Direct conversion of CO2 to Î±-farnesene using metabolically

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Letter 2

Direct conversion of CO to #-farnesene using metabolically engineered Synechococcus elongatus PCC 7942 Hyun Jeong Lee, Jiwon Lee, Sun-Mi Lee, Youngsoon Um, Yunje Kim, Sang Jun Sim, Jong-il Choi, and Han Min Woo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03625 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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Journal of Agricultural and Food Chemistry

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Letter

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Direct conversion of CO2 to α-farnesene using metabolically engineered

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Synechococcus elongatus PCC 7942

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Hyun Jeong Lee1,2, Jiwon Lee3, Sun-Mi Lee3, Youngsoon Um3, Yunje Kim3, Sang Jun Sim4,

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Jong-il Choi5, Han Min Woo1*

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Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066

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Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

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3

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gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea

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4

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Seongbuk-gu, Seoul 02841, Republic of Korea

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Yongbong-ro, Buk-gu, Gwangju 61186, Republic of Korea

Institute of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), 2066

Clean Energy Research Center, Korea Institute of Science and Technology, Hwarang-ro 14-

Department of Chemical and Biological Engineering, Korea University, 145 Anam-ro,

Department of Biotechnology and Bioengineering, Chonnam National University, 77

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*Corresponding author at Department of Food Science and Biotechnology, Sungkyunkwan

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University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea; Tel.: +82

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31 290 7810; E-mail address: [email protected] (H. M. Woo)

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Abstract: Direct conversion of carbon dioxide (CO2) to value-added chemicals by

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engineering of cyanobacteria has received attention as a sustainable strategy in food and

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chemical industries. Herein, Synechococcus elongatus PCC 7942, a model cyanobacterium,

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was engineered to produce α-farnesene from CO2. Due to the lack of farnesene synthase (FS)

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activity in the wild-type cyanobacterium, we metabolically engineered S. elongatus PCC

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7942 to express heterologous FS from either Norway spruce or apple fruit, resulting in

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detectable peaks of α-farnesene. To enhance α-farnesene production, an optimized

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methylerythritol phosphate (MEP) pathway was introduced in the farnesene-producing strain

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to supply farnesyl diphosphate. Subsequent cyanobacterial culture with a dodecane overlay

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resulted in photosynthetic production of α-farnesene (4.6 ± 0.4 mg/L in 7 days) from CO2. To

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the best of our knowledge, this is the first report of the photosynthetic production of α-

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farnesene from CO2 in the unicellular cyanobacterium S. elongatus PCC 7942.

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Keywords: cyanobacteria, metabolic engineering, farnesene, CO2 conversion

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INTRODUCTION

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Solar-to-chemical and solar-to-fuel production from CO2 have been developed using

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engineered microorganisms that assimilate CO2 and convert target chemicals and fuels using

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solar energy.1 Besides integrated bio-electrochemical systems, photosynthetic cyanobacteria

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have been metabolically engineered to re-direct CO2 to value-added chemicals as bio-solar

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cell factories. In cyanobacteria, approximately 5% of photosynthetic carbon flux is

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distributed to the terpenoid biosynthetic pathway.2 However, the corresponding free terpenes

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are not accumulated. Thus, to re-direct the terpenoid biosynthesis flux to free terpenes in

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cyanobacteria, a methylerythritol phosphate (MEP) pathway that initiates from central

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intermediates (pyruvate and glyceraldehyde-3-phosphate) must be engineered, and the

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cyanobacteria must express the corresponding heterologous terpene synthase gene. The

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cyanobacterial terpene production platform has been developed through metabolic

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engineering and synthetic biology, based on a number of isoprene units: hemiterpene (C5;

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isoprene3,4; 57.4 mg/L/d), monoterpene (C10; limonene5; 0.05 mg/L/d), sesquiterpene (C15;

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amorpha-4,11-diene6; 1.98 mg/L/d), diterpene (C20; manoyl oxide7; 0.45 mg/gDCW; volume

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concentration data not presented), triterpene (C30; squalene8; 5.3 mg/L/d), and tetraterpene

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(not reported yet).

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Specifically, α-farnesene (C15H24), which belongs to sesquiterpenes found in apple peels

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and plays a role in plant defense, has been known as a precursor of high-performance

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polymers9 and a promising bio-jet fuel candidate10. To produce α-farnesene, metabolic

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engineering has been successfully applied to generate α-farnesene in microbial hosts such as

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Escherichia coli11 (0.38 mg/g glycerol), Saccharomyces cerevisiae12 (0.57 mg/g glucose), or

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Yarrowia lipolytica13 (6.5 mg/g glucose and fructose). The combination of heterologous gene

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expression of farnesene synthase and the reconstitution of the terpenoid biosynthesis pathway

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was key to increasing microbial α-farnesene production levels. Recently, the filamentous 3 ACS Paragon Plus Environment


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cyanobacterium Anabaena sp. PCC 7129, harboring a plasmid (pFaS, the codon-optimized

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farnesene synthase gene from Norway spruce), was constructed and yielded 305.4 µg/L

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farnesene in 15 days by expressing only the heterologous farnesene synthase gene.14

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Here, we report the metabolic engineering of unicellular Synechococcus elongatus

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PCC 7942 to improve the photosynthetic production of α-farnesene from CO2. In this study,

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both heterologous gene expression and optimization of the terpenoid biosynthesis pathway

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led to substantial levels of α-farnesene production from CO2. This engineered strain could be

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further optimized to accelerate the development of bio-solar cell factories to convert CO2 to

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α-farnesene as a value-added bio-product (fragrance, surfactants, etc.) or α-farnesane, a

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potential bio-jet fuel.

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MATERIALS AND METHODS

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Strains and plasmid construction. All bacterial strains and plasmids used in this study are

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listed in Table 1. The E. coli strain DH5α was used for gene cloning and was grown in Luria-

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Bertani medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) at 37°C. When

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appropriate, the medium was supplemented with 50 µg/mL kanamycin and 100 µg/mL

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spectinomycin. All plasmids were derived from SyneBrick expression plasmids (standard

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vectors for chromosomal integration at neutral site I (NSI) or neutral site II (NSII),

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pSe1Bb1s-GFP and pSe2Bb1k-GFP)15,16 using the BglBrick standard cloning method17 as a

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synthetic platform for gene expression in S. elongatus PCC 7942. The α-farnesene synthase

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genes from Picea abies L. Karst (Norway spruce; the NSFS gene)18 and Malus × domestica

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Borkh. (apple fruit; the AFS gene; GenBank accession number AY182241)19 were codon-

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optimized using Gene Designer 2.0 software (DNA2.0; ATUM, Menlo Park, CA) (see details

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in the Supporting Information). They were then synthesized (Genscript Inc., Piscataway, NJ)

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for heterologous expression in S. elongatus PCC 7942. The NSFS or AFS genes were also 4 ACS Paragon Plus Environment

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cloned into pSe2Bb1k-GFP to construct pSe2Bb1k-NSFS and pSe2Bb1k-AFS for production

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of α-farnesene.

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Transformation of engineered S. elongatus PCC 7942. Transformation of S. elongatus

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PCC 7942 was performed as described previously.20 The SyneBrick vectors constructed in

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this study were transferred for chromosomal integration. First, SeHL01FS and SeHL02FS

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were constructed with pSe2Bb1k-NSFS and pSe2Bb1k-AFS, respectively. Furthermore, four

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engineered strains were constructed by transforming the two different farnesene synthase

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strains with plasmids pSe1Bb1s-dxs or pSe1Bb1s-dxs-idi-ispA for the optimized MEP

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pathway, as previously described.6 The genotypes of recombinant S. elongatus strains are

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listed in Table 1. The strains were confirmed by PCR to verify chromosomal integration of

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targets into either NSI or NSII (Figure 1). DNA sequences were also verified using Se1-fw

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(5’-AAG CGC TCC GCA TGG ATC TG-3’) and Se1-rv (5’-CAA GGC AGC TTG GAA

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GGG CG-3’) for NSI and Se2-fw (5’-GGC TAC GGT TCG TAA TGC CA-3’) and Se2-rv

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(5’-GAG ATC AGG GCT GTA CTT AC-3’) for NSII.

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Cyanobacterial growing conditions. Recombinant S. elongatus PCC 7942 used for the

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production of α-farnesene was cultivated at 30°C under continuous fluorescent light

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(100 µE/m2/s), measured using a LightScout Quantum meter (3415FXSE; Spectrum

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Technologies, Aurora, IL). The cultivation was conducted using a sample volume of 100 mL

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(Duran bottle with a three-port cap) in BG-11 medium supplemented with 10 mM MOPS at

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pH 7.5, with 5% (v/v) CO2 gas (monitored by online gas analyzer) and 95% (v/v) filtered air,

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supplied at a constant flow rate of 10 cc/min into the medium.6,15,21 In addition, 10 µg/mL

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spectinomycin and 10 µg/mL kanamycin were supplemented for selection pressure. Next,

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1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added into the culture medium 5 ACS Paragon Plus Environment


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24 h after inoculation for induction. Samples were collected in 20% (v/v) dodecane overlay

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for in situ α-farnesene extraction.6,21

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Quantification of photosynthetic α-farnesene. To quantify the secreted α-farnesene,

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samples (200 µL) from the dodecane layer in the culture were collected and diluted with ethyl

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acetate (800 µL) containing 5 mg/L β-caryophyllene as an internal standard. Samples from

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dodecane/ethyl acetate were analyzed using gas chromatography–mass spectrometry (GC-MS;

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Agilent 6890N GC series, coupled with TOF-MS (LECO)). The sample solution (2 µL) was

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injected in split mode (5:1) at an injector temperature of 250°C and was separated in an

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Ultra-2 capillary column (17 m × 0.2 mm i.d., 0.11 µm film thickness; Agilent Technologies,

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Santa Clara, CA). The oven temperature was 50°C for 1.50 min, ramped to 200°C at

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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.

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Helium (99.9999%) was used as the carrier gas (1.0 mL/min). The mass spectrometer was

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operated at 70 eV, and the mass range was 50-650 m/z. The ion source temperature was

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maintained at 220°C. Trans-β-farnesene (No. 73492; Sigma-Aldrich, St. Louis, MO) was

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used as an authentic standard for quantitative analysis.

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RESULTS AND DISCUSSION

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The wild-type unicellular cyanobacterium S. elongatus PCC 7942 does not produce farnesene

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due to the lacking activity of farnesene synthase and its corresponding gene. Thus, we

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metabolically engineered S. elongatus PCC 7942 to convert CO2 directly to α-farnesene

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(Figure 1).

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First, two different farnesene synthase genes (NSFS and AFS) were codon-optimized and

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chromosomally integrated into the wild type, yielding the strains SeHL01FS and SeHL02FS,

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respectively. Instead of using an adsorption column for volatile α-farnesene, an in situ 6 ACS Paragon Plus Environment

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dodecane overlay was applied to extract farnesene from the cultures (Figure 2). The dodecane

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overlay has been chosen for not having a toxic effect on cyanobacterial cell growth and for

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being widely used for isoprenoid extraction from microbial cultures.6,11 As a result,

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photosynthetic α-farnesene was not detected in the dodecane layer of the cultures, of which

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the strain SeHL01FS overexpressed the same farnesene synthase gene (NSFS) that was used

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in the recombinant Anabaena sp. PCC 7129. However, α-farnesene was detected in the strain

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SeHL02FS overexpressing the AFS gene (lower than limit of quantification, < 0.136 mg/L),

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although the production level was significantly lower than that by the recombinant Anabaena

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sp. PCC 7129. The reason could be the low level of farnesene synthase gene expression or

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low levels of farnesyl diphosphate (FPP) pools, which are crucial factors for high-level

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conversion of FPP to farnesene. The level of overexpression of the target gene in S. elongatus

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PCC 7942 using the SyneBrick vector16 could be lower than in Anabaena sp. PCC 7129 due

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to the chromosomal gene integration of the NSFS or AFS genes, instead of gene expression in

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a high-copy plasmid (pZR1188)14. Nonetheless, a plasmid-bearing host has instability issues

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due to long-term expression of the heterologous genes.22 Also, the strain SeHL01FS and

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SeHL02FS showed growth inhibitions due to possible depletion of the FPP levels (Figure 3).

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Thus, we focused on metabolic engineering to increase pools of FPP, the key intermediate for

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improving the production level of α-farnesene.

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Previously, FPP-derived phytochemicals, including amorpha-4,11-diene (19.8 mg/L)6 and

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squalene (11.98 mg/L)6,8, have been successfully overproduced from CO2 in engineered S.

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elongatus PCC 7942 constructed from the parental strain (SeHL33; OverMEP module), in

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which the MEP pathway was optimized. Two different parental strains were used for the

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production improvement: the MEP-basic strain, SeHL11 and the MEP-optimized strain,

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SeHL33. The dxs gene is overexpressed in SeHL11, and the combinatorial expressions of the

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dxs, idi, and ispA genes are allowed in SeHL33 (Table 1). As a result, four strains 7 ACS Paragon Plus Environment


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(SeHL11FS, SeHL12FS, SeHL31FS, and SeHL32FS) were cultivated with a dodecane

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overlay (Figure 3). MEP-basic strains with heterologous NSFS or ASF genes (SeHL11FS and

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SeHL12FS, respectively) failed to produce quantifiable levels of α-farnesene. However,

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SeHL31FS and SeHL32FS, for which the MEP-optimized strain (SeHL33) was used as a

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parental strain, produced farnesene from CO2 (0.4 ± 0.01 mg/L and 4.6 ± 0.4 mg/L in 7 days,

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respectively). Indeed, the OverMEP module increased the FPP pools and alleviated the

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growth inhibitions. Subsequently, the farnesene productions were significantly enhanced with

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the insertion of the NSFS or ASF genes. The heterologous expression of the AFS gene in

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SeHL33 resulted in higher farnesene production than that of the NSFS gene. None of the

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engineered strains was shown for α-farnesene accumulation inside a cell.

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Each α-farnesene synthase (AFS and NSFS) used in this study has the RR(x8)W motif, a

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characteristic that plays a role in the reaction mechanism of two sesquiterpene synthases that

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yield an acyclic product, i.e. α-farnesene in this study (see details in the Supporting

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Information; Figure S1). An absolutely conserved aspartate-rich motif (DDxxD) was also

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present in both enzymes. However, the AFS and NSFS genes have been classified as the

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TPBS-b23 and TPS-d18, respectively, based on the phylogenetic analysis of terpene synthases

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(by sharing a 31.7% identity between AFS and NSFS). The difference in α-farnesene

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production levels between the AFS and NSFS in S. elongatus PCC 7942 remains unclear

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owing to missing information on the evolutionary relationship of the terpene synthases, such

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as the specific selective pressure to maintain native α-farnesene production.23,24

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In this study, the best photosynthetic farnesene producer was the strain SeHL32FS, which

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overexpressed the ASF gene in SeHL33. Compared to the farnesene production level of the

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recombinant Anabaena sp. PCC 7129 (305.4 µg/L farnesene in 15 days, equivalent to

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20.4 µg/L/d)14, the production level by SeHL32FS was an order higher (4.6 ± 0.4 mg/L in 7

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days, equivalent to 600 µg/L/d). In addition, the specific farnesene productivity of SeHL32FS 8 ACS Paragon Plus Environment

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(232.2 ± 17.0 µg/L/OD730/d) during the first 3 days was 3.4-fold higher than the maximum

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specific productivity of the recombinant Anabaena sp. PCC 7129 (69.1 µg/L/OD730/d) during

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the same culturing period. The main reasons for the success could be the selection of the

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terpene synthase (the AFS gene) and an increased supply of the FPP pool in engineered

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cyanobacteria.

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Furthermore, specific farnesene production rates of SeHL32FS were calculated during 7

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days of growth (three different periods) (see details in Supporting Information; Figure S2).

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The production rates continued to increase during the growth period to 480.3 µg/L/OD730/d

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from days 3-5 and to 625.2 µg/L/OD730/d from days 5-7. The increased productivities differ

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from the results of recombinant Anabaena sp. PCC 712914, where the rates decreased rapidly.

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Despite the cyanobacterial lifestyles of Anabaena sp. and S. elongatus being significantly

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different, the expression of chromosomally-integrated target genes in S. elongatus PCC 7942

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could be more stable than plasmid-borne genes in Anabaena sp. PCC 7129.

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We demonstrated direct conversion of CO2 to α-farnesene using metabolically engineered

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cyanobacteria at the highest rate reported so far (4.6 ± 0.4 mg/L in 7 days). The OverMEP

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module for MEP pathway optimization has again proven to increase the carbon flux toward

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FPP. Combined with the expression of any terpene synthases, FPP-derived chemical

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production can be achieved directly from CO2 in S. elongatus PCC 7942. Combined with the

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pathway optimization, the activity of farnesene synthase can be improved by increasing gene

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dosages8 or expressing the gene with fusion partners.3,8,25 Further optimization of our

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cyanobacterial strains is necessary to enable a higher production of farnesene from CO2

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through the recent programmable RNA-guided genome engineering.26,27

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Funding



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This work was supported by Korea CCS R&D Center (KCRC) (2017M1A8A1072034) and

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Basic Science Research Program (2017R1A2B2002566, 2017R1A6A3A01011460) through

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the National Research Foundation of Korea, funded by the Korean Government (Ministry of

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Science and ICT). In addition, this work was partially supported by the Golden Seed Project

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(213008-05-1-WT911) grant, funded by the Ministry of Agriculture and the Ministry of

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Oceans and Fisheries.

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Acknowledgements

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The authors thank M. S. Jaeyeon Choi at the Korean Institute of Science and Technology for

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technical support.

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at

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DOI:

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Codon-optimized DNA sequence for each target gene and its original GenBank accession

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number. Figure S1. Protein sequence alignment of NSFS and AFS. Figure S2. Specific

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productivity of α-farnesene in the cyanobacterial culture.

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Notes

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The authors declare that they have no conflict of interest.

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(17) 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, 1. (18) Martin, D. M.; Faldt, J.; Bohlmann, J., Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily. Plant Physiol. 2004, 135, 1908-1927. (19) Pechous, S. W.; Whitaker, B. D., Cloning and functional expression of an ( E, E)alpha-farnesene synthase cDNA from peel tissue of apple fruit. Planta 2004, 219, 84-94. (20) Golden, S. S.; Brusslan, J.; Haselkorn, R., Genetic engineering of the cyanobacterial chromosome. Methods Enzymol. 1986, 153, 215-231. (21) Bentley, F. K.; Melis, A., Diffusion-based process for carbon dioxide uptake and isoprene emission in gaseous/aqueous two-phase photobioreactors by photosynthetic microorganisms. Biotechnol. Bioeng. 2012, 109, 100-109. (22) Tyo, K. E.; Ajikumar, P. K.; Stephanopoulos, G., Stabilized gene duplication enables long-term selection-free heterologous pathway expression. Nat. Biotechnol. 2009, 27, 760765. (23) Nieuwenhuizen, N. J.; Green, S. A.; Chen, X.; Bailleul, E. J.; Matich, A. J.; Wang, M. Y.; Atkinson, R. G., Functional genomics reveals that a compact terpene synthase gene family can account for terpene volatile production in apple. Plant Physiol. 2013, 161, 787804. (24) Bohlmann, J.; Meyer-Gauen, G.; Croteau, R., Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 4126-4133. (25) Formighieri, C.; Melis, A., A phycocyanin.phellandrene synthase fusion enhances recombinant protein expression and beta-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria). Metab. Eng. 2015, 32, 116-124. (26) Gordon, G. C.; Korosh, T. C.; Cameron, J. C.; Markley, A. L.; Begemann, M. B.; Pfleger, B. F., CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab. Eng. 2016, 38, 170-179. (27) Wendt, K. E.; Ungerer, J.; Cobb, R. E.; Zhao, H.; Pakrasi, H. B., CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. Microb. Cell Fact. 2016, 15, 115. (28) Choi, S. Y.; Park, B.; Choi, I. G.; Sim, S. J.; Lee, S. M.; Um, Y.; Woo, H. M., Transcriptome landscape of Synechococcus elongatus PCC 7942 for nitrogen starvation responses using RNA-seq. Sci. Rep. 2016, 6, 30584.

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Figure Legends

318

Figure 1. Scheme of photosynthetic farnesene production from CO2 in engineered S.

319

elongatus PCC 7942. (A) A schematic pathway for farnesene production by expressing

320

heterologous farnesene synthase in the MEP-optimized recombinant S. elongatus PCC 7942

321

(SeHL33). The strain SeHL33 overexpressing the key genes (dxs, idi, and ispA) of the MEP

322

has been developed to supply the pool of FPP6, which is shown in black. FPP is converted to

323

farnesene by heterologous farnesene synthase (shown in red). (B) Schematic diagrams of the

324

construction of S. elongatus PCC 7942 strains that produce farnesene. The dxs gene or the

325

dxs-idi-ispA genes have been introduced to neutral site I (NSI) for supplying the FPP

326

intermediate from CO2 [OverMEP module]. Subsequently, two different heterologous

327

farnesene synthase genes (NSFS or AFS, from Picea abies L. Karst18 or Malus × domestica

328

Borkh19, respectively) were introduced to neutral site II (NSII) of S. elongatus genomic DNA

329

[FS module]. A gel image shows colony-PCR results verifying recombinant S. elongatus

330

strains using a pair of Se1-fw/Se1-rv and Se2-fw/Se2-rv for the NSI and NSII integrations,

331

respectively. The DNA sequences were also verified. The target size of each PCR product for

332

wild-type or mutant cyanobacteria was: wild type (1.6 kb), SeHL11FS (6.4 kb), SeHL12FS

333

(6.4 kb), SeHL31FS (7.9 kb), and SeHL32FS (7.9 kb) at NSI and wild type (2.8 kb),

334

SeHL01FS (6.8 kb), SeHL02FS (6.7 kb), SeHL11FS (6.8 kb), SeHL12FS (6.7 kb),

335

SeHL31FS (6.8 kb), and SeHL32FS (6.7 kb) at NSII. The genotypes of the recombinant

336

strains SeHL01FS, SeHL02FS, SeHL11FS, SeHL12FS, SeHL31FS, and SeHL32FS are

337

presented in Table 1. Abbreviations of the corresponding enzymes are: Dxs (Ec), 1-deoxy-D-

338

xylulose-5-phosphate synthase of E. coli; Idi, isopentenyl diphosphate isomerase of E. coli;

339

IspA (Ec), farnesyl diphosphate synthase (IspA) of E. coli; FS (Apple fruit), farnesene

340

synthase of Malus × domestica Borkh.; G3P, glyceraldehyde 3-phosphate; DXP, 1-deoxy-D-



MEP,

2-C-methyl-D-erythritol-4-phosphate;

Page 14 of 19

341

xylulose-5-phosphate;

342

diphosphocytidyl-2-C-methyl-D-erythritol; CDP-MEP,

343

erythritol-2-phosphate; MEcPP, 2C-methyl-D-erythritol-2,4-cyclodiphosphate; HMBPP, (E)-

344

4-hydroxy-3-methylbut-2-enyl-diphosphate;

345

dimethylallyl diphosphate; and FPP, farnesyl diphosphate.

IPP,

CDP-ME,

4-

4-diphosphocytidyl-2C-methyl-D-

isopentenyl

diphosphate;

DMAPP,

346 347

Figure 2. Measurement of extracellular farnesene from engineered S. elongatus PCC 7942

348

strains. The recombinant strain was cultivated and measured at OD730 under 5% (v/v) CO2

349

bubbling with a 20% (v/v) dodecane overlay. Photographic images and schematic diagram

350

are show in the upper panel. The samples from the dodecane overlay were analyzed by GC-

351

MS for quantification of farnesene. Authentic standard (β-farnesene) and internal standard

352

(IS; β-caryophyllene) were used. Retention times and relative abundances are shown for both

353

the standard and the sample.

354 355

Figure 3. Photosynthetic farnesene produced from CO2 in engineered S. elongatus PCC

356

7942. Engineered S. elongatus PCC 7942 strains were cultivated under 5% (v/v) CO2

357

bubbling in the presence of a 20% (v/v) dodecane overlay. Measurements of cell growth and

358

farnesene production (mg/L) were performed for the recombinant strains. The genotypes of

359

the recombinant strains are presented in Table 1. All data are expressed as mean ± standard

360

deviation from cultures run in triplicate. N.D., not detected; LOD, 0.045 mg/L; LOQ,

361

0.136 mg/L.


Page 15 of 19


Table 1. Bacterial strains and plasmids used in this study.

Table 1. Bacterial strains and plasmids used in this study. Strain or plasmid

Relevant characteristics

References

Strains E. coli HIT-DH5α S. elongatus PCC 7942

F-(80d lacZ M15) (lacZYA-argF) U169 hsdR17(r– m+) recA1 endA1 relA1 deoR96 Wild type (ATCC 33912)

RBC Bioscience ATCC

SeHL11

S. elongatus PCC 7942 NSI::Bb1s-dxs

SeHL33

S. elongatus PCC 7942 NSI::Bb1s-dxs-idi-ispA

SeHL01FS

S. elongatus PCC 7942 NSII::Bb1k-NSFS

This study

SeHL02FS

S. elongatus PCC 7942 NSII::Bb1k-AFS

This study

SeHL11FS SeHL12FS SeHL31FS SeHL32FS

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

This study 28

This study This study This study This study

Plasmid pUC, Kmr, LacI, Ptrc, BglBrick sites, NSII target 15 sites, SyneBrick Vector pUC, Kmr, LacI, Ptrc, NSII target sites, pSe2Bb1k-NSFS the farnesene synthase gene originated from Picea This study abies L. Karst18; the NSFS gene(se.co) pUC, Kmr, LacI, Ptrc, NSII target sites, the farnesene synthase gene (AFS) originated from This study pSe2Bb1k-AFS Malus × domestica Borkh19.; the AFS gene(se.co) Note: (se.co) represents that the gene sequence is codon-optimized to S. elongatus PCC 7942. pSe2Bb1k-GFP



Figure 1.


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Figure 2.



Figure 3.


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Graphic for table of contents


Direct conversion of CO2 to Î±-farnesene using metabolically

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