biosynthesis flux to free terpenes in cyanobacteria, a methylerythritol phosphate (MEP) pathway that initiates from central intermediates (pyruvate and glyceraldehyde3-phosphate) must be engineered and the cyanobacteria must express the corresponding heterologous terpene synthase gene. The cyanobacterial terpene production platform has been developed through metabolic engineering and synthetic biology on the basis of a number of isoprene units: hemiterpene (C5, isoprene,3,4 57.4 mg L−1 day−1), monoterpene (C10, limonene,5 0.05 mg L−1 day−1), sesquiterpene (C15, amorpha-4,11-diene,6 1.98 mg L−1 day−1), diterpene [C20, manoyl oxide,7 0.45 mg/g of dry cell weight (DCW), volume concentration data not presented], triterpene (C30, squalene,8 5.3 mg L−1 day−1), and tetraterpene (not yet reported). Specifically, α-farnesene (C15H24), which belongs to sesquiterpenes found in apple peels and plays a role in plant defense, has been known as a precursor of high-performance polymers9 and a promising biojet fuel candidate.10 To produce α-farnesene, metabolic engineering has been successfully applied to generate α-farnesene in microbial hosts, such as Escherichia coli11 (0.38 mg/g of glycerol), Saccharomyces cerevisiae12 (0.57 mg/g of glucose), or Yarrowia lipolytica13 (6.5 mg/g of glucose and fructose). The combination of heterologous gene expression of farnesene synthase and the reconstitution of the terpenoid biosynthesis pathway were key to increasing microbial α-farnesene production levels. Recently, the filamentous cyanobacterium Anabaena sp. PCC 7129, harboring a plasmid (pFaS, the codonoptimized farnesene synthase gene from Norway spruce), was constructed and yielded 305.4 μg/L farnesene in 15 days by expressing only the heterologous farnesene synthase gene.14 © 2017 American Chemical Society
Here, we report the metabolic engineering of unicellular Synechococcus elongatus PCC 7942 to improve the photosynthetic production of α-farnesene from CO2. In this study, both heterologous gene expression and optimization of the terpenoid biosynthesis pathway led to substantial levels of α-farnesene production from CO2. This engineered strain could be further optimized to accelerate the development of biosolar cell factories to convert CO2 to α-farnesene as a value-added bioproduct (fragrance, surfactants, etc.) or α-farnesane, a potential biojet fuel.
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MATERIALS AND METHODS
Strains and Plasmid Construction. All bacterial strains and plasmids used in this study are listed in Table 1. The 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. 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]15,16 using the BglBrick standard cloning method17 as a synthetic platform for gene expression in S. elongatus PCC 7942. The α-farnesene synthase genes from Picea abies L. Karst. (Norway spruce, the NSFS gene)18 and Malus × domestica Borkh. (apple fruit, the AFS gene, GenBank accession number AY182241)19 were codon-optimized using Gene Designer 2.0 software (DNA2.0, ATUM, Menlo Park, CA, U.S.A.) (see details in the Supporting Information). They were then synthesized (Genscript, Inc., Piscataway, NJ, U.S.A.) for heterologous expression in S. elongatus PCC 7942. The NSFS or AFS genes were also cloned into pSe2Bb1k-GFP to construct pSe2Bb1k-NSFS and pSe2Bb1kAFS for production of α-farnesene. Transformation of Engineered 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. First, SeHL01FS and SeHL02FS were constructed with pSe2Bb1k-NSFS and pSe2Bb1k-AFS, respectively. Furthermore, four engineered strains were constructed by transforming the two different farnesene synthase strains with plasmids pSe1Bb1s-dxs or pSe1Bb1s-dxs-idi-ispA for the optimized MEP pathway, Received: Revised: Accepted: Published: 10424
August 4, 2017 October 23, 2017 October 25, 2017 October 25, 2017 DOI: 10.1021/acs.jafc.7b03625 J. Agric. Food Chem. 2017, 65, 10424−10428
Letter
Journal of Agricultural and Food Chemistry Table 1. Bacterial Strains and Plasmids Used in This Study strain or plasmid E. coli HIT-DH5α S. elongatus PCC 7942 SeHL11 SeHL33 SeHL01FS SeHL02FS SeHL11FS SeHL12FS SeHL31FS SeHL32FS pSe2Bb1k-GFP pSe2Bb1k-NSFS pSe2Bb1k-AFS a
relevant characteristic
reference
Strains F−(80d lacZ M15) (lacZYA-argF) U169 hsdR17(r− m+) recA1 endA1 relA1 deoR96 wild type (ATCC 33912) S. elongatus PCC 7942 NSI::Bb1s-dxs S. elongatus PCC 7942 NSI::Bb1s-dxs-idi-ispA S. elongatus PCC 7942 NSII::Bb1k-NSFS S. elongatus PCC 7942 NSII::Bb1k-AFS S. elongatus PCC 7942 NSI::Bb1s-dxs, NSII::Bb1k-NSFS S. elongatus PCC 7942 NSI::Bb1s-dxs, NSII::Bb1k-AFS S. elongatus PCC 7942 NSI::Bb1s-dxs-idi-ispA, NSII::Bb1k-NSFS S. elongatus PCC 7942 NSI::Bb1s-dxs-idi-ispA, NSII::Bb1k-AFS Plasmid pUC, Kmr, LacI, Ptrc, BglBrick sites, NSII target sites, SyneBrick vector pUC, Kmr, LacI, Ptrc, NSII target sites, the farnesene synthase gene originated from P. abies L. Karst.,18 the NSFS gene (se.co)a pUC, Kmr, LacI, Ptrc, NSII target sites, the farnesene synthase gene (AFS) originated from M. × domestica Borkh.19, the AFS gene (se.co)a
RBC Bioscience ATCC this study 6,28 this study this study this study this study this study this study 15 this study this study
(se.co) represents that the gene sequence is codon-optimized to S. elongatus PCC 7942.
as previously described.6 The genotypes of recombinant S. elongatus strains are listed in Table 1. The strains were confirmed by polymerase chain reaction (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. Cyanobacterial Growing Conditions. Recombinant S. elongatus PCC 7942 used for the production of α-farnesene was cultivated at 30 °C under continuous fluorescent light (100 μE m−2 s−1), measured using a LightScout quantum meter (3415FXSE, Spectrum Technologies, Aurora, IL, U.S.A.). The cultivation was conducted using a sample volume of 100 mL (Duran bottle with a three-port cap) in BG-11 medium supplemented with 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.5, with 5% (v/v) CO2 gas (monitored by an online gas analyzer) and 95% (v/v) filtered air, supplied at a constant flow rate of 10 cm3/min into the medium.6,15,21 In addition, 10 μg/mL spectinomycin and 10 μg/mL kanamycin were supplemented for selection pressure. Next, 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to the culture medium 24 h after inoculation for induction. Samples were collected in 20% (v/v) dodecane overlay for in situ α-farnesene extraction.6,21 Quantification of Photosynthetic α-Farnesene. To quantify the secreted α-farnesene, samples (200 μL) from the dodecane layer in the culture were collected and diluted with ethyl acetate (800 μL) containing 5 mg/L β-caryophyllene as an internal standard. Samples from dodecane/ethyl acetate were analyzed using gas chromatography−mass spectrometry [GC−MS, Agilent 6890N GC series, coupled with timeof-flight−mass spectrometry (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 inner diameter, 0.11 μm film thickness, Agilent Technologies, Santa Clara, CA, U.S.A.). The oven temperature was 50 °C for 1.50 min, ramped to 200 °C at 20 °C/min, 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). The mass spectrometer was operated at 70 eV, and the mass range was m/z 50−650. The ion source temperature was maintained at 220 °C. trans-β-Farnesene (73492, Sigma-Aldrich, St. Louis, MO, U.S.A.) was used as an authentic standard for quantitative analysis.
farnesene synthase and its corresponding gene. Thus, we metabolically engineered S. elongatus PCC 7942 to convert CO2 directly to α-farnesene (Figure 1). First, two different farnesene synthase genes (NSFS and AFS) were codon-optimized and chromosomally integrated into the wild type, yielding the strains SeHL01FS and SeHL02FS, respectively. Instead of using an adsorption column for volatile α-farnesene, an in situ dodecane overlay was applied to extract farnesene from the cultures (Figure 2). The dodecane overlay has been chosen for not having a toxic effect on cyanobacterial cell growth and for being widely used for isoprenoid extraction from microbial cultures.6,11 As a result, photosynthetic α-farnesene was not detected in the dodecane layer of the cultures, of which the strain SeHL01FS overexpressed the same farnesene synthase gene (NSFS) that was used in the recombinant Anabaena sp. PCC 7129. However, α-farnesene was detected in the strain SeHL02FS overexpressing the AFS gene [lower than the limit of quantification (LOQ),
